Chronic lymphocytic leukemia (CLL) growth is dependent on both B cell receptor (BCR) signaling and signals from microenvironmental T helper (Th) cells. We previously described a mechanism where Th cells enhance BCR signaling and proliferation through CD45 phosphatase activity regulation via galectin-1 and CD43. The CLL negative prognostic indicator CD38 is linked to BCR signaling and proliferation, with its expression induced by Th cells. Here, we explore a link between CD38 and CD45 phosphatase activity regulation using patient-derived material in a Th-CLL cell co-culture model. Results demonstrate CD43 and galectin-1 are co-expressed with CD38, defining proliferative CLL cells with augmented CD45 activity. CD38 enzymatic and receptor inhibition regulated CD43 and galectin-1 expression, CD45 activityhi populations, and CLL proliferation, while leaving Th cells largely unaffected. Mechanistically, CD38- or LGALS1 (galectin-1)-deficient malignant B cell lines further confirmed CD38-mediated regulation of CD45 activity and BCR signaling through CD43 expression and galectin-1 surface binding, while galectin-1 contributed to CD43/CD45 colocalization. Together, this highlights CD38 as an important regulator of CD45 activity via CD43 and galectin-1, in turn acting as a positive modulator of CLL proliferation. Ultimately, the CD38/CD45 molecular hub could be an important therapeutic target in CLL.

Coronavirus disease 2019, or COVID-19, is a major challenge facing scientists worldwide. Alongside the lungs, the system of organs comprising the GI tract is commonly targeted by COVID-19. The dysbiotic modulations in the intestine influence the disease severity, potentially due to the ability of the intestinal microbiota to modulate T lymphocyte functions, i.e., to suppress or activate T cell subpopulations. The interplay between the lungs and intestinal microbiota is named the gut–lung axis. One of the most usual comorbidities in COVID-19 patients is type 2 diabetes, which induces changes in intestinal microbiota, resulting in a pro-inflammatory immune response, and consequently, a more severe course of COVID-19. However, changes in the microbiota in this comorbid pathology remain unclear. Metformin is used as a medication to treat type 2 diabetes. The use of the type 2 diabetes drug metformin is a promising treatment for this comorbidity because, in addition to its hypoglycemic action, it can increase amount of intestinal bacteria that induce regulatory T cell response. This dual activity of metformin can reduce lung damage and improve the course of the COVID-19 disease.

Chronic lymphocytic leukaemia (CLL) is characterised by malignant mature-like B cells. Supportive to CLL cell survival is chronic B-cell receptor (BCR) signalling; however, emerging evidence demonstrates CLL cells proliferate in response to T-helper (Th) cells in a CD40L-dependent manner. We showed provision of Th stimulation via CD40L upregulated CD45 phosphatase activity and BCR signalling in non-malignant B cells. Consequently, we hypothesised Th cell upregulation of CLL cell CD45 activity may be an important regulator of CLL BCR signalling and proliferation. Using patient-derived CLL cells in a culture system with activated autologous Th cells, results revealed increases in both Th and CLL cell CD45 activity, which correlated with enhanced downstream antigen receptor signalling and proliferation. Concomitantly increased was the surface expression of Galectin-1, a CD45 ligand, and CD43, a CLL immunophenotypic marker. Galectin-1/CD43 double expression defined a proliferative CLL cell population with enhanced CD45 activity. Targeting either Galectin-1 or CD43 using silencing, pharmacology, or monoclonal antibody strategies dampened CD45 activity and CLL cell proliferation. These results highlight a mechanism where activated Th cells drive CLL cell BCR signalling and proliferation via Galectin-1 and CD43-mediated regulation of CD45 activity, identifying modulation of CD45 phosphatase activity as a potential therapeutic target in CLL.

Summary Humoral immunity relies on the efficient differentiation of memory B cells (MBCs) into antibody-secreting cells (ASCs). T helper (Th) signals upregulate B cell receptor (BCR) signaling by potentiating Src family kinases through increasing CD45 phosphatase activity (CD45 PA). In this study, we show that high CD45 PA in MBCs enhances BCR signaling and is essential for their effective ASC differentiation. Mechanistically, Th signals upregulate CD45 PA through intensifying the surface binding of a CD45 ligand, Galectin-1. CD45 PA works as a sensor of T cell help and defines high-affinity germinal center (GC) plasma cell (PC) precursors characterized by IRF4 expression in vivo. Increasing T cell help in vitro results in an incremental CD45 PA increase and enhances ASC differentiation by facilitating effective induction of the transcription factors IRF4 and BLIMP1. This study connects Th signals with BCR signaling through Galectin-1-dependent regulation of CD45 PA and provides a mechanism for efficient ASC differentiation of MBCs.

Coordination of intracellular Ca2+ signaling in parotid acini is crucial for controlling the secretion of primary saliva. Previous work from our lab has demonstrated acidic-organelle Ca2+ release as a participant in agonist-evoked signaling dynamics of the parotid acinar cell. Furthermore, results implicated a potential role for the potent Ca2+ releasing second messenger NAADP in these events. The current study interrogated a direct role of NAADP for Ca2+ signaling in the parotid salivary gland acinar cell. Use of live-cell Ca2+ imaging, patch-clamp methods, and confocal microscopy revealed for the first time NAADP can evoke or enhance Ca2+ dynamics in parotid acini. These results were compared with pancreatic acini, a morphologically similar cell type previously shown to display NAADP-dependent Ca2+ signals. Findings presented here may be relevant in establishing new therapeutic targets for those suffering from xerostomia produced by hypofunctioning salivary glands.

Autonomic neural activation of intracellular Ca 2+ release in parotid acinar cells induces the secretion of the fluid and protein components of primary saliva critical for maintaining overall oral homeostasis. In the current study, we profiled the role of acidic organelles in shaping the Ca 2+ signals of parotid acini using a variety of imaging and pharmacological approaches. Results demonstrate that zymogen granules predominate as an apically polarized population of acidic organelles that contributes to the initial Ca 2+ release. Moreover, we provide evidence that indicates a role for the intracellular messenger NAADP in the release of Ca 2+ from acidic organelles following elevation of cAMP. Our data are consistent with the “trigger” hypothesis where localized release of Ca 2+ sensitizes canonical intracellular Ca 2+ channels to enhance signals from the endoplasmic reticulum. Release from acidic stores may be important for initiating saliva secretion at low levels of stimulation and a potential therapeutic target to augment secretory activity in hypofunctioning salivary glands.

Imbery JF, Bhattacharya S, Khuder S, Weiss A, Goswamee P, Iqbal AK, Giovannucci DR. cAMP-dependent recruitment of acidic organelles for Ca signaling in the salivary gland. Am J Physiol Cell Physiol 311: C697–C709, 2016. First published September 7, 2016; doi:10.1152/ajpcell.00010.2016.—Autonomic neural activation of intracellular Ca release in parotid acinar cells induces the secretion of the fluid and protein components of primary saliva critical for maintaining overall oral homeostasis. In the current study, we profiled the role of acidic organelles in shaping the Ca signals of parotid acini using a variety of imaging and pharmacological approaches. Results demonstrate that zymogen granules predominate as an apically polarized population of acidic organelles that contributes to the initial Ca release. Moreover, we provide evidence that indicates a role for the intracellular messenger NAADP in the release of Ca from acidic organelles following elevation of cAMP. Our data are consistent with the “trigger” hypothesis where localized release of Ca sensitizes canonical intracellular Ca channels to enhance signals from the endoplasmic reticulum. Release from acidic stores may be important for initiating saliva secretion at low levels of stimulation and a potential therapeutic target to augment secretory activity in hypofunctioning salivary glands.

Calcium signaling in response to autonomic neural input drives fluid and protein secretion within salivary glands. Previous research focused primarily on the canonical IP3-mediated calcium release from ER stores. However, other important reservoirs for calcium release exist such as lysosomes, endosomes, and secretory granules. Whether and how these acidic stores are activated remains largely undefined in terms of their contributions to global calcium signals, protein exocytosis and saliva production in salivary tissue. To address this question we used lysotracker red, a fluorescent dye that labels acidic organelles. Confocal images demonstrated that mouse parotid acini had abundant stores that were apically located and whose pH gradients were dissipated by treatment with the vacuolar H-ATPase inhibitor bafilomycin A1. In contrast, parotid ductal cells displayed only a punctate and dispersed or perinuclear labeling. The functional effect of bafilomycin treatment was tested using live cell imaging methods. Bafilomycin treatment resulted in a two-thirds diminishment of peak calcium release amplitudes evoked by a threshold level of carbachol stimulation. The diminishment in peak amplitude and in the rate of rise of the evoked calcium signal was only evident in cells where cAMP was first elevated by either forskolin or the beta-adrenergic agonist isoproterenol. There is evidence that acidic store calcium release can be triggered by the dinucleotide second messenger NAADP. Consistent with this, NED19, a NAADP receptor antagonist, diminished by half the peak calcium response. This data indicates that calcium release from acidic stores in mouse parotid acinar cells is cAMP-dependent and may be mediated by NAADP. In addition, bafilomycin and NED19 treatment significantly reduced exocytotic secretory activity, even without cAMP elevation, supporting the idea that acidic stores may contribute to vesicle fusion and protein release.

The subthalamic nucleus (STN) is a part of the basal ganglia (BG) and thus has a role in modulating voluntary motor function. Dysfunction of STN neuronal activity is thought to underlie a variety of movement disorders. For example, in Parkinson's disease (PD), abnormalities in STN activity correlate

Isolated clusters of mouse parotid acinar cells in combination with live cell imaging were used to explore the crosstalk in molecular signaling between purinergic, cholinergic and adrenergic pathways that integrate to control fluid and protein secretion. This crosstalk was manifested by (1) β-adrenergic receptor activation and amplification of P2X4R evoked Ca2+ signals, (2) β-adrenergic-induced amplification of P2X7R-evoked Ca2+ signals and (3) muscarinic receptor induced activation of P2X7Rs via exocytotic activity. The findings from our study reveal that purinoceptor-mediated Ca2+ signaling is modulated by crosstalk with canonical signaling pathways in parotid acinar cells. Integration of these signals are likely important for dynamic control of saliva secretion to match physiological demand in the parotid gland.