Leishmania disease expression has been linked to IL-10. In this study, we investigated the regulation of IL-10 production by macrophages infected with Leishmania donovani. Infection of either murine or human macrophages brought about selective phosphorylation of Akt-2 in a PI3K-dependent manner. These events were linked to phosphorylation and inactivation of glycogen synthase kinase-3β (GSK-3β) at serine 9, as the latter was abrogated by inhibition of either PI3K or Akt. One of the transcription factors that is negatively regulated by GSK-3β is CREB, which itself positively regulates IL-10 expression. Infection of macrophages with leishmania induced phosphorylation of CREB at serine 133, and this was associated with enhanced CREB DNA binding activity and induction of IL-10. Similar to phosphorylation of GSK-3β, both phosphorylation of CREB at serine 133 and CREB DNA binding activity were abrogated in cells treated with inhibitors of either PI3K or Akt prior to infection. Furthermore, disruption of this pathway either by inhibition of Akt or by overexpression of GSK-3β markedly attenuated IL-10 production in response to leishmania. Thus, GSK-3β negatively regulates myeloid cell IL-10 production in response to leishmania. Switching off GSK-3β promotes disease pathogenesis.

Mononuclear phagocytes are critical modulators and effectors of innate and adaptive immune responses, and PI-3Ks have been shown to be multifunctional monocyte regulators. The PI-3K family includes eight catalytic isoforms, and only limited information is available about how these contribute to fine specificity in monocyte cell regulation. We examined the regulation of phagocytosis, the phagocyte oxidative burst, and LPSinduced cytokine production by human monocytic cells deficient in p110␣ PI-3K. We observed that p110␣ PI-3K was required for phagocytosis of IgG-opsonized and nonopsonized zymosan in differentiated THP-1 cells, and the latter was inhibitable by mannose. In contrast, p110␣ PI-3K was not required for ingestion serum-opsonized zymosan. Taken together, these results suggest that Fc␥R-and mannose receptor-mediated phagocytosis are p110␣-dependent, whereas CR3-mediated phagocytosis involves a distinct isoform. It is notable that the phagocyte oxidative burst induced in response to PMA or opsonized zymosan was also found to be dependent on p110␣ in THP-1 cells. Furthermore, p110␣ was observed to exert selective and bidirectional effects on the secretion of pivotal cytokines. Incubation of p110␣-deficient THP-1 cells with LPS showed that p110␣ was required for IL-12p40 and IL-6 production, whereas it negatively regulated the production of TNF-␣ and IL-10. Cells deficient in p110␣ also exhibited enhanced p38 MAPK, JNK, and NF-B phosphorylation. Thus, p110␣ PI-3K appears to uniquely regulate important monocyte functions, where other PI-3K isoforms are uninvolved or unable to fully compensate. J. Leukoc. Biol. 81: 1548 -1561; 2007.

Islet transplantation is a promising treatment for diabetes. However, it faces several challenges including requirement of systemic immunosuppression. Indoleamine 2,3-dioxygenase (IDO), a tryptophan degrading enzyme, is a potent immunomodulatory factor. Local expression of IDO in bystander fibroblasts suppresses islet allogeneic immune response in vitro. The aim of the present study was to investigate the impact of IDO on viability and function of mouse islets embedded within IDO-expressing fibroblastpopulated collagen scaffold. Mouse islets were embedded within collagen matrix populated with IDO adenovector-transduced or control fibroblasts. Proliferation, insulin content, glucose responsiveness, and activation of general control nonderepressible-2 kinase stress-responsive pathway were then measured in IDOexposed islets. In vivo viabilities of composite islet grafts were also tested in a syngeneic diabetic animal model. No reduction in islet cells proliferation was detected in both IDO-expressing and control composites compared to the baseline rates. Islet functional studies showed normal insulin content and secretion in both preparations. In contrast to lymphocytes, general control nonderepressible-2 kinase pathway was not activated in islets cocultured with IDO-expressing fibroblasts. When transplanted to diabetic mice, syngeneic IDO-expressing composite islet grafts were functional up to 100 days tested. These findings collectively confirm normal viability and functionality of islets cocultured with IDO-expressing cells and indicate the feasibility of development of a functional nonrejectable islet graft.

Immature cells of the mononuclear phagocyte series differentiate in response to calcitriol. This is accompanied by increased expression of both CD11b and CD14 and has been shown to be phosphatidylinositol 3-kinase (PI3K) dependent. The events downstream of PI3K that regulate mononuclear phagocyte gene expression, however, remain to be fully understood. In the present study, we show that incubation of THP-1 cells with calcitriol brings about activation of the myeloid zinc finger-1 (MZF-1) transcription factor dependent upon PI3K. In addition, we show that the proximal promoter regions of both CD11b and CD14 contain functional MZF-1 binding sites that are calcitriol responsive. Site-directed mutagenesis of the putative MZF-1 elements abolished MZF-1 binding to the promoters of both CD11b and CD14. Not only did calcitriol treatment increase MZF-1 DNA binding activity to these sites, but it also up-regulated cellular levels of MZF-1. Silencing of MZF-1 resulted in a markedly blunted response to calcitriol for induction of both CD11b and CD14 mRNA transcript levels. Cell surface expression of CD11b and CD14 was also reduced, but to a lesser extent. Taken together, these results show that MZF-1 is involved downstream of PI3K in a calcitriol-induced signaling pathway leading to myeloid cell differentiation and activation of CD11b and CD14.

Many of the biological activities of IFN-γ are mediated through the IFN-γR3-linked Jak-Stat1α pathway. However, regulation of IFN-γ signaling is not fully understood, and not all responses to IFN-γ are Stat1α dependent. To identify novel elements involved in IFN-γ cell regulation, the cytoplasmic domain of the R2 subunit of the human IFN-γR was used as bait in a yeast two-hybrid screen of a human monocyte cDNA library. This identified annexin A5 (AxV) as a putative IFN-γR binding protein. The interaction was confirmed in pull-down experiments in which a GST-R2 cytoplasmic domain fusion protein was incubated with macrophage lysates. Furthermore, immunoprecipitation using anti-IFN-γR2 Abs showed that AxV interacted with IFN-γR2 to form a stable complex following incubation of cells with IFN-γ. In 293T cells with reduced expression of AxV, brought about by small interfering RNA targeting, activation of Jak2 and Stat1α in response to IFN-γ was enhanced. Inhibition of cell proliferation, a hallmark of the IFN-γ response, also was potentiated in HeLa cells treated with small interfering RNA directed at AxV. Taken together, these results suggest that through an inducible association with the R2 subunit of the IFN-γR, AxV modulates cellular responses to IFN-γ by modulating signaling through the Jak-Stat1 pathway.