The ARP2/3 complex,acting cooperatively with Class I formins,modulates penetration resistance in Arabidopsis against powdery mildew invasion

The actin cytoskeleton regulates an array of diverse cellular activities that support the establishment of plant–microbe interactions and plays a critical role in the execution of plant immunity. However, molecular and cellular mechanisms regulating the assembly and rearrangement of actin filaments (AFs) at plant–pathogen interaction sites remain largely elusive. Here, using live-cell imaging, we show that one of the earliest cellular responses in Arabidopsis thaliana upon powdery mildew attack is the formation of patch-like AF structures beneath fungal invasion sites. The AFs constituting actin patches undergo rapid turnover, which is regulated by the actin-related protein (ARP)2/3 complex and its activator, the WAVE/SCAR regulatory complex (W/SRC). The focal accumulation of phosphatidylinositol-4,5-bisphosphate at fungal penetration sites appears to be a crucial upstream modulator of the W/SRC–ARP2/3 pathway-mediated actin patch formation. Knockout of W/SRC–ARP2/3 pathway subunits partially compromised penetration resistance with impaired endocytic recycling of the defense-associated t-SNARE protein PEN1 and its deposition into apoplastic papillae. Simultaneously knocking out ARP3 and knocking down the Class I formin (AtFH1) abolished actin patch formation, severely impaired the deposition of cell wall appositions, and promoted powdery mildew entry into host cells. Our results demonstrate that the ARP2/3 complex and formins, two actin-nucleating systems, act cooperatively and contribute to Arabidopsis penetration resistance to fungal invasion.

ARP2/3 complex, acting cooperatively with Class I formins, modulates actin patch formation beneath fungal penetration sites, contributing to the penetration resistance of Arabidopsis against powdery mildew invasion.     

Control of Dendritic Cell Migration,T Cell-Dependent Immunity,and Autoimmunity by Protein Tyrosine Phosphatase PTPN12 Expressed in Dendritic Cells

Dendritic cells (DCs) capture and process antigens in peripheral tissues, migrate to lymphoid tissues, and present the antigens to T cells. PTPN12, also known as PTP-PEST, is an intracellular protein tyrosine phosphatase (PTP) involved in cell-cell and cell-substratum interactions. Herein, we examined the role of PTPN12 in DCs, using a genetically engineered mouse lacking PTPN12 in DCs. Our data indicated that PTPN12 was not necessary for DC differentiation, DC maturation, or cytokine production in response to inflammatory stimuli. However, it was needed for full induction of T cell-dependent immune responses in vivo. This function largely correlated with the need of PTPN12 for DC migration from peripheral sites to secondary lymphoid tissues. Loss of PTPN12 in DCs resulted in hyperphosphorylation of the protein tyrosine kinase Pyk2 and its substrate, the adaptor paxillin. Pharmacological inhibition of Pyk2 or downregulation of Pyk2 expression also compromised DC migration, suggesting that Pyk2 deregulation played a pivotal role in the migration defect caused by PTPN12 deficiency. Together, these findings identified PTPN12 as a key regulator in the ability of DCs to induce antigen-induced T cell responses. This is due primarily to the role of PTPN12 in DC migration from peripheral sites to secondary lymphoid organs through regulation of Pyk2.     

PPARγ and LXR Signaling Inhibit Dendritic Cell-Mediated HIV-1 Capture and trans-Infection

Dendritic cells (DCs) contribute to human immunodeficiency virus type 1 (HIV-1) transmission and dissemination by capturing and transporting infectious virus from the mucosa to draining lymph nodes, and transferring these virus particles to CD4+ T cells with high efficiency. Toll-like receptor (TLR)-induced maturation of DCs enhances their ability to mediate trans-infection of T cells and their ability to migrate from the site of infection. Because TLR-induced maturation can be inhibited by nuclear receptor (NR) signaling, we hypothesized that ligand-activated NRs could repress DC-mediated HIV-1 transmission and dissemination. Here, we show that ligands for peroxisome proliferator-activated receptor gamma (PPARγ) and liver X receptor (LXR) prevented proinflammatory cytokine production by DCs and inhibited DC migration in response to the chemokine CCL21 by preventing the TLR-induced upregulation of CCR7. Importantly, PPARγ and LXR signaling inhibited both immature and mature DC-mediated trans-infection by preventing the capture of HIV-1 by DCs independent of the viral envelope glycoprotein. PPARγ and LXR signaling induced cholesterol efflux from DCs and led to a decrease in DC-associated cholesterol, which has previously been shown to be required for DC capture of HIV-1. Finally, both cholesterol repletion and the targeted knockdown of the cholesterol transport protein ATP-binding cassette A1 (ABCA1) restored the ability of NR ligand treated cells to capture HIV-1 and transfer it to T cells. Our results suggest that PPARγ and LXR signaling up-regulate ABCA1-mediated cholesterol efflux from DCs and that this accounts for the decreased ability of DCs to capture HIV-1. The ability of NR ligands to repress DC mediated trans-infection, inflammation, and DC migration underscores their potential therapeutic value in inhibiting HIV-1 mucosal transmission.     

MICAL2 fine-tunes Arp2/3 for actin branching

The ARP2/3 complex promotes branched actin networks, but the importance of specific subunit isoforms is unclear. In this issue, Galloni, Carra, et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202102043) show that MICAL2 mediates methionine oxidation of ARP3B, thus destabilizing ARP2/3 complexes and leading to disassembly of branched actin filaments.

Remodeling of branched actin networks enables cell protrusion and sensing of the environment and is essential for cell motility. Migrating cells such as fibroblasts, immune cells, and metastatic cancer cells rely on actin dynamics to generate pushing, pulling, and squeezing forces to propel themselves. Therefore, studying the processes regulating assembly and disassembly of actin filaments is key to understanding cell locomotion in health and disease. One of the most important catalyzers of actin assembly is the Arp2/3 complex, which drives lamellipodia formation and cell protrusion. Arp2/3-generated actin networks are also important for endocytic trafficking, membrane remodeling during vesicle internalization, cargo sorting, and membrane excision (1). The seven-protein ARP2/3 complex contains two unconventional actin-related proteins (ARP2 and ARP3) and five additional subunits (ARPC1–5). Mammals express two isoforms of three of the subunits (ARP3/ARP3B, ARPC1A/ARPC1B, and ARPC5/ARPC5L), resulting in functional diversity depending on the specific isoforms incorporated into the ARP2/3 complex; however, despite some intriguing roles described in muscle development (2) and platelet function (3), little is known about the biological significance of these isoforms.The nucleation activity of ARP2/3 complex is regulated at multiple levels to ensure that new actin generation is spatially and temporally controlled. Activation is controlled by Wiskott Aldrich Syndrome Protein (WASP)–family proteins, which are themselves part of multi-protein complex machines (4). WASP-family protein complexes detect multiple inputs such as membrane phospholipids, protein–protein interactions, or post-translational modifications, and act as signaling hubs to regulate branched actin nucleation. Other proteins, such as cortactin or coronin, also modulate branch stability in an antagonistic manner (5). ARP2/3 can be post-translationally modified by phosphorylation and interaction with negative regulators, whereas actin itself is regulated by targeted oxidation of methionine residues (6). How these feedback loops that control ARP2/3 activity are coordinated with cell function is an intense area of research.Molecule interacting with CasL (MICAL) proteins have emerged as important mediators of targeted protein oxidation (6). MICAL proteins (MICAL1–3) are flavin adenine dinucleotide–binding monooxygenases capable of oxidizing target proteins (including actin), either directly or through generation of diffusible H₂O₂, which in turn oxidizes proteins in close proximity. Actin oxidation occurs on two methionine residues (Met44 and Met47), resulting in F-actin disassembly and increased cofilin-mediated F-actin severing. Although actin is the best characterized MICAL substrate, there remains the intriguing possibility of the existence of additional targets that regulate cytoskeleton dynamics.In this issue, Galloni, Carra, et al. evaluated the ability of ARP2/3 complexes, containing either ARP3 or the ARP3B isoform (i.e., isocomplexes), to promote actin assembly, and determined isoform-specific differences in their activity and molecular regulation (7). As a model system, the authors used HeLa cells infected with vaccinia virus to study actin branching, given that this virus induces actin tail nucleation in the host cells. They noticed that in cells lacking ARP3, the localization of GFP-ARP3 or GFP-ARP3B to actin tails was comparable, and both isoforms were similarly incorporated into ARP2/3 complexes (Fig. 1). However, the length of the actin tails in ARP3B-expressing cells was shorter than in ARP3-expressing counterparts. Given that ARP3 and ARP3B isocomplexes were equivalent in their ability to induce actin polymerization in vitro, these data pointed to a faster disassembly rate as the potential cause underlying shorter actin tails in ARP3B-expressing cells. Indeed, by tracking photoactivatable actin to study its dynamics, the researchers confirmed that the rate of filament disassembly was faster in ARP3B-expressing cells.Open in a separate windowFigure 1.Vaccinia virus surfs on the outside of the cell, forming an actin tail in the cytoplasm that aids its propulsion. Arp2/3 complex is involved in initiating the branched actin structures and shows slow dissociation from the branches when it is stabilized by the linker protein cortactin. When an Arp2/3 complex containing the ARP3B isoform of ARP3 forms, the dissociation is enhanced, as ARP3B is subject to oxidation by MICAL2, which is recruited to branches by coronin, causing cortactin displacement and rapid branch dissociating leading to shorter actin tails.To identify the molecular basis for the differences between ARP3 and ARP3B, the authors tested a series of ARP3 and ARP3B chimeric proteins, which revealed the importance of ARP3B amino acids 281–418 in mediating the functional differences with ARP3. In particular, Met293 was essential for ARP3B to generate short actin tails. Given that MICAL enzymes promote actin filament disassembly through oxidation of actin Met44 and Met47, Galloni, Carra, et al. decided to investigate the possibility that MICAL-induced oxidation of Met293 in ARP3B inhibits ARP3B activity. Fluorescently tagged MICAL2, but not MICAL1, was recruited to vaccinia-induced actin tails at a position relatively distant from the virus itself, similar to the actin-binding protein coronin (8). Down-regulation of MICAL2, but not MICAL1, increased actin tail stability and suppressed the short actin tail phenotype induced by ARP3B overexpression. Using an antibody raised against oxidized Met293, the researchers confirmed that ARP3B oxidation was reduced following MICAL2 knockdown. Recruitment of MICAL2 to actin tails was dependent on coronin 1C expression, and silencing of coronin 1C resulted in actin filament stabilization and reversal of ARP3B-induced actin tail shortening comparable to MICAL2 knockdown. Thus, coronin 1C recruitment of MICAL2 results in ARP3B oxidation on Met293, leading to dissociation of ARP2/3B isocomplexes and consequent actin networks destabilization.Interestingly, the authors noted that the actin nucleation promoting factor cortactin, which stabilizes ARP2/3-mediated branch points along actin filaments, was required for actin tail destabilization in ARP3B overexpressing cells but was not necessary for localization of coronin 1C or MICAL2 to actin tails. One possibility is that cortactin supports local MICAL2-mediated oxidation of ARP3B at branch points to induce filament de-branching, rather than bulk actin filament depolymerization that would result from direct actin oxidation. Since MICAL proteins are directed to specific cytoskeleton locations by interacting with Myosin 5A (9) and Myosin 15 (10), the consequences of MICAL activity on actin cytoskeleton organization and function may be fine-tuned by specific MICAL subcellular localization and interacting partners.Given that actin binds directly to the catalytic monooxygenase and calponin homology domains of MICAL proteins to increase enzyme activity and promote methionine oxidation, it is not entirely surprising that the actin-related ARP3B protein can be oxidized by MICAL2. However, the location of Met293 in ARP3B is not analogous to the Met44 or Met47 residues of actin, which raises questions regarding the mechanism of ARP3B oxidation by MICAL2. Structural modeling of the MICAL3–actin complex positions the actin loop containing Met44 and Met47 near the enzyme active site (11). ARP3B may interact with MICAL2 differently to bring Met293 close to the active site for direct oxidation, or H₂O₂ produced by MICAL2 might diffuse and oxidize highly concentrated nearby proteins. If this second possibility were true, then it is also possible that additional protein targets (e.g., coronin 1C, cortactin, additional ARP2/3 subunits) might also be oxidized on Met or Cys residues. Since the effects of MICAL1 on actin are counteracted via reduction of the oxidized Met residues by the sulfoxide reductase enzyme SelR (12), it remains to be determined if ARP3B can be similarly reactivated.     

Tumour-Derived Reg3A Educates Dendritic Cells to Promote Pancreatic Cancer Progression

As a pancreatic inflammatory marker, regenerating islet-derived protein 3A (Reg3A) plays a key role in inflammation-associated pancreatic carcinogenesis by promoting cell proliferation, inhibiting apoptosis, and regulating cancer cell migration and invasion. This study aimed to reveal a novel immuno-regulatory mechanism by which Reg3A modulates tumour-promoting responses during pancreatic cancer (PC) progression. In an in vitro Transwell system that allowed the direct co-culture of human peripheral blood-derived dendritic cells (DCs) and Reg3A-overexpressing/ silenced human PC cells, PC cell-derived Reg3A was found to downregulate CD80, CD83 and CD86 expression on educated DCs, increase DC endocytic function, inhibit DC-induced T lymphocyte proliferation, reduce IL-12p70 production, and enhance IL-23 production by DCs. The positive effect of tumour-derived Reg3A-educated human DCs on PC progression was demonstrated in vivo by intraperitoneally transferring them into PC-implanted severe combined immunodeficiency (SCID) mice reconstituted with human T cells. A Reg3A-JAK2/STAT3 positive feedback loop was identified in DCs educated with Reg3A. In conclusion, as a tumour-derived factor, Reg3A acted to block the differentiation and maturation of the most important antigen-presenting cells, DCs, causing them to limit their potential anti-tumour responses, thus facilitating PC escape and progression.     

Ascaris lumbricoides pseudocoelomic body fluid induces a partially activated dendritic cell phenotype with Th2 promoting ability in vivo

Dendritic cells (DCs) matured with helminth-derived molecules that promote Th2 immune responses do not follow conventional definitions of DC maturation processes. While a number of models of DC maturation by Th2 stimuli are postulated, further studies are required if we are to clearly define DC maturation processes that lead to Th2 immune responses. In this study, we examine the interaction of Th2-inducing molecules from the parasitic helminth Ascaris lumbricoides with the maturation processes and function of DCs. Here we show that murine bone marrow-derived DCs are partially matured by A. lumbricoides pseudocoelomic body fluid (ABF) as characterised by the production of IL-6, IL-12p40 and macrophage inflammatory protein 2 (MIP-2) but no enhanced expression of cluster of differentiation (CD)-14, T-cell co-stimulatory markers CD80, CD86, CD40, OX40L and major histocompatibility complex class II was observed. Despite these phenotypic characteristics, ABF-stimulated DCs displayed the functional hallmarks of fully matured cells, enhancing DC phagocytosis and promoting Th2-type responses in skin-draining lymph node cells in vivo. ABF activated Th2-associated extracellular signal-regulated kinase-1 and nuclear factor-kB intracellular signalling pathways independently of toll-like receptor 4. Taken together, we believe this is the first paper to demonstrate A. lumbricoides murine DC-Th cell-driven responses shedding further light on DC maturation processes by helminth antigens.     

Hematopoietic lineage cell-specific protein 1 functions in concert with the Wiskott-Aldrich syndrome protein to promote podosome array organization and chemotaxis in dendritic cells

Dendritic cells (DCs) are professional APCs that reside in peripheral tissues and survey the body for pathogens. Upon activation by inflammatory signals, DCs undergo a maturation process and migrate to lymphoid organs, where they present pathogen-derived Ags to T cells. DC migration depends on tight regulation of the actin cytoskeleton to permit rapid adaptation to environmental cues. We investigated the role of hematopoietic lineage cell-specific protein 1 (HS1), the hematopoietic homolog of cortactin, in regulating the actin cytoskeleton of murine DCs. HS1 localized to lamellipodial protrusions and podosomes, actin-rich structures associated with adhesion and migration. DCs from HS1(-/-) mice showed aberrant lamellipodial dynamics. Moreover, although these cells formed recognizable podosomes, their podosome arrays were loosely packed and improperly localized within the cell. HS1 interacts with Wiskott-Aldrich syndrome protein (WASp), another key actin-regulatory protein, through mutual binding to WASp-interacting protein. Comparative analysis of DCs deficient for HS1, WASp or both proteins revealed unique roles for these proteins in regulating podosomes with WASp being essential for podosome formation and with HS1 ensuring efficient array organization. WASp recruitment to podosome cores was independent of HS1, whereas HS1 recruitment required Src homology 3 domain-dependent interactions with the WASp/WASp-interacting protein heterodimer. In migration assays, the phenotypes of HS1- and WASp-deficient DCs were related, but distinct. WASp(-/y) DCs migrating in a chemokine gradient showed a large decrease in velocity and diminished directional persistence. In contrast, HS1(-/-) DCs migrated faster than wild-type cells, but directional persistence was significantly reduced. These studies show that HS1 functions in concert with WASp to fine-tune DC cytoarchitecture and direct cell migration.     

A critical role for prostaglandin E2 in podosome dissolution and induction of high-speed migration during dendritic cell maturation

Dendritic cells (DCs) are professional APCs of the immune system that play a key role in regulating T cell-based immunity. The capacity of DCs to activate T cells depends on their maturation state as well as their ability to migrate to the T cell areas of draining lymph nodes. In this study, we investigated the effects of DC maturation stimuli on the actin cytoskeleton and beta(1) integrin-dependent adhesion and migration. Podosomes, specialized adhesion structures found in immature monocyte-derived DCs as well as myeloid DCs, rapidly dissolve in response to maturation stimuli such as TNF-alpha and PGE(2), whereas the TLR agonist LPS induces podosome dissolution only after a long lag time. We demonstrate that LPS-mediated podosome disassembly as well as the onset of high-speed DC migration are dependent on the production of PGs by the DCs. Moreover, both of these processes are inhibited by Ab-induced activation of beta(1) integrins. Together, these results show that maturation-induced podosome dissolution and loss of alpha(5)beta(1) integrin activity allow human DCs to undergo the transition from an adhesive to a highly migratory phenotype.     

Catching the WAVEs of Plant Actin Regulation

Plants, as all other eukaryotic organisms, depend on a dynamic actin cytoskeleton for proper function and development. Actin dynamics is a complex process, regulated by a number of actin-binding proteins and large multiprotein complexes like ARP2/3 and WAVE. The ARP2/3 complex is recognized as a nucleator of actin filaments, and it generates a highly branched network of interlaced microfilaments. Results from multiple organisms show that ARP2/3 activity is regulated through multiple pathways. Recent results from plants point to a signaling pathway leading from the small GTPase RAC/ROP through a protein complex containing the ARP2/3-activating protein WAVE. This signaling pathway appears to be evolutionarily conserved. Support for this regulatory mechanism comes from studies of mutations in genes encoding subunits of the putative ARP2/3 complex and the WAVE complex in Arabidopsis. Several such mutants have defects of actin filament organization, leading to a conspicuous “distorted” trichome phenotype. Multiple growth and developmental phenotypes reported for napp/gnarled/atnap, pirp/pirogi/atpir, and distorted3 mutants reveal that these WAVE proteins are also required for a wider variety of cellular functions in addition to regulating trichome cell growth. These results have implications for the current view on cell morphogenesis in plants.     

Comparison of media and serum supplementation for generation of monophosphoryl lipid A/interferon-γ–matured type I dendritic cells for immunotherapy

Background aimsEx vivo–generated monocyte-derived dendritic cells (DCs) matured with monophosphoryl lipid A (MPLA) and interferon-γ (IFN-γ) can be used as cancer immunotherapy. MPLA/IFN-γ DCs induce Th1 T cell responses and have migratory capacity. Different culture regimens have been used for generation of immunotherapeutic DCs, with varying results. In the present study, culture conditions for MPLA/IFN-γ–matured type I DCs were optimized for clinical application.MethodsDCs were generated from monocytes in the clinical grade culture media CellGro DC, AIM V or X-VIVO 15 in the absence or presence of 2% human serum (HS) and matured with the use of MPLA/IFN-γ. DC yield and DC functionality were assessed. DC functionality was determined by means of analysis of cytokines in culture supernatant, migratory capacity, expression of co-stimulatory molecules, T cell stimulatory capacity of DCs and T helper cell (Th) polarization by the DCs.ResultsDCs generated in the presence of 2% HS produced low amounts of pro-inflammatory cytokines and could not migrate irrespective of the medium used. In the absence of HS, MPLA/IFN-γ DCs generated in X-VIVO did not migrate either. MPLA/IFN-γ DCs generated in AIM V have slightly lower capacity to induce Th1 cells than do DCs generated in CellGro or X-VIVO.ConclusionsAddition of HS to different GMP culture media is detrimental for pro-inflammatory DC maturation and migration. In the absence of serum, CellGro is the most optimal medium tested for generation of migratory and Th1-inducing MPLA/IFN-γ DCs for cancer immunotherapy.     

Infection by Toxoplasma gondii Induces Amoeboid-Like Migration of Dendritic Cells in a Three-Dimensional Collagen Matrix

Toxoplasma gondii, an obligate intracellular parasite of humans and other warm-blooded vertebrates, invades a variety of cell types in the organism, including immune cells. Notably, dendritic cells (DCs) infected by T. gondii acquire a hypermigratory phenotype that potentiates parasite dissemination by a ‘Trojan horse’ type of mechanism in mice. Previous studies have demonstrated that, shortly after parasite invasion, infected DCs exhibit hypermotility in 2-dimensional confinements in vitro and enhanced transmigration in transwell systems. However, interstitial migration in vivo involves interactions with the extracellular matrix in a 3-dimensional (3D) space. We have developed a collagen matrix-based assay in a 96-well plate format that allows quantitative locomotion analyses of infected DCs in a 3D confinement over time. We report that active invasion of DCs by T. gondii tachyzoites induces enhanced migration of infected DCs in the collagen matrix. Parasites of genotype II induced superior DC migratory distances than type I parasites. Moreover, Toxoplasma-induced hypermigration of DCs was further potentiated in the presence of the CCR7 chemotactic cue CCL19. Blocking antibodies to integrins (CD11a, CD11b, CD18, CD29, CD49b) insignificantly affected migration of infected DCs in the 3D matrix, contrasting with their inhibitory effects on adhesion in 2D assays. Morphological analyses of infected DCs in the matrix were consistent with the acquisition of an amoeboid-like migratory phenotype. Altogether, the present data show that the Toxoplasma-induced hypermigratory phenotype in a 3D matrix is consistent with integrin-independent amoeboid DC migration with maintained responsiveness to chemotactic and chemokinetic cues. The data support the hypothesis that induction of amoeboid hypermigration and chemotaxis/chemokinesis in infected DCs potentiates the dissemination of T. gondii.     

MIF synergizes with Trypanosoma cruzi antigens to promote efficient dendritic cell maturation and IL-12 production via p38 MAPK

Macrophage migration inhibitory factor (MIF) has been found to be involved in host resistance to several parasitic infections. To determine the mechanisms of the MIF-dependent responses to Trypanosoma cruzi, we investigated host resistance in MIF-/- mice (on the BALB/c background) during an intraperitoneal infection. We focused on the potential involvement of MIF in dendritic cell (DC) maturation and cytokine production. Following a challenge with 5 x 10³ T. cruzi parasites, wild type (WT) mice developed a strong IL-12 response and adequate maturation of the draining mesenteric lymph node DCs and were resistant to infection. In contrast, similarly infected MIF^-/- mice mounted a weak IL-12 response, displayed immature DCs in the early phases of infection and rapidly succumbed to T. cruzi infection. The lack of maturation and IL-12 production by the DCs in response to total T. cruzi antigen (TcAg) was confirmed by in vitro studies. These effects were reversed following treatment with recombinant MIF. Interestingly, TcAg-stimulated bone marrow-derived DCs from both WT and MIF^-/- mice had increased ERK1/2 MAPK phosphorylation. In contrast, p38 phosphorylation was only upregulated in WT DCs. Reconstitution of MIF to MIF^-/- DCs upregulated p38 phosphorylation. The MIF-p38 pathway affected MHC-II and CD86 expression as well as IL-12 production. These findings demonstrate that the MIF-induced early DC maturation and IL-12 production mediates resistance to T. cruzi infection, probably by activating the p38 pathway.     

Mycobacterium abscessus MAB2560 induces maturation of dendritic cells via Toll-like receptor 4 and drives Th1 immune response

In this study, we showed that Mycobacterium abscessus MAB2560 induces the maturation of dendritic cells (DCs), which are representative antigen-presenting cells (APCs). M. abscessus MAB2560 stimulate the production of pro-inflammatory cytokines [interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β, and IL-12p70] and reduce the endocytic capacity and maturation of DCs. Using TLR4^-/- DCs, we found that MAB2560 mediated DC maturation via Toll-like receptor 4 (TLR4). MAB2560 also activated the MAPK signaling pathway, which was essential for DC maturation. Furthermore, MAB2560-treated DCs induced the transformation of naïve T cells to polarized CD4⁺ and CD8⁺ T cells, which would be crucial for Th1 polarization of the immune response. Taken together, our results indicate that MAB2560 could potentially regulate the host immune response to M. abscessus and may have critical implications for the manipulation of DC functions for developing DC-based immunotherapy. [BMB Reports 2014;47(9): 512-517]     

Supernatant from bifidobacterium differentially modulates transduction signaling pathways for biological functions of human dendritic cells

Background

Probiotic bacteria have been shown to modulate immune responses and could have therapeutic effects in allergic and inflammatory disorders. However, the signaling pathways engaged by probiotics are poorly understood. We have previously reported that a fermentation product from Bifidobacterium breve C50 (BbC50sn) could induce maturation, high IL-10 production and prolonged survival of DCs via a TLR2 pathway. We therefore studied the roles of mitogen-activated protein kinases (MAPK), glycogen synthase kinase-3 (GSK3) and phosphatidylinositol 3-kinase (PI3K) pathways on biological functions of human monocyte-derived DCs treated with BbC50sn.

Methodology/Principal Findings

DCs were differentiated from human monocytes with IL-4 and GM-CSF for 5 days and cultured with BbC50sn, lipopolysaccharide (LPS) or Zymosan, with or without specific inhibitors of p38MAPK (SB203580), ERK (PD98059), PI3K ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) and GSK3 (SB216763). We found that 1) the PI3K pathway was positively involved in the prolonged DC survival induced by BbC50sn, LPS and Zymosan in contrast to p38MAPK and GSK3 which negatively regulated DC survival; 2) p38MAPK and PI3K were positively involved in DC maturation, in contrast to ERK and GSK3 which negatively regulated DC maturation; 3) ERK and PI3K were positively involved in DC-IL-10 production, in contrast to GSK3 that was positively involved in DC-IL-12 production whereas p38MAPK was positively involved in both; 4) BbC50sn induced a PI3K/Akt phosphorylation similar to Zymosan and a p38MAPK phosphorylation similar to LPS.

Conclusion/Significance

We report for the first time that a fermentation product of a bifidobacteria can differentially activate MAPK, GSK3 and PI3K in order to modulate DC biological functions. These results give new insights on the fine-tuned balance between the maintenance of normal mucosal homeostasis to commensal and probiotic bacteria and the specific inflammatory immune responses to pathogen bacteria.     

Loss of Jak2 Selectively Suppresses DC-Mediated Innate Immune Response and Protects Mice from Lethal Dose of LPS-Induced Septic Shock

Given the importance of Jak2 in cell signaling, a critical role for Jak2 in immune cells especially dendritic cells (DCs) has long been proposed. The exact function for Jak2 in DCs, however, remained poorly understood as Jak2 deficiency leads to embryonic lethality. Here we established Jak2 deficiency in adult Cre^+/+Jak2^fl/fl mice by tamoxifen induction. Loss of Jak2 significantly impaired DC development as manifested by reduced BMDC yield, smaller spleen size and reduced percentage of DCs in total splenocytes. Jak2 was also crucial for the capacity of DCs to mediate innate immune response. Jak2^−/− DCs were less potent in response to inflammatory stimuli and showed reduced capacity to secrete proinflammatory cytokines such as TNFα and IL-12. As a result, Jak2^−/− mice were defective for the early clearance of Listeria after infection. However, their potency to mediate adaptive immune response was not affected. Unlike DCs, Jak2^−/− macrophages showed similar capacity secretion of proinflammatory cytokines, suggesting that Jak2 selectively modulates innate immune response in a DC-dependent manner. Consistent with these results, Jak2^−/− mice were remarkably resistant to lethal dose of LPS-induced septic shock, a deadly sepsis characterized by the excessive innate immune response, and adoptive transfer of normal DCs restored their susceptibility to LPS-induced septic shock. Mechanistic studies revealed that Jak2/SATA5 signaling is pivotal for DC development and maturation, while the capacity for DCs secretion of proinflammatory cytokines is regulated by both Jak2/STAT5 and Jak2/STAT6 signaling.