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Mitochondrial Ca2+ handling is accomplished by balancing Ca2+ uptake, primarily via the Ru360-sensitive mitochondrial calcium uniporter (MCU), Ca2+ buffering in the matrix and Ca2+ efflux mainly via Ca2+ ion exchangers, such as the Na+/Ca2+ exchanger (NCLX) and the Ca2+/H+ exchanger (CHE). The mechanism of CHE in cardiac mitochondria is not well-understood and its contribution to matrix Ca2+ regulation is thought to be negligible, despite higher expression of the putative CHE protein, LETM1, compared to hepatic mitochondria. In this study, Ca2+ efflux via the CHE was investigated in isolated rat cardiac mitochondria and permeabilized H9c2 cells. Mitochondria were exposed to (a) increasing matrix Ca2+ load via repetitive application of a finite CaCl2 bolus to the external medium and (b) change in the pH gradient across the inner mitochondrial membrane (IMM). Ca2+ efflux at different matrix Ca2+ loads was revealed by inhibiting Ca2+ uptake or reuptake with Ru360 after increasing number of CaCl2 boluses. In Na+-free experimental buffer and with Ca2+ uptake inhibited, the rate of Ca2+ efflux and steady-state free matrix Ca2+ [mCa2+]ss increased as the number of administered CaCl2 boluses increased. ADP and cyclosporine A (CsA), which are known to increase Ca2+ buffering while maintaining a constant [mCa2+]ss, decreased the rate of Ca2+ efflux via the CHE, with a significantly greater decrease in the presence of ADP. ADP also increased Ca2+ buffering rate and decreased [mCa2+]ss. A change in the pH of the external medium to a more acidic value from 7.15 to 6.8∼6.9 caused a twofold increase in the Ca2+ efflux rate, while an alkaline change in pH from 7.15 to 7.4∼7.5 did not change the Ca2+ efflux rate. In addition, CHE activation was associated with membrane depolarization. Targeted transient knockdown of LETM1 in permeabilized H9c2 cells modulated Ca2+ efflux. The results indicate that Ca2+ efflux via the CHE in cardiac mitochondria is modulated by acidic buffer pH and by total matrix Ca2+. A mechanism is proposed whereby activation of CHE is sensitive to changes in both the matrix Ca2+ buffering system and the matrix free Ca2+ concentration. [ABSTRACT FROM AUTHOR]

STAT4 is critical for the production of IFN-γ during the generation of Th1 immune responses. We investigated the role of STAT4 in mediating Th1-inducing activity of a vaccine adjuvant monophosphoryl lipid A (MPL-A) using the standard antigen ovalbumin (OVA) in STAT4KO mice. Our results show that splenocytes from STAT4KO mice displayed lower OVA-specific T-cell proliferation and IL-2 production compared with wild-type (WT) mice. Further, IFN-γ production was diminished in STAT4KO-derived splenocytes but the levels of IL-12 and TNF-α were similar compared with WT mice. Interestingly, STAT4 deficiency also led to a decrease in IL-10 and Th2 cytokines such as IL-4 and IL-13 upon MPL-A immunization, although IL-17 production was similar between WT- and STAT4KOderived splenocytes. Our observations for defective Th1 and Th2 responses in STAT4KO mice were further supported by the low levels of Th1-associated IgG2a and Th2-associated IgG1 in the sera of these mice. Taken together, our results show that STAT4 plays a critical role in mediating both Th1 and Th2 responses upon immunization with MPL-A. Our study provides a better understanding of how MPL-A mediates T-cell activation which will be critical for future vaccine development. [ABSTRACT FROM AUTHOR]

Ibrutinib (PCI-32765) is an irreversible dual Btk/Itk inhibitor shown to be effective in treating several B cell malignancies. However, limited studies have been conducted to study the effect of this drug on myeloid cell function. Hence, we studied the effect of ibrutinib treatment on TLR-4 mediated activation of bone marrow derived dendritic cell culture (DCs). Upon ibrutinib treatment, LPS-treated DCs displayed lower synthesis of TNF-α and nitric oxide (NO) and higher induction of IL-6, TGF-β, IL-10 and IL-18. While ibrutinib dampened MHC-II and CD86 expression on DCs, CD80 expression was upregulated. Further, ibrutinib-treated DCs promoted T cell proliferation and enhanced IL-17 production upon co-culture with nylon wool enriched T cells. Taken together, our results indicate that ibrutinib modulates TLR-4 mediated DC activation to promote an IL-17 response. We describe a novel mode of action for ibrutinib on DCs which should be explored to treat other forms of cancer besides B cell malignancies. [ABSTRACT FROM PUBLISHER]

Mitochondrial Ca 2+ handling is accomplished by balancing Ca 2+ uptake, primarily via the Ru360-sensitive mitochondrial calcium uniporter (MCU), Ca 2+ buffering in the matrix and Ca 2+ efflux mainly via Ca 2+ ion exchangers, such as the Na + /Ca 2+ exchanger (NCLX) and the Ca 2+ /H + exchanger (CHE). The mechanism of CHE in cardiac mitochondria is not well-understood and its contribution to matrix Ca 2+ regulation is thought to be negligible, despite higher expression of the putative CHE protein, LETM1, compared to hepatic mitochondria. In this study, Ca 2+ efflux via the CHE was investigated in isolated rat cardiac mitochondria and permeabilized H9c2 cells. Mitochondria were exposed to (a) increasing matrix Ca 2+ load via repetitive application of a finite CaCl 2 bolus to the external medium and (b) change in the pH gradient across the inner mitochondrial membrane (IMM). Ca 2+ efflux at different matrix Ca 2+ loads was revealed by inhibiting Ca 2+ uptake or reuptake with Ru360 after increasing number of CaCl 2 boluses. In Na + -free experimental buffer and with Ca 2+ uptake inhibited, the rate of Ca 2+ efflux and steady-state free matrix Ca 2+ [mCa 2+ ] ss increased as the number of administered CaCl 2 boluses increased. ADP and cyclosporine A (CsA), which are known to increase Ca 2+ buffering while maintaining a constant [mCa 2+ ] ss , decreased the rate of Ca 2+ efflux via the CHE, with a significantly greater decrease in the presence of ADP. ADP also increased Ca 2+ buffering rate and decreased [mCa 2+ ] ss. A change in the pH of the external medium to a more acidic value from 7.15 to 6.8∼6.9 caused a twofold increase in the Ca 2+ efflux rate, while an alkaline change in pH from 7.15 to 7.4∼7.5 did not change the Ca 2+ efflux rate. In addition, CHE activation was associated with membrane depolarization. Targeted transient knockdown of LETM1 in permeabilized H9c2 cells modulated Ca 2+ efflux. The results indicate that Ca 2+ efflux via the CHE in cardiac mitochondria is modulated by acidic buffer pH and by total matrix Ca 2+ . A mechanism is proposed whereby activation of CHE is sensitive to changes in both the matrix Ca 2+ buffering system and the matrix free Ca 2+ concentration., (Copyright © 2020 Natarajan, Glait, Mishra, Stowe, Camara and Kwok.)