Control of GM-CSF-Dependent Dendritic Cell Differentiation and Maturation by DEF6 and SWAP-70.

Although GM-CSF has been widely used in dendritic cell (DC) research, the mechanisms, factors, and signals regulating steady-state differentiation and maturation of GM-CSF-dependent DCs are insufficiently known. We found that the absence, individually or combined, of the related proteins DEF6 and SWAP-70 strongly enhances differentiation of murine GM-CSF-derived DCs. Contrasting SWAP-70, control through DEF6 does not depend on RHOA activation. DEF6 deficiency leads to expression of the DC-specific transcription factor ZBTB46 and prolonged STAT5 activation in GM-CSF cultures. SWAP-70 and DEF6-mediated restriction of DC differentiation converges mechanistically at the NF-κB pathway. DEF6 acts at early stages of DC differentiation in CD115-cKIT+ myeloid DC progenitors, whereas SWAP-70 acts subsequently. SWAP-70 and DEF6 regulate steady-state DC cytokine expression as well as in vivo accumulation in lymphatic tissue of migratory DCs. Our studies thus elucidate previously unknown roles of two closely related factors with distinct and complementary activities in DC differentiation and steady-state DC function.

Tolerogenic versus Immunogenic Lipidomic Profiles of CD11c+ Immune Cells and Control of Immunogenic Dendritic Cell Ceramide Dynamics.

Lipids affect the membrane properties determining essential biological processes. Earlier studies have suggested a role of switch-activated protein 70 (SWAP-70) in lipid raft formation of dendritic cells. We used lipidomics combined with genetic and biochemical assays to analyze the role of SWAP-70 in lipid dynamics. TLR activation using LPS as a ligand represented a pathogenic immunogenic stimulus, physical disruption of cell-cell contacts a tolerogenic stimulus. Physical disruption, but not LPS, caused an increase of phosphatidylcholine ether and cholesteryl esters in CD11c+ immune cells. An increase of ceramide (Cer) was a hallmark for LPS activation. SWAP-70 was required for regulating the increase and localization of Cers in the cell membrane. SWAP-70 controls Cer accumulation through the regulation of pH-dependent acid-sphingomyelinase activity and of RhoA-dependent transport of endosomal contents to the plasma membrane. Poor accumulation of Cers in Swap70-/- cells caused decreased apoptosis. This shows that two different pathways of activation, immunogenic and tolerogenic, induce different changes in the lipid composition of cultured CD11c+ cells, and highlights the important role of SWAP-70 in Cer dynamics in dendritic cells.

SWAP-70 restricts spontaneous maturation of dendritic cells.

Spontaneous maturation observed in dendritic cell (DC) cultures has been linked to their capacity to induce immune responses. Despite several recent studies, the mechanisms and signals triggering spontaneous maturation of DCs are largely unknown. We found that the absence of SWAP-70 causes spontaneous maturation of spleen- and bone marrow-derived DCs and, in vivo, of spleen-resident CD11c(+)CD11b(+)CD8α(-) DCs. Activation markers, cross-presentation of exogenous Ags, and activation of CD8(+) T cells are much increased in Swap-70(-/-) DCs. Spontaneous maturation of Swap-70(-/-) DCs depends on cell-cell contact and does not involve ß-catenin signaling. SWAP-70 is known to regulate integrin activity. Signaling through the integrin CD11b (αM) subunit increases spontaneous maturation of wild-type (wt), but not of Swap-70(-/-) DCs. Signaling through the CD18 (ß2) subunit decreases spontaneous maturation of wt and Swap-70(-/-) DCs. Constitutive activation of RhoA in Swap-70(-/-) DCs was determined as a key mechanism causing the increased spontaneous maturation. Inhibition of RhoA early, but not late, in the activation process reduces spontaneous maturation in Swap-70(-/-) DCs to wt levels. Inhibition of RhoA activation during CD11b integrin activation had a significant effect only in Swap-70(-/-) but not in wt DCs. Together, our data suggest that integrin-mediated spontaneous maturation of wt DCs does not depend on active RhoA, whereas the increase in spontaneous maturation of Swap-70(-/-) DCs is supported by integrin CD11b and by hyperactive RhoA. Thus, SWAP-70 deficiency reveals two pathways that contribute to spontaneous maturation of DCs.

Sphingosine 1-phosphate-induced motility and endocytosis of dendritic cells is regulated by SWAP-70 through RhoA.

The phospholipid mediator sphingosine 1-phosphate (S1P) enhances motility and endocytosis of mature dendritic cells (DCs). We show that in vitro migration of Swap-70(-/-) bone marrow-derived DCs (BMDCs) in response to S1P and S1P-induced upregulation of endocytosis are significantly reduced. S1P-stimulated movement of Swap-70(-/-) BMDCs, specifically retraction of their trailing edge, in a collagen three-dimensional environment is impaired. These in vitro observations correlate with delayed entry into lymphatic vessels and migration to lymph nodes of skin DCs in Swap-70(-/-) mice. Expression of S1P receptors (S1P(1-3)) by wild-type and Swap-70(-/-) BMDCs is similar, but Swap-70(-/-) BMDCs fail to activate RhoA and to localize Rac1 and RhoA into areas of actin polymerization after S1P stimulus. The Rho-activating G protein Gα(i) interacts with SWAP-70, which also supports the localization of Gα(13) to membrane rafts in BMDCs. LPS-matured Swap-70(-/-) BMDCs contain significantly more active RhoA than wild-type DCs. Preinhibition of Rho activation restored migration to S1P, S1P-induced upregulation of endocytosis in mature Swap-70(-/-) BMDCs, and localization of Gα(13) to membrane rafts. These data demonstrate SWAP-70 as a novel regulator of S1P signaling necessary for DC motility and endocytosis.

SWAP-70 regulates RhoA/RhoB-dependent MHCII surface localization in dendritic cells.

Stimulated dendritic cells (DCs) mature and migrate to lymphoid organs to prime naive T cells. DC maturation augments antigen-presentation capacity of DCs by increasing peptide loading, half-life, and cell surface localization of MHC molecules. Activated SWAP-70(-/-) DCs fail to properly localize MHCII molecules in the plasma membrane, are strongly impaired in T-cell activation, and are altered in F-actin rearrangement. MHCII synthesis, invariant chain removal, and MHCII internalization, however, are unaffected. MHCII surface localization is known to require RhoGTPases. Surprisingly, SWAP70, hitherto known to bind F-actin and Rac, also binds RhoA-GTP. In SWAP-70(-/-) DCs, RhoA and RhoB are stimulus-independent and constitutively active. Surface localization of MHCII molecules and T-cell activation can be restored by blocking RhoA and RhoB before but not during DC activation. Thus, contrasting positive regulation of Rac, SWAP-70 negatively regulates RhoA and-indirectly-RhoB, preventing premature RhoA/RhoB activation. Through RhoA/RhoB regulation, SWAP-70 defines a new pathway to control surface localization of MHCII, a critical element in DC-dependent immune responses.

Malaria blood stage suppression of liver stage immunity by dendritic cells.

Malaria starts with Plasmodium sporozoites infection of the host's liver, where development into blood stage parasites occurs. It is not clear why natural infections do not induce protection against the initial liver stage and generate low CD8+ T cell responses. Using a rodent malaria model, we show that Plasmodium blood stage infection suppresses CD8+ T cell immune responses that were induced against the initial liver stage. Blood stage Plasmodium affects dendritic cell (DC) functions, inhibiting maturation and the capacity to initiate immune responses and inverting the interleukin (IL)-12/IL-10 secretion pattern. The interaction of blood stage parasites with DCs induces the secretion of soluble factors that inhibit the activation of CD8+ T cells in vitro and the suppression of protective CD8+ T cell responses against the liver stage in vivo. We propose that blood stage infection induces DCs to suppress CD8+ T cell responses in natural malaria infections. This evasion mechanism leaves the host unprotected against reinfection by inhibiting the immune response against the initial liver stage of the disease.

Dual effect of Plasmodium-infected erythrocytes on dendritic cell maturation.

BACKGROUND: Infection with Plasmodium is the cause of malaria, a disease characterized by a high inflammatory response in the blood. Dendritic cells (DC) participate in both adaptive and innate immune responses, influencing the generation of inflammatory responses. DC can be activated through different receptors, which recognize specific molecules in microbes and induce the maturation of DC. METHODS: Using Plasmodium yoelii, a rodent malaria model, the effect of Plasmodium-infected erythrocytes on DC maturation and TLR responses have been analysed. RESULTS: It was found that intact erythrocytes infected with P. yoelii do not induce maturation of DC unless they are lysed, suggesting that accessibility of parasite inflammatory molecules to their receptors is a key issue in the activation of DC by P. yoelii. This activation is independent of MyD88. It was also observed that pre-incubation of DC with intact P. yoelii-infected erythrocytes inhibits the maturation response of DC to other TLR stimuli. The inhibition of maturation of DC is reversible, parasite-specific and increases with the stage of parasite development, with complete inhibition induced by schizonts (mature infected erythrocytes). Plasmodium yoelii-infected erythrocytes induce a broad inhibitory effect rendering DC non-responsive to ligands for TLR2, TLR3, TLR4, TLR5, TLR7 and TLR9. CONCLUSIONS: Despite the presence of inflammatory molecules within Plasmodium-infected erythrocytes, which are probably responsible for DC maturation induced by lysates, intact Plasmodium-infected erythrocytes induce a general inhibition of TLR responsiveness in DC. The observed effect on DC could play an important role in the pathology and suboptimal immune response observed during the disease. These results help to explain why immune functions are altered during malaria, and provide a system for the identification of a parasite-derived broad inhibitor of TLR-mediated signaling pathways.

Interactions between dendritic cells and CD4+ T cells during Plasmodium infection.

BACKGROUND: During infection, dendritic cells (DCs) encounter pathogenic microorganisms that can modulate their function and shape the T cell responses generated. During the process of T cell activation, DCs establish strong, long-lasting interactions with naïve T cells. METHODS: Using a mouse malaria model, the interactions of DCs and naïve CD4+ T cells have been analysed. RESULTS: DCs, either incubated in vitro with infected erythrocytes or isolated from infected mice, are able to present exogenous antigens by MHC-II, but are not able to establish prolonged effective interactions with naïve CD4+ T cells and do not induce T cell activation. It was also found that effective T cell activation of naïve CD4+ T cells is impaired during late Plasmodium yoelii infection. CONCLUSION: These data may provide a mechanism for the lack of effective adaptive immune responses induced by the Plasmodium parasite.

A Plasmodium yoelii soluble factor inhibits the phenotypic maturation of dendritic cells.

BACKGROUND: Infection with the protozoan parasite Plasmodium is the cause of malaria. Plasmodium infects host erythrocytes causing the pathology of the disease. Plasmodium-infected erythrocytes can modulate the maturation of dendritic cells (DCs) and alter their capacity to activate T cells. METHODS: Mice infected with Plasmodium yoelii and isolated P. yoelii-infected erythrocytes were used to study their effect on the maturation of mouse dendritic cells. RESULTS: DCs are not able to mature in response to LPS injection during the late stage of P. yoelii infection in mice, indicating impaired functionality of these cells in vivo. P. yoelii- infected erythrocytes inhibit the maturation of DCs in vitro in a dose-dependent manner, which is consistent with the inhibition found during late infection when parasite burden is highest. The inhibition of DC maturation and the cytokine secretion profile of DCs are modulated by soluble factors released by P. yoelii-infected erythrocytes. A small, heat-stable, non-hydrophobic molecule of P. yoelii-infected erythrocytes rapidly inhibits the LPS induced phenotypic maturation of DCs in a reversible manner. CONCLUSION: These findings add evidence to the malaria associated immune suppression in vivo and in vitro and provide insight into the nature and mechanism of the Plasmodium factor(s) responsible for altering DC functions.

The F-actin modulator SWAP-70 controls podosome patterning in osteoclasts.

Osteoclasts are bone resorbing cells acting as key mediators of bone disorders. Upon adhesion to bone, osteoclasts polarize and reorganize their cytoskeleton to generate a ring-like F-actin-rich structure, the sealing zone, wherein the osteoclast's resorptive organelle, the ruffled border, is formed. The dynamic self-organization of actin-rich adhesive structures, the podosomes, from clusters to belts is crucial for osteoclast-mediated bone degradation. Mice lacking the protein SWAP-70 display an osteopetrotic phenotype due to defective bone resorption caused by impaired actin ring formation in Swap-70-/- osteoclasts. To further elucidate the mechanisms underlying this defect, we investigated the specific function of SWAP-70 in the organization and dynamics of podosomes. These detailed studies show that the transition from podosome clusters to rings is impaired in Swap-70-/- osteoclasts. Live cell imaging of dynamic F-actin turnover and SWAP-70 localization during podosome patterning indicate that SWAP-70 is dispensable for cluster formation but plays a key role in F-actin ring generation. Our data provide insights in the role of SWAP-70's F-actin binding domain and pleckstrin homology (PH) domain in the proper localization of SWAP-70 and formation of a peripheral podosome belt, respectively. Ex vivo bone analyses revealed that SWAP-70-deficient osteoclasts exhibit defective ruffled border formation and V-ATPase expression. Our findings suggest an important role of membrane binding of SWAP-70 for the regulation of actin dynamics, which is essential for podosome patterning, and thus for the resorptive activity of osteoclasts.

Role of TGF-beta and PGE2 in T cell responses during Plasmodium yoelii infection.

During an acute blood-stage malaria infection, T cell responses to malaria and other bystander antigens are inhibited. Plasmodium infection induces strong cytokine responses that facilitate parasite clearance but may interfere with T cell functions, as some of the soluble immune mediators induced are also general inhibitors of T cell responses. Using a malaria mouse model, we have analyzed the cytokines produced by dendritic cells in response to P. yoelii infection that have potential T cell inhibitory activity. We found that during acute infection DC migrate to the spleen and secrete TGF-beta, prostaglandin E2 (PGE2) and IL-10. We have analyzed the role of these general T cell inhibitors in a particular T cell response of evident importance in malaria infections: the CD8+ T cells generated against the liver-stage of the disease. During blood-stage infection, inhibition of the activity of TGF-beta and PGE2 restores the CD8+ T cell responses generated by sporozoites, increasing protection against re-infection. Our findings suggest that the strong cytokine response induced by blood-stage P. yoelii infection affects host T cell responses, inhibiting protective CD8+ T cells against the liver-stage of the disease.