Human dendritic cells discriminate between viable and killed Toxoplasma gondii tachyzoites: dendritic cell activation after infection with viable parasites results in CD28 and CD40 ligand signaling that controls IL-12-dependent and -independent T cell production of IFN-gamma

We studied how the interaction between human dendritic cells (DC) and Toxoplasma gondii influences the generation of cell-mediated immunity against the parasite. We demonstrate that viable, but not killed, tachyzoites of T. gondii altered the phenotype of immature DC. DC infected with viable parasites up-regulated the expression of CD40, CD80, CD86, and HLA-DR and down-regulated expression of CD115. These changes are indicative of DC activation induced by T. gondii. Viable and killed tachyzoites had contrasting effects on cytokine production. DC infected with viable T. gondii rather than DC that phagocytosed killed parasites induced secretion of high amounts of IFN-gamma by T cells from T. gondii-seronegative donors. IFN-gamma production in response to DC infected with viable parasites required CD28 and CD40 ligand (CD40L) signaling. In addition, this IFN-gamma response was dependent in part on IL-12 secretion. Production of IL-12 p70 occurred after interaction between T cells and DC infected with viable T. gondii, but not after incubation of T cells with DC plus killed tachyzoites. IL-12 synthesis was inhibited by blockade of CD40L signaling. IL-12-independent IFN-gamma production required CD80/CD86-CD28 interaction and, to a lesser extent, CD40-CD40L signaling. Taken together, T. gondii-induced activation of human DC is associated with T cell production of IFN-gamma through CD40-CD40L-dependent release of IL-12 and through CD80/CD86-CD28 and CD40-CD40L signaling that mediate IFN-gamma secretion even in the absence of bioactive IL-12.     

PI3K regulates the activation of NLRP3 inflammasome in atherosclerosis through part-dependent AKT signaling pathway

PI3K is a downstream target of multiple cell-surface receptors, which acts as a crucial modulator of both cell polarization and survival. PI3K/AKT signaling pathway is commonly involved in cancer, atherosclerosis, and other diseases. However, its role in cardiovascular diseases, especially in atherosclerosis, remains to be further investigated. To determine the effect of PI3K/AKT signaling pathway on cellular inflammatory response and oxidative stress, PI3K inhibitor (GDC0941) and AKT inhibitor (MK2206) were used. First, THP-1 cells were incubated with ox-LDL (100 µg/ml) to establish an in vitro atherosclerosis model. The inflammatory factors and foam cell formation were then evaluated to ascertain and compare the effects of PI3K and AKT inhibition. ApoE^−/− mice fed a high-fat diet were used to assess the roles of PI3K and AKT in aortic plaque formation. Our results showed that the inhibition of PI3K or AKT could suppress the activation of NLRP3, decreased the expression levels of p-p65/p65 and reduced the production of mitochondrial reaction oxygen species (mitoROS) in THP-1 cells. Inhibition of PI3K or AKT could also reduced atherosclerosis lesion and plaque area, and decreased the levels of NLRP3 and IL-1β in ApoE^−/− mice. The effect of PI3K inhibition was more significant than AKT. Therefore, PI3K inhibition can retard the progress of atherosclerosis. Besides, there may be other AKT-independent pathways that regulate the formation of atherosclerosis.     

Modulation of the development of human monocyte‐derived dendritic cells by lithium chloride

Lithium has been used or explored to treat psychiatric and neurodegenerative diseases that are frequently associated with an abnormal immune status. It is likely that lithium may work through modulation of immune responses in these patients. Because dendritic cells (DC) play a central role in regulating immune responses, this study investigated the influence of lithium chloride (LiCl) on the development and function of DC. Exposure to LiCl during the differentiation of human monocyte‐derived immature DCs (iDC) enhances CD86 and CD83 expression and increases the production of IL‐1β, IL‐6, IL‐8, IL‐10, and TNF‐α. However, the presence of LiCl during LPS‐induced maturation of iDC has the opposite effect. During iDC differentiation, LiCl suppresses the activity of glycogen synthase kinase (GSK)‐3β, and activates PI3K and MEK. In addition, LiCl activates peroxisome proliferator‐activated receptor γ (PPARγ) during iDC differentiation, a pathway not described before. Each of these signaling pathways appears to have distinct impact on the differentiating iDC. The enhanced CD86 expression by LiCl involves the PI3K/AKT and GSK‐3β pathway. LiCl modulates the expression of CD83 in iDC mainly through MEK/ERK, PI3K/AKT, and PPARγ pathways, while the increased production of IL‐1β and TNF‐α mainly involves the MEK/ERK pathway. The effect of LiCl on IL‐6/IL‐8/IL‐10 secretion in iDC is mediated through inhibition of GSK‐3β. We have also demonstrated that PPARγ is downstream of GSK‐3β and is responsible for the LiCl‐mediated modulation of CD86/83 and CD1 expression, but not IL‐6/8/10 secretion. The combined influence of these molecular signaling pathways may account for certain clinical effect of lithium. J. Cell. Physiol. 226: 424–433, 2011. © 2010 Wiley‐Liss, Inc.     

Detailed role of microRNA-mediated regulation of PI3K/AKT axis in human tumors

The regulation of signal transmission and biological processes, such as cell proliferation, apoptosis, metabolism, migration, and angiogenesis are greatly influenced by the PI3K/AKT signaling pathway. Highly conserved endogenous non-protein-coding RNAs known as microRNAs (miRNAs) have the ability to regulate gene expression by inhibiting mRNA translation or mRNA degradation. MiRNAs serve key role in PI3K/AKT pathway as upstream or downstream target, and aberrant activation of this pathway contributes to the development of cancers. A growing body of research shows that miRNAs can control the PI3K/AKT pathway to control the biological processes within cells. The expression of genes linked to cancers can be controlled by the miRNA/PI3K/AKT axis, which in turn controls the development of cancer. There is also a strong correlation between the expression of miRNAs linked to the PI3K/AKT pathway and numerous clinical traits. Moreover, PI3K/AKT pathway-associated miRNAs are potential biomarkers for cancer diagnosis, therapy, and prognostic evaluation. The role and clinical applications of the PI3K/AKT pathway and miRNA/PI3K/AKT axis in the emergence of cancers are reviewed in this article.     

Extracellular acidosis triggers the maturation of human dendritic cells and the production of IL-12

Although the development of an acidic tissue environment or acidosis is a hallmark of inflammatory processes, few studies analyze the effect of extracellular pH on immune cells. We have previously shown that exposure of murine dendritic cells (DCs) to pH 6.5 stimulates macropinocytosis and cross-presentation of extracellular Ags by MHC class I molecules. We report that the transient exposure of human DCs to pH 6.5 markedly increases the expression of HLA-DR, CD40, CD80, CD86, CD83, and CCR7 and improves the T cell priming ability of DCs. Incubation of DCs at pH 6.5 results in the activation of the PI3K/Akt and the MAPK pathways. Using specific inhibitors, we show that the maturation of DCs induced by acidosis was strictly dependent on the activation of p38 MAPK. DC exposure to pH 6.5 also induces a dramatic increase in their production of IL-12, stimulating the synthesis of IFN-gamma, but not IL-4, by Ag-specific CD4(+) T cells. Interestingly, we find that suboptimal doses of LPS abrogated the ability of pH 6.5 to induce DC maturation, suggesting a cross-talk between the activation pathways triggered by LPS and extracellular protons in DCs. We conclude that extracellular acidosis in peripheral tissues may contribute to the initiation of adaptive immune responses by DCs, favoring the development of Th1 immunity.     

The TRAF3 adaptor protein drives proliferation of anaplastic large cell lymphoma cells by regulating multiple signaling pathways

T cells devoid of tumor necrosis factor receptor associated factor-3 (Traf3) exhibit decreased proliferation, sensitivity to apoptosis, and an improper response to antigen challenge. We therefore hypothesized that TRAF3 is critical to the growth of malignant T cells. By suppressing TRAF3 protein in different cancerous T cells, we found that anaplastic large cell lymphoma (ALCL) cells require TRAF3 for proliferation. Since reducing TRAF3 results in aberrant activation of the noncanonical nuclear factor-κB (NF-κB) pathway, we prevented noncanonical NF-κB signaling by suppressing RelB together with TRAF3. This revealed that TRAF3 regulates proliferation independent of the noncanonical NF-κB pathway. However, suppression of NF-κB-inducing kinase (NIK) along with TRAF3 showed that high levels of NIK have a partial role in blocking cell cycle progression. Further investigation into the mechanism by which TRAF3 regulates cell division demonstrated that TRAF3 is essential for continued PI3K/AKT and JAK/STAT signaling. In addition, we found that while NIK is dispensable for controlling JAK/STAT activity, NIK is critical to regulating the PI3K/AKT pathway. Analysis of the phosphatase and tensin homolog (PTEN) showed that NIK modulates PI3K/AKT signaling by altering the localization of PTEN. Together our findings implicate TRAF3 as a positive regulator of the PI3K/AKT and JAK/STAT pathways and reveal a novel function for NIK in controlling PI3K/AKT activity. These results provide further insight into the role of TRAF3 and NIK in T cell malignancies and indicate that TRAF3 differentially governs the growth of B and T cell cancers.     

Reactivation of AKT signaling following treatment of cancer cells with PI3K inhibitors attenuates their antitumor effects

Targeting the phosphatidylinositol-3-kinase (PI3K) is a promising approach in cancer therapy. In particular, PI3K blockade leads to the inhibition of AKT, a major downstream effector responsible for the oncogenic activity of PI3K. However, we report here that small molecule inhibitors of PI3K only transiently block AKT signaling. Indeed, treatment of cancer cells with PI3K inhibitors results in a rapid inhibition of AKT phosphorylation and signaling which is followed by the reactivation of AKT signaling after 48 h as observed by Western blot. Reactivation of AKT signaling occurs despite effective inhibition of PI3K activity by PI3K inhibitors. In addition, wortmannin, a broad range PI3K inhibitor, did not block AKT reactivation suggesting that AKT signals independently of PI3K. In a therapeutical perspective, combining AKT and PI3K inhibitors exhibit stronger anti-proliferative and pro-apoptotic effects compared to AKT or PI3K inhibitors alone. Similarly, in a tumor xenograft mouse model, concomitant PI3K and AKT blockade results in stronger anti-cancer activity compared with either blockade alone. This study shows that PI3K inhibitors only transiently inhibit AKT which limits their antitumor activities. It also provides the proof of concept to combine PI3K inhibitors with AKT inhibitors in cancer therapy.     

Immunotherapy with costimulatory dendritic cells to control autoimmune inflammation

Costimulation-deficient dendritic cells (DCs) prevent autoimmune disease in mouse models. However, autoimmune-prone mice and humans fail to control expansion of peripheral autoreactive effector memory T cells (T(EMs)), which resist immunoregulation by costimulation-deficient DCs. In contrast, activation of DC costimulation may be coupled with regulatory capacity. To test whether costimulatory DCs control T(EMs) and attenuate established autoimmune disease, we used RelB-deficient mice, which have multiorgan inflammation, expanded peripheral autoreactive T(EMs), and dysfunctional Foxp3(+) regulatory T cells (Tregs) cells and conventional DCs. T(EMs) were regulated by Foxp3(+) Tregs when costimulated by CD3/CD28-coated beads or wild-type DCs but not DCs deficient in RelB or CD80/CD86. After transfer, RelB and CD80/CD86-sufficient DCs restored tolerance and achieved a long-term cure of autoimmune disease through costimulation of T(EM) and Foxp3(+) Treg IFN-γ production, as well as induction of IDO by host APCs. IDO was required for regulation of T(EMs) and suppression of organ inflammation. Our data challenge the paradigm that costimulation-deficient DCs are required to regulate established autoimmune disease to avoid T(EM) activation and demonstrate cooperative cross-talk between costimulatory DCs, IFN-γ, and IDO-dependent immune regulation. IFN-γ and IDO activity may be good surrogate biomarkers measured against clinical efficacy in trials of autoimmune disease immunoregulation.     

The role of the p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and phosphoinositide-3-OH kinase signal transduction pathways in CD40 ligand-induced dendritic cell activation and expansion of virus-specific CD8+ T cell memory responses

Mature dendritic cells (DCs) are central to the development of optimal T cell immune responses. CD40 ligand (CD40L, CD154) is one of the most potent maturation stimuli for immature DCs. We studied the role of three signaling pathways, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and phosphoinositide-3-OH kinase (PI3K), in CD40L-induced monocyte-derived DC activation, survival, and expansion of virus-specific CD8(+) T cell responses. p38 MAPK pathway was critical for CD40L-mediated up-regulation of CD83, a marker of DC maturation. CD40L-induced monocyte-derived DC IL-12 production was mediated by both the p38 MAPK and PI3K pathways. CD40L-mediated DC survival was mostly mediated by the PI3K pathway, with smaller contributions by p38 MAPK and ERK pathways. Finally, the p38 MAPK pathway was most important in mediating CD40L-stimulated DCs to induce strong allogeneic responses as well as expanding virus-specific memory CD8(+) T cell responses. Thus, although the p38 MAPK, PI3K, and ERK pathways independently affect various parameters of DC maturation induced by CD40L, the p38 MAPK pathway within CD40L-conditioned DCs is the most important pathway to maximally elicit T cell immune responses. This pathway should be exploited in vivo to either completely suppress or enhance CD8(+) T cell immune responses.     

Rewiring of sIgM-Mediated Intracellular Signaling through the CD180 Toll-like Receptor

Chronic lymphocytic leukemia (CLL) development and progression are thought to be driven by unknown antigens/autoantigens through the B cell receptor (BCR) and environmental signals for survival and expansion including toll-like receptor (TLR) ligands. CD180/RP105, a membrane-associated orphan receptor of the TLR family, induces normal B cell activation and proliferation and is expressed by approximately 60% of CLL samples. Half of these respond to ligation with anti-CD180 antibody by increased activation/phosphorylation of protein kinases associated with BCR signaling. Hence CLL cells expressing both CD180 and the BCR could receive signals via both receptors. Here we investigated cross-talk between BCR and CD180-mediated signaling on CLL cell survival and apoptosis. Our data indicate that ligation of CD180 on responsive CLL cells leads to activation of either prosurvival Bruton tyrosine kinase (BTK)/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT-mediated, or proapoptotic p38 mitogen-activated protein kinase (p38MAPK)-mediated signaling pathways, while selective immunoglobulin M (sIgM) ligation predominantly engages the BTK/PI3K/AKT pathway. Furthermore, pretreatment of CLL cells with anti-CD180 redirects IgM-mediated signaling from the prosurvival BTK/PI3K/AKT toward the proapoptotic p38MAPK pathway. Thus preengaging CD180 could prevent further prosurvival signaling mediated via the BCR and, instead, induce CLL cell apoptosis, opening the door to therapeutic profiling and new strategies for the treatment of a substantial cohort of CLL patients.     

Selective regulation of interleukin-10 production via Janus kinase pathway in murine conventional dendritic cells

In this study, we examined the role of JAKs in regulation of inflammatory versus anti-inflammatory cytokine balance in murine conventional dendritic cells (DCs). Highly purified lipopolysaccharide (upLPS) combined with imiquimod (IQ) synergistically induced IL-10 production by DCs, while each ligand alone showed a slight effect on the IL-10 production. Marked phosphorylation of JAK2, STAT1 and STAT3 was detected in DCs following upLPS plus IQ stimulation. Blocking the JAK pathway by JAK inhibitor I (JAKi) resulted in significant inhibition of IL-10 production by the DCs. However, JAKi showed negligible effect on the DC production of IL-12, IL-6 and TNF-α. JAKi completely blocked the TLR-mediated STATs activation, and attenuated the activation of Akt, a downstream effector of PI3K, in DCs stimulated by upLPS plus IQ. LY294002, a specific inhibitor of PI3K, markedly inhibited the DC production of IL-10. Thus, JAK-PI3K axis appeared to be responsible for the IL-10 production by DCs.     

Reciprocal modulation of Toll-like receptor-4 signaling pathways involving MyD88 and phosphatidylinositol 3-kinase/AKT by saturated and polyunsaturated fatty acids

Toll-like receptor-4 (TLR4) can be activated by nonbacterial agonists, including saturated fatty acids. However, downstream signaling pathways activated by nonbacterial agonists are not known. Thus, we determined the downstream signaling pathways derived from saturated fatty acid-induced TLR4 activation. Saturated fatty acid (lauric acid)-induced NFkappaB activation was inhibited by a dominant-negative mutant of TLR4, MyD88, IRAK-1, TRAF6, or IkappaBalpha in macrophages (RAW264.7) and 293T cells transfected with TLR4 and MD2. Lauric acid induced the transient phosphorylation of AKT. LY294002, dominant-negative (DN) phosphatidylinositol 3-kinase (PI3K), or AKT(DN) inhibited NFkappaB activation, p65 transactivation, and cyclooxygenase-2 (COX-2) expression induced by lauric acid or constitutively active (CA) TLR4. AKT(DN) blocked MyD88-induced NFkappaB activation, suggesting that AKT is a MyD88-dependent downstream signaling component of TLR4. AKT(CA) was sufficient to induce NFkappaB activation and COX-2 expression. These results demonstrate that NFkappaB activation and COX-2 expression induced by lauric acid are at least partly mediated through the TLR4/PI3K/AKT signaling pathway. In contrast, docosahexaenoic acid (DHA) inhibited the phosphorylation of AKT induced by lipopolysaccharide or lauric acid. DHA also suppressed NFkappaB activation induced by TLR4(CA), but not MyD88(CA) or AKT(CA), suggesting that the molecular targets of DHA are signaling components upstream of MyD88 and AKT. Together, these results suggest that saturated and polyunsaturated fatty acids reciprocally modulate the activation of TLR4 and its downstream signaling pathways involving MyD88/IRAK/TRAF6 and PI3K/AKT and further suggest the possibility that TLR4-mediated target gene expression and cellular responses are also differentially modulated by saturated and unsaturated fatty acids.     

CD154 plays a central role in regulating dendritic cell activation during infections that induce Th1 or Th2 responses

We compared splenic DC activation during infection with either the Th2 response-inducing parasite Schistosoma mansoni or with the Th1 response-inducing parasite Toxoplasma gondii. CD8alpha(+) DC from schistosome-infected mice exhibited a 2- to 3-fold increase in the expression of MHC class II, CD80, and CD40 (but not CD86) compared with DC from uninfected control animals, while CD8alpha(-) DC exhibited a 2- to 3-fold increase in the expression of MHC class II and CD80 and no alteration, compared with DC from uninfected mice, in the expression of CD86 or CD40. Intracellular staining revealed that DC did not produce IL-12 during infection with S. mansoni. In contrast, infection with T. gondii resulted in a more pronounced increase in the expression of activation-associated molecules (MHC class II, CD80, CD86, and CD40) on both CD8alpha(-) and CD8alpha(+) splenic DC and promoted elevated IL-12 production by DC. Analysis of MHC class I and of additional costimulatory molecules (ICOSL, ICAM-1, OX40L, 4-1BBL, and B7-DC) revealed a generally similar pattern, with greater indication of activation in T. gondii-infected mice compared with S. mansoni-infected animals. Strikingly, the activation of DC observed during infection with either parasite was not apparent in DC from infected CD154(-/-) mice, indicating that CD40/CD154 interactions are essential for maintaining DC activation during infection regardless of whether the outcome is a Th1 or a Th2 response. However, the ability of this activation pathway to induce IL-12 production by DC is restrained in S. mansoni-infected, but not T. gondii-infected, mice by Ag-responsive CD11c(-) cells.     

Involvement of PI3K/AKT and MAPK Pathways for TNF-α Production in SiHa Cervical Mucosal Epithelial Cells Infected with Trichomonas vaginalis

Trichomonas vaginalis; induces proinflammation in cervicovaginal mucosal epithelium. To investigate the signaling pathways in TNF-α production in cervical mucosal epithelium after T. vaginalis infection, the phosphorylation of PI3K/AKT and MAPK pathways were evaluated in T. vaginalis-infected SiHa cells in the presence and absence of specific inhibitors. T. vaginalis increased TNF-α production in SiHa cells, in a parasite burden-dependent and incubation time-dependent manner. In T. vaginalis-infected SiHa cells, AKT, ERK1/2, p38 MAPK, and JNK were phosphorylated from 1 hr after infection; however, the phosphorylation patterns were different from each other. After pretreatment with inhibitors of the PI3K/AKT and MAPK pathways, TNF-α production was significantly decreased compared to the control; however, TNF-α reduction patterns were different depending on the type of PI3K/MAPK inhibitors. TNF-α production was reduced in a dose-dependent manner by treatment with wortmannin and PD98059, whereas it was increased by SP600125. These data suggested that PI3K/AKT and MAPK signaling pathways are important in regulation of TNF-α production in cervical mucosal epithelial SiHa cells. However, activation patterns of each pathway were different from the types of PI3K/MAPK pathways.