Commensal fungi and their cell-wall ß-glucans direct differential responses in human intestinal epithelial cells.

Intestinal epithelial cells (IECs) are the first to encounter luminal antigens and play an active role in intestinal immune responses. We previously reported that ß-glucans, major fungal cell-wall glycans, induced chemokine secretion by IEC lines in a Dectin-1- and Syk-dependent manner. Here, we show that in contrast to ß-glucans, stimulation of IEC lines with Candida albicans and Saccharomyces cerevisiae did not induce secretion of any of the proinflammatory cytokines IL-8, CCL2, CXCL1, and GM-CSF. Commensal fungi and ß-glucans activated Syk and ERK in IEC lines. However, only ß-glucans activated p38, JNK, and the transcription factors NF-κB p65 and c-JUN, which were necessary for cytokine secretion. Furthermore, costimulation of IEC lines with ß-glucans and C. albicans yielded decreased cytokine secretion compared to stimulation with ß-glucans alone. Finally, ex vivo stimulation of human colonic mucosal explants with zymosan and C. albicans, leads to epithelial Syk and ERK phosphorylation, implying recognition of fungi and similar initial signaling pathways as in IEC lines. Lack of cytokine secretion in response to commensal fungi may reflect IECs' response to fungal glycans, other than ß-glucans, that contribute to mucosal tolerance. Skewed epithelial response to commensal fungi may impair homeostasis and contribute to intestinal inflammation.

Anti-Inflammatory and Proresolving Effects of the Omega-6 Polyunsaturated Fatty Acid Adrenic Acid.

Polyunsaturated fatty acids (PUFAs) and their metabolites are potent regulators of inflammation. Generally, omega (n)-3 PUFAs are considered proresolving whereas n-6 PUFAs are classified as proinflammatory. In this study, we characterized the inflammatory response in murine peritonitis and unexpectedly found the accumulation of adrenic acid (AdA), a poorly studied n-6 PUFA. Functional studies revealed that AdA potently inhibited the formation of the chemoattractant leukotriene B4 (LTB4), specifically in human neutrophils, and this correlated with a reduction of its precursor arachidonic acid (AA) in free form. AdA exposure in human monocyte-derived macrophages enhanced efferocytosis of apoptotic human neutrophils. In vivo, AdA treatment significantly alleviated arthritis in an LTB4-dependent murine arthritis model. Our findings are, to our knowledge, the first to indicate that the n-6 fatty acid AdA effectively blocks production of LTB4 by neutrophils and could play a role in resolution of inflammation in vivo.

The pterocarpanquinone LQB 118 inhibits inflammation triggered by zymosan in vivo and in vitro.

LQB 118, a hydride molecule, has been described as an antineoplastic and antiparasitic drug. Recently, LQB118 was also shown to display anti-inflammatory properties using an LPS-induced lung inflammation model. However, LQB 118 effects on the inflammatory response induced by zymosan has not been demonstrated. In this study, swiss mice were LQB 118 intraperitoneally (i.p.) treated and zymosan was used to induce peritoneal inflammation. Peritoneal fluid was collected and used for cell counting and proinflammatory cytokines quantification (IL-1ß, IL-6, and TNF-α) by immunoenzymatic assay (ELISA). For in vitro studies, peritoneal macrophages zymosan-stimulated were used. Results demonstrated that LQB 118 treatment reduced polymorphonuclear cell migration and TNF-α, IL-1ß, and IL-6 levels in the peritoneal cavity. In macrophages, LQB 118 treatment display no cytotoxic effect and is also able to reduce cytokines levels. To investigate LQB 118 putative mechanism of action, TLR2, CD69, and P-p38 MAPK expression were evaluated. LQB 118 treatment reduced CD69 expression and p38 phosphorylation induced by zymosan. Furthermore, LQB 118 was able to negatively modulate TLR2 expression in the presence of inflammatory stimulus. Thus, our study provide new evidences for the mechanisms related to the anti-inflammatory effect of LQB 118 in vivo and in vitro.

Reporters of TCR signaling identify arthritogenic T cells in murine and human autoimmune arthritis.

How pathogenic cluster of differentiation 4 (CD4) T cells in rheumatoid arthritis (RA) develop remains poorly understood. We used Nur77-a marker of T cell antigen receptor (TCR) signaling-to identify antigen-activated CD4 T cells in the SKG mouse model of autoimmune arthritis and in patients with RA. Using a fluorescent reporter of Nur77 expression in SKG mice, we found that higher levels of Nur77-eGFP in SKG CD4 T cells marked their autoreactivity, arthritogenic potential, and ability to more readily differentiate into interleukin-17 (IL-17)-producing cells. The T cells with increased autoreactivity, nonetheless had diminished ex vivo inducible TCR signaling, perhaps reflective of adaptive inhibitory mechanisms induced by chronic autoantigen exposure in vivo. The enhanced autoreactivity was associated with up-regulation of IL-6 cytokine signaling machinery, which might be attributable, in part, to a reduced amount of expression of suppressor of cytokine signaling 3 (SOCS3)-a key negative regulator of IL-6 signaling. As a result, the more autoreactive GFPhi CD4 T cells from SKGNur mice were hyperresponsive to IL-6 receptor signaling. Consistent with findings from SKGNur mice, SOCS3 expression was similarly down-regulated in RA synovium. This suggests that despite impaired TCR signaling, autoreactive T cells exposed to chronic antigen stimulation exhibit heightened sensitivity to IL-6, which contributes to the arthritogenicity in SKG mice, and perhaps in patients with RA.

Protective effects of dioscin against systemic inflammatory response syndromevia adjusting TLR2/MyD88/NF­κb signal pathway.

Development of active compounds to control inflammation against systemic inflammatory response syndrome (SIRS) is critical important. Dioscin shows anti-inflammatory effects in our previous works. However, the action of the compound on SIRS still remained unknown. In the present paper, zymosan induced generalized inflammation (ZIGI) models in mice and rats, and PMA-differentiated THP­1 cells stimulated by lipopolysaccharide (LPS) and Pam3-Cys-Ser-Lys4 (Pam3CSK4) were used. The results showed that dioscin significantly inhibited the proliferation of THP­1 cells stimulated by LPS and Pam3CSK4, obviously reduced the soakage of inflammatory cells and necrosis in liver, kidney and intestine of rats and mice, and reduced peritoneal ascites fluid compared with ZIGI model groups. In addition, dioscin significantly declined the levels of alanine transaminase (ALT), aspartate transaminase (AST), creatinine (Cr), blood urea nitrogen (BUN), malondialdehyde (MDA) and myeloperoxidase (MPO), increased the levels of superoxide dismutase (SOD) in rats and mice. The migration of macrophages in tissues was also suppressed by dioscin. Mechanism investigation showed that dioscin significantly inhibited the expression levels of TLR2, MyD88, NF­κb, HMGB­1, increased the expression levels of IKBα, and decreased the mRNA levels of interleukin­1 beta (IL­1ß), interleukin­6 (IL­6) and tumor necrosis factor­alpha (TNF­α) in liver, kidney, intestine tissues of rats and mice, and in PMA-differentiated THP­1 cells, which were further confirmed by TLR2 siRNA silencing in vitro. In conclusion, our data confirmed that dioscin exhibited protective effects against SIRS via adjusting TLR2/MyD88 signal pathway, which should be developed as one potent candidate to treat SIRS in the future.

Immune synergistic oligodeoxynucleotide from Lactobacillus rhamnosus GG enhances the immune response upon co-stimulation by bacterial and fungal cell wall components.

Bacterial genomic DNA has recently been shown to elicit a highly evolved immune defense. This response can be selectively triggered for a wide range of therapeutic applications, including use as a vaccine adjuvant to immunotherapies for allergy, cancer, and infectious diseases. Previously, we identified a low-concentration immune synergistic oligodeoxynucleotide (iSN-ODN, named iSN34) from Lactobacillus rhamnosus GG that has immunosynergistic activity upon costimulation of target cells with ligands of Toll-like receptor 9 (TLR9). Here, we extend that observation by demonstrating the synergistic induction (in mouse splenocytes) of IL-6 by the combination of iSN34 with cell wall components of bacteria and fungi. We observed that splenocytes pretreated with iSN34 and then costimulated with agonists for TLR1/2 (Pam3 CSK4 ), TLR4 (lipopolysaccharide), or TLR2/6 (Zymosan) exhibited enhanced accumulation of IL-6. These results suggested that the combination of iSN34 with TLR1/2, TLR4, or TLR2/6 agonists may permit the induction of a potent immune response.

Myc binding protein 2 suppresses M2-like phenotypes in macrophages during zymosan-induced inflammation in mice.

MYCBP2 is an E3 ubiquitin ligase, which is well characterized as a key element in the inhibition of neuronal growth, synapse formation and synaptic strength by regulating several signaling pathways. Although MYCBP2 was suspected to be expressed also in immune cells, to date nothing is known about its role in inflammation. We used Multi-epitope ligand cartography (MELC), a method for multiple sequential immunohistology, to show that MYCBP2 is strongly expressed in monocyte-derived macrophages during zymosan-induced inflammation. We generated a myeloid-specific knockout mouse and found that loss of MYCBP2 in myeloid cells reduced nociceptive (painful) behavior during the resolution phase (1-3 days after zymosan injection). Quantitative MELC analyses and flow cytometric analysis showed an increased number of CD206-expressing macrophages in the inflamed paw tissue. Fittingly, CD206 and arginase 1 expression was upregulated in MYCBP2-deficient bone marrow-derived macrophages after polarization with IL10 or IL4. The regulation of protein expression in these macrophages by MYCBP2 varied depending on the polarization signal. The increased IL10-induced CD206 expression in MYCBP2-deficient macrophages was mediated by p38 MAPK, while IL4-induced CD206 expression in MYCBP2-deficient macrophages was mediated by protein kinase A.

DEK-targeting DNA aptamers as therapeutics for inflammatory arthritis.

Novel therapeutics are required for improving the management of chronic inflammatory diseases. Aptamers are single-stranded RNA or DNA molecules that have recently shown utility in a clinical setting, as they can specifically neutralize biomedically relevant proteins, particularly cell surface and extracellular proteins. The nuclear chromatin protein DEK is a secreted chemoattractant that is abundant in the synovia of patients with juvenile idiopathic arthritis (JIA). Here, we show that DEK is crucial to the development of arthritis in mouse models, thus making it an appropriate target for aptamer-based therapy. Genetic depletion of DEK or treatment with DEK-targeted aptamers significantly reduces joint inflammation in vivo and greatly impairs the ability of neutrophils to form neutrophil extracellular traps (NETs). DEK is detected in spontaneously forming NETs from JIA patient synovial neutrophils, and DEK-targeted aptamers reduce NET formation. DEK is thus key to joint inflammation, and anti-DEK aptamers hold promise for the treatment of JIA and other types of arthritis.

Fungal pattern receptors down-regulate the inflammatory response by a cross-inhibitory mechanism independent of interleukin-10 production.

Cyclic AMP regulatory element binding protein and signal transducer and activator of transcription 3 (STAT3) may control inflammation by several mechanisms, one of the best characterized is the induction of the expression of the anti-inflammatory cytokine interleukin-10 (IL-10). STAT3 also down-regulates the production of pro-inflammatory cytokines induced by immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, a mechanism termed cross-inhibition. Because signalling via ITAM-dependent mechanisms is a hallmark of fungal pattern receptors, STAT3 activation might be involved in the cross-inhibition associated with invasive fungal infections. The fungal surrogate zymosan produced the phosphorylation of Y705-STAT3 and the expression of Ifnb1 and Socs3, but did not induce the interferon (IFN)-signature cytokines Cxcl9 and Cxcl10 in bone marrow-derived dendritic cells. Unlike lipopolysaccharide (LPS), zymosan induced IL-10 and phosphorylated Y705-STAT3 to a similar extent in Irf3 and Ifnar1 knockout and wild-type mice. Human dendritic cells showed similar results, although the induction of IFNB1 was less prominent. These results indicate that LPS and zymosan activate STAT3 through different routes. Whereas type I IFN is the main effector of LPS effect, the mechanism involved in Y705-STAT3 phosphorylation by zymosan is more complex, cannot be associated with type I IFN, IL-6 or granulocyte-macrophage colony-stimulating factor, and seems dependent on several factors given that it was partially inhibited by the platelet-activating factor antagonist WEB2086 and high concentrations of COX inhibitors, p38 mitogen-activate protein kinase inhibitors, and blockade of tumour necrosis factor-α function. Altogether, these results indicate that fungal pattern receptors share with other ITAM-coupled receptors the capacity to produce cross-inhibition through a mechanism involving STAT3 and induction of SOCS3 and IL-10, but that cannot be explained through type I IFN signalling.

CD44 Antibody Inhibition of Macrophage Phagocytosis Targets Fcγ Receptor- and Complement Receptor 3-Dependent Mechanisms.

Targeting CD44, a major leukocyte adhesion molecule, using specific Abs has been shown beneficial in several models of autoimmune and inflammatory diseases. The mechanisms contributing to the anti-inflammatory effects of CD44 Abs, however, remain poorly understood. Phagocytosis is a key component of immune system function and can play a pivotal role in autoimmune states where CD44 Abs have shown to be effective. In this study, we show that the well-known anti-inflammatory CD44 Ab IM7 can inhibit murine macrophage phagocytosis of RBCs. We assessed three selected macrophage phagocytic receptor systems: Fcγ receptors (FcγRs), complement receptor 3 (CR3), and dectin-1. Treatment of macrophages with IM7 resulted in significant inhibition of FcγR-mediated phagocytosis of IgG-opsonized RBCs. The inhibition of FcγR-mediated phagocytosis was at an early stage in the phagocytic process involving both inhibition of the binding of the target RBC to the macrophages and postbinding events. This CD44 Ab also inhibited CR3-mediated phagocytosis of C3bi-opsonized RBCs, but it did not affect the phagocytosis of zymosan particles, known to be mediated by the C-type lectin dectin-1. Other CD44 Abs known to have less broad anti-inflammatory activity, including KM114, KM81, and KM201, did not inhibit FcγR-mediated phagocytosis of RBCs. Taken together, these findings demonstrate selective inhibition of FcγR and CR3-mediated phagocytosis by IM7 and suggest that this broadly anti-inflammatory CD44 Ab inhibits these selected macrophage phagocytic pathways. The understanding of the immune-regulatory effects of CD44 Abs is important in the development and optimization of therapeutic strategies for the potential treatment of autoimmune conditions.

TLR2, TLR4 and Dectin-1 signalling in hematopoietic stem and progenitor cells determines the antifungal phenotype of the macrophages they produce.

TLRs represent an attractive target for the stimulation of myeloid cell production by HSPCs. We have previously demonstrated that HSPCs use TLR2 to sense Candida albicans in vivo and induce the production of macrophages. In this work, we used an in vitro model of HSPCs differentiation to investigate the functional consequences for macrophages of exposure of HSPCs to various PAMPs and C. albicans cells. Mouse HSPCs (Lin(-) cells) were cultured with M-CSF to induce macrophage differentiation, in the presence or absence of the following PRR agonists: Pam3CSK4 (TLR2 ligand), LPS (TLR4 ligand), depleted zymosan (which only activates Dectin-1), or C. albicans yeasts (which activate several PRRs, but principally TLR2 and Dectin-1). Our data show that these PAMPs differentially impact the anti-microbial function of the macrophages produced by the exposed HSPCs. Pure TLR2 and TLR4 ligands generate macrophages with a diminished ability to produce inflammatory cytokines. In contrast, HSPCs activation in response to C. albicans leads to the generation of macrophages that are better prepared to deal with the infection, as they produce higher amounts of inflammatory cytokines and have higher fungicidal capacity than control macrophages. Therefore, the tailored manipulation of the differentiation process may help to boost the innate immune response to infection.

Platelets attenuate production of cytokines and nitric oxide by macrophages in response to bacterial endotoxin.

Considerable evidence has been accumulated concerning the roles of platelets in immune responses. In the present study, we examined the functional modulation of macrophages by platelets. When mouse bone marrow-derived macrophages (BMDMs) were co-cultured with platelets, BMDMs produced lower levels of nitric oxide (NO), tumor necrosis factor-α (TNF)-α, and interleukin (IL)-6 in response to a bacterial endotoxin (LPS) and zymosan. The attenuation in the macrophage susceptibility to LPS appeared to be mediated by soluble factors secreted from platelets. The mRNA levels of NOS2 (iNOS), TNF-α, and IL-6 in LPS-stimulated BMDMs that had been cultured with a conditioned medium of platelets were also decreased as analyzed by RT-qPCR. The ability of the platelet-conditioned medium to suppress macrophage NO production was recovered in a high-molecular-weight fraction (>670 kDa) after gel-filtration chromatography on a Superose 6 column. These results suggest that platelets control the susceptibility of macrophages to prevent excessive responses to LPS and provide mechanistic insight into a previous report that experimental thrombocytopenia aggravated organ failure in LPS-induced endotoxemia.

Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages.

Phagocytosis of zymosan by phagocytes is a widely used model of microbial recognition by the innate immune system. Live-cell imaging showed that fluorescent protein-fused Rab35 accumulated in the membranes of phagocytic cups and then dissociated from the membranes of newly formed phagosomes. By our novel pull-down assay for Rab35 activity, we found that Rab35 is deactivated immediately after zymosan internalization into the cells. Phagosome formation was inhibited in cells expressing the GDP- or GTP-locked Rab35 mutant. Moreover, the simultaneous expression of ACAP2-a Rab35 effector protein-with GTP-locked Rab35 or the expression of plasma membrane-targeted ACAP2 showed a marked inhibitory effect on phagocytosis through ARF6 inactivation by the GAP activity of ACAP2. ARF6, a substrate for ACAP2, was also localized on the phagocytic cups and dissociated from the membranes of internalized phagosomes. In support of the microscopic observations, ARF6-GTP pull-down experiments showed that ARF6 is transiently activated during phagosome formation. Furthermore, the expression of GDP- or GTP-locked ARF6 mutants also suppresses the uptake of zymosan. These data suggest that the activation-inactivation cycles of Rab35 and ARF6 are required for the uptake of zymosan and that ACAP2 is an important component that links Rab35/ARF6 signaling during phagocytosis of zymosan.

C-Type Lectin Receptor MCL Facilitates Mincle Expression and Signaling through Complex Formation.

C-type lectin receptors expressed in APCs are recently defined pattern recognition receptors that play a crucial role in immune responses against pathogen-associated molecular patterns. Among pathogen-associated molecular patterns, cord factor (trehalose-6,6'-dimycolate [TDM]) is the most potent immunostimulatory component of the mycobacterial cell wall. Two C-type lectin receptors, macrophage-inducible C-type lectin (Mincle) and macrophage C-type lectin (MCL), are required for immune responses against TDM. Previous studies indicate that MCL is required for TDM-induced Mincle expression. However, the mechanism by which MCL induces Mincle expression has not been fully understood. In this study, we demonstrate that MCL interacts with Mincle to promote its surface expression. After LPS or zymosan stimulation, MCL-deficient bone marrow-derived dendritic cells (BMDCs) had a lower level of Mincle protein expression, although mRNA expression was comparable with wild-type BMDCs. Meanwhile, BMDCs from MCL transgenic mice showed an enhanced level of Mincle expression on the cell surface. MCL was associated with Mincle through the stalk region and this region was necessary and sufficient for the enhancement of Mincle expression. This interaction appeared to be mediated by the hydrophobic repeat of MCL, as substitution of four hydrophobic residues within the stalk region with serine (MCL(4S)) abolished the function to enhance the surface expression of Mincle. MCL(4S) mutant failed to restore the defective TDM responses in MCL-deficient BMDCs. These results suggest that MCL positively regulates Mincle expression through protein-protein interaction via its stalk region, thereby magnifying Mincle-mediated signaling.

Proresolving actions of a new resolvin D1 analog mimetic qualifies as an immunoresolvent.

Resolution of inflammation is an active process driven by several new families of endogenous lipid mediators collectively coined specialized proresolving mediators (SPM). Here, we report a synthetic analog of resolvin D1 (RvD1) and aspirin-triggered RvD1, benzo-diacetylenic-17R-RvD1-methyl ester (BDA-RvD1), which was prepared using fewer steps than required for total organic synthesis of natural SPM. BDA-RvD1 was resistant to further metabolism by human recombinant 15-prostaglandin dehydrogenase, a major inactivation pathway for RvD1. In ischemia-reperfusion-initiated second organ injury, BDA-RvD1 intravenously (1 µg) reduced neutrophil infiltration into the lungs by 58 ± 9% and was significantly more potent than native RvD1. BDA-RvD1 at 100 ng/mouse also shortened the resolution interval, Ri, of Escherichia coli peritonitis with a similar potency as RvD1, by ~57%, from Ri 10.5 h to 4.5 h. With isolated human phagocytes, BDA-RvD1 at picomolar concentrations (10(-12) M) stimulated phagocytosis of zymosan A particles. BDA-RvD1 activated human recombinant G protein-coupled receptor 32/DRV1, an RvD1 receptor, in a dose-dependent manner. These results indicate that, both in vivo in mice and with isolated human cells, BDA-RvD1 shares defining proresolving actions of RvD1, including inhibiting leukocyte infiltration and stimulating phagocytosis. Moreover, they provide evidence for a new analog mimetic and example of an immunoresolvent, namely an agent that stimulates active resolution of inflammation, for a potential new therapeutic class.

Aging delays resolution of acute inflammation in mice: reprogramming the host response with novel nano-proresolving medicines.

Aging is associated with an overt inflammatory phenotype and physiological decline. Specialized proresolving lipid mediators (SPMs) are endogenous autacoids that actively promote resolution of inflammation. In this study, we investigated resolution of acute inflammation in aging and the roles of SPMs. Using a self-resolving peritonitis and resolution indices coupled with lipid mediator metabololipidomics, we found that aged mice had both delayed resolution and reduced SPMs. The SPM precursor docosahexaenoic acid accelerated resolution via increased SPMs and promoted human monocyte reprogramming. In aged mice, novel nano-proresolving medicines carrying aspirin-triggered resolvins D1 and D3 reduced inflammation by promoting efferocytosis. These findings provide evidence for age-dependent resolution pathways in acute inflammation and novel means to activate resolution.

TLR2-dependent activation of ß-catenin pathway in dendritic cells induces regulatory responses and attenuates autoimmune inflammation.

Dendritic cells (DCs) sense microbes via multiple innate receptors. Signals from different innate receptors are coordinated and integrated by DCs to generate specific innate and adaptive immune responses against pathogens. Previously, we have shown that two pathogen recognition receptors, TLR2 and dectin-1, which recognize the same microbial stimulus (zymosan) on DCs, induce mutually antagonistic regulatory or inflammatory responses, respectively. How diametric signals from these two receptors are coordinated in DCs to regulate or incite immunity is not known. In this study, we show that TLR2 signaling via AKT activates the ß-catenin/T cell factor 4 pathway in DCs and programs them to drive T regulatory cell differentiation. Activation of ß-catenin/T cell factor 4 was critical to induce regulatory molecules IL-10 (Il-10) and vitamin A metabolizing enzyme retinaldehyde dehydrogenase 2 (Aldh1a2) and to suppress proinflammatory cytokines. Deletion of ß-catenin in DCs programmed them to drive Th17/Th1 cell differentiation in response to zymosan. Consistent with these findings, activation of the ß-catenin pathway in DCs suppressed chronic inflammation and protected mice from Th17/Th1-mediated autoimmune neuroinflammation. Thus, activation of ß-catenin in DCs via the TLR2 receptor is a novel mechanism in DCs that regulates autoimmune inflammation.

Canonical and noncanonical g-protein signaling helps coordinate actin dynamics to promote macrophage phagocytosis of zymosan.

Both chemotaxis and phagocytosis depend upon actin-driven cell protrusions and cell membrane remodeling. While chemoattractant receptors rely upon canonical G-protein signaling to activate downstream effectors, whether such signaling pathways affect phagocytosis is contentious. Here, we report that Gαi nucleotide exchange and signaling helps macrophages coordinate the recognition, capture, and engulfment of zymosan bioparticles. We show that zymosan exposure recruits F-actin, Gαi proteins, and Elmo1 to phagocytic cups and early phagosomes. Zymosan triggered an increase in intracellular Ca(2+) that was partially sensitive to Gαi nucleotide exchange inhibition and expression of GTP-bound Gαi recruited Elmo1 to the plasma membrane. Reducing GDP-Gαi nucleotide exchange, decreasing Gαi expression, pharmacologically interrupting Gßγ signaling, or reducing Elmo1 expression all impaired phagocytosis, while favoring the duration that Gαi remained GTP bound promoted it. Our studies demonstrate that targeting heterotrimeric G-protein signaling offers opportunities to enhance or retard macrophage engulfment of phagocytic targets such as zymosan.

Differential stimulation of peripheral blood mononuclear cells in Crohn's disease by fungal glycans.

BACKGROUND AND AIM: Crohn's disease (CD) is characterized by loss of tolerance to intestinal microorganisms. This is reflected by serological responses to fungal glycans such as mannan and ß-glucans. Fungal glycans have various effects on immune cells. However, the evidence for their effects in CD is vague. This study aimed to assess the effects of fungal cell wall glycans on human peripheral blood mononuclear cells (PBMCs) from CD and control patients. METHODS: Human PBMCs from CD and control patients were stimulated by fungal cell wall glycans. Cytokine secretion was detected by ELISA and glycan receptor expression by flow cytometry. RESULTS: Mannan, ß-glucans (curdlan), chitosan, and zymosan induced the secretion of interleukin (IL)-1ß, IL-6, IL-23, IL-10, and tumor necrosis factor-α by PBMCs. Spleen tyrosin kinase and Src tyrosine kinase were involved in the response to mannan and ß-glucans. Mannan and whole yeast cells induced a significantly higher pro-inflammatory cytokine response in CD compared with control patients. CONCLUSIONS: The results may suggest that CD is characterized by hyperresponsiveness to fungal glycans. Thus, glycans may potentially be triggering or perpetuating inflammation.

MiR-146a negatively regulates TLR2-induced inflammatory responses in keratinocytes.

Keratinocytes represent the first line of defense against pathogens in the skin and have important roles in initiating and regulating inflammation during infection and autoimmunity. Here we investigated the role of miR-146a in the regulation of the innate immune response of keratinocytes. Toll-like receptor 2 (TLR2) stimulation of primary human keratinocytes resulted in an NF-κB- and mitogen-activated protein kinase-dependent upregulation of miR-146a expression, which was surprisingly long lasting, contrasting with the rapid and transient induction of inflammatory mediators. Overexpression of miR-146a significantly suppressed the production of IL-8, CCL20, and tumor necrosis factor-α, which functionally suppressed the chemotactic attraction of neutrophils by keratinocytes. Inhibition of endogenous miR-146a induced the production of inflammatory mediators even in nonstimulated keratinocytes, and potentiated the effect of TLR2 stimulation. Transcriptomic profiling revealed that miR-146a suppresses the expression of a large number of immune-related genes in keratinocytes. MiR-146a downregulated interleukin-1 receptor-associated kinase 1 and TNF receptor-associated factor 6, two key adapter molecules downstream of TLR signaling, and suppressed NF-κB promoter-binding activity as shown by promoter luciferase experiments. Together, these data identify miR-146a as a regulatory element in keratinocyte innate immunity, which prevents the production of inflammatory mediators under homeostatic conditions and serves as a potent negative feedback regulator after TLR2 stimulation.