OK‐432‐stimulated chemokine secretion from human monocytes depends on MEK1/2, and involves p38 MAPK and NF‐κB phosphorylation,in vitro

Interaction between the immune system and cancer cells allows for the use of biological response modifiers, like OK‐432, in cancer therapy. We have studied the involvement of monocytes (MOs) in the immune response to OK‐432 by examining MCP‐1, MIP‐1α and MIP‐1β secretion, in vitro. OK‐432‐induced IL‐6/TNF‐α secretion has previously been shown to depend on mitogen‐activated protein kinases (MAPKs) ERK1/2 and p38, and we therefore investigated the role of these MAPKs in OK‐432‐induced chemokine secretion. Here we demonstrate that pharmacological MEK1/2 kinase inhibition generally impaired chemokine secretion from MOs, whereas p38 MAPK inhibition in particular reduced MIP‐1α production. Furthermore, simultaneous inhibition of MEK1/2 and Syk kinase was seen to have an additive impact on reduced MCP‐1, MIP‐1α and MIP‐1β secretion. Based on single cell flow cytometry analyses, OK‐432, lipoteichoic acid (LTA) and lipopolysaccharide (LPS) were seen to induce p38 MAPK and NF‐κB phosphorylation in MOs with different time kinetics. LTA and LPS have been shown to induce ERK1/2 phosphorylation, whereas the levels of phosphorylated ERK1/2 remained constant following OK‐432 treatment at the time points tested. Toll‐like receptors (TLRs) recognize pathogen‐associated molecular patterns, and we demonstrate increased TLR2 cell surface levels on the MO population, most profoundly following stimulation with LTA and OK‐432. Together these results indicate that modulation of MEK1/2 and p38 MAPK signalling could affect the response to OK‐432 treatment, having the potential to improve its therapeutic potential within cancer and lymphangioma treatment.     

Tolerogenic dendritic cells produced by lentiviral‐mediated CD40‐ and interleukin‐23p19‐specific shRNA can ameliorate experimental autoimmune encephalomyelitis by suppressing T helper type 17 cells

Down‐regulation of soluble or membrane‐bound co‐stimulatory molecules by RNAi in dendritic cells can prevent the activation of immune responses. Therefore, this study was designed to evaluate the therapeutic efficacy of bone marrow‐derived DCs (BMDCs) transduced with lentiviral vectors to permanently expressed shRNA specific for CD40 (CD40LV‐DCs) and/or p19 subunit of interleukin (IL)‐23 (p19LV‐DCs) mRNAs in experimental autoimmune encephalomyelitis (EAE). In‐vitro studies showed that double‐transduced BMDCs (CD40⁺p19LV‐DCs) resemble tolerogenic DCs due to profound down‐regulation of CD40, lower expression of proinflammatory cytokines (IL‐6 and IL‐12), increased IL‐10 production and stronger stimulation of myelin oligodendrocyte glycoprotein (MOG)_35–55‐specific T cells for production of IL‐10 compared with CD40LV‐DCs, p19LV‐DCs and BMDCs transduced with control lentiviral vector (CoLV‐DCs). Moreover, injection of transduced CD40⁺p19LV‐ BMDCs in EAE mice resulted in more reduction in clinical score, significant reduction in IL‐17 or increased production of IL‐10 by mononuclear cells derived from the lymph nodes or spinal cord compared with CoLV‐DCs‐treated EAE mice. In conclusion, simultaneous knock‐down of CD40 and IL‐23 production by BMDCs may represent a promising therapeutic tool for the treatment of IL‐17‐dependent autoimmune diseases, including multiple sclerosis.     

Mycobacterium tuberculosis RpfE promotes simultaneous Th1‐ and Th17‐type T‐cell immunity via TLR4‐dependent maturation of dendritic cells

Reciprocal induction of the Th1 and Th17 immune responses is essential for optimal protection against Mycobacterium tuberculosis (Mtb); however, only a few Mtb antigens are known to fulfill this task. A functional role for resuscitation‐promoting factor (Rpf) E, a latency‐associated member of the Rpf family, in promoting naïve CD4⁺ T‐cell differentiation toward both Th1 and Th17 cell fates through interaction with dendritic cells (DCs) was identified in this study. RpfE induces DC maturation by increasing expression of surface molecules and the production of IL‐6, IL‐1β, IL‐23p19, IL‐12p70, and TNF‐α but not IL‐10. This induction is mediated through TLR4 binding and subsequent activation of ERK, p38 MAPKs, and NF‐κB signaling. RpfE‐treated DCs effectively caused naïve CD4⁺ T cells to secrete IFN‐γ, IL‐2, and IL‐17A, which resulted in reciprocal expansions of the Th1 and Th17 cell response along with activation of T‐bet and RORγt but not GATA‐3. Furthermore, lung and spleen cells from Mtb‐infected WT mice but not from TLR4^?/? mice exhibited Th1 and Th17 polarization upon RpfE stimulation. Taken together, our data suggest that RpfE has the potential to be an effective Mtb vaccine because of its ability to activate DCs that simultaneously induce both Th1‐ and Th17‐polarized T‐cell expansion.     

A novel IL‐23p19/Ebi3 (IL‐39) cytokine mediates inflammation in Lupus‐like mice

Interleukin‐12 family cytokines have emerged as critical regulators of immunity with some members (IL‐12, IL‐23) associated with disease pathogenesis while others (IL‐27, IL‐35) mitigate autoimmune diseases. Each IL‐12 family member is comprised of an α and a β chain, and chain‐sharing is a key feature. Although four bona fide members have thus far been described, promiscuous chain‐pairing between alpha (IL‐23p19, IL‐27p28, IL‐12/IL‐35p35) and beta (IL‐12/IL‐23p40, IL‐27/IL‐35Ebi3) subunits, predicts six possible heterodimeric IL‐12 family cytokines. Here, we describe a new IL‐12 member composed of IL‐23p19 and Ebi3 heterodimer (IL‐39) that is secreted by LPS‐stimulated B cells and GL7⁺ activated B cells of lupus‐like mice. We further show that IL‐39 mediates inflammatory responses through activation of STAT1/STAT3 in lupus‐like mice. Taken together, our results show that IL‐39 might contribute to immunopathogenic mechanisms of systemic lupus erythematosus, and could be used as a possible target for its treatment.     

Immunotherapy using lipopolysaccharide‐stimulated bone marrow‐derived dendritic cells to treat experimental autoimmune encephalomyelitis

Lipopolysaccharide (LPS) produced by Gram‐negative bacteria induces tolerance and suppresses inflammatory responses in vivo; however, the mechanisms are poorly understood. In this study we show that LPS induces apoptosis of bone marrow‐derived dendritic cells (DCs) and modulates phenotypes of DCs. LPS treatment up‐regulates expression of tolerance‐associated molecules such as CD205 and galectin‐1, but down‐regulates expression of Gr‐1 and B220 on CD11c⁺ DCs. Moreover, LPS treatment regulates the numbers of CD11c⁺CD8⁺, CD11c⁺CD11b^low and CD11c⁺CD11b^hi DCs, which perform different immune functions in vivo. Our data also demonstrated that intravenous transfer of LPS‐treated DCs blocks experimental autoimmune encephalomyelitis (EAE) development and down‐regulates expression of retinoic acid‐related orphan receptor gamma t (ROR‐γt), interleukin (IL)‐17A, IL‐17F, IL‐21, IL‐22 and interferon (IFN)‐γ in myelin oligodendrocyte glycoprotein (MOG)‐primed CD4⁺ T cells in the peripheral environment. These results suggest that LPS‐induced apoptotic DCs may lead to generation of tolerogenic DCs and suppress the activity of MOG‐stimulated effector CD4⁺ T cells, thus inhibiting the development of EAE in vivo. Our results imply a potential mechanism of LPS‐induced tolerance mediated by DCs and the possible use of LPS‐induced apoptotic DCs to treat autoimmune diseases such as multiple sclerosis.     

Children with atopic histories exhibit impaired lipopolysaccharide‐induced Toll‐like receptor‐4 signalling in peripheral monocytes

Background The hygiene hypothesis states that early exposure to bacterial products such as lipopolysaccharide (LPS) may be protective against the development of allergic diseases. Whether atopic disease affects the ability of immune cells to respond to LPS is unclear. Our laboratory has demonstrated previously that children express high levels of Toll‐like receptor (TLR)‐4 on CD4⁺ cells in nasal mucosa. Objective To determine if children with a history of allergic disease have impaired responses to LPS on circulating CD4⁺ leucocytes. Methods Peripheral blood mononuclear cells from children (aged 2–18) and adults with or without a history of atopic conditions were cultured with/without IL‐4 or LPS for up to 24 h. Expression of surface TLR‐4, CD14, CD4, CD3, as well as of intracellular phosphorylated ^p42/p44ERK and p38 mitogen‐activated protein kinase (MAPK) were assessed by flow cytometry. Results A history of atopy in children was associated with impaired LPS‐induced TLR‐4‐dependent phosphorylation of ^p42/44ERK and p38 MAPK by CD4⁺ monocytes. Decreased LPS signalling was reproduced by pre‐incubation of control cells with recombinant IL‐4. LPS stimulation also decreased TLR‐4 expression on monocytes from children without atopic histories but not from atopic subjects. CD4⁺ T lymphocytes showed limited LPS responsiveness, regardless of atopic status. In contrast with non‐atopic children, TLR‐4 expression on monocytes of children with atopic histories decreased as a function of age. Conclusions This study provides evidence for defective LPS recognition on circulating CD4⁺ leucocytes of subjects with atopic histories compared with those from non‐atopic children. CD4⁺TLR4⁺ monocytes from children with atopic histories failed to phosphorylate MAPKs. Our results suggest that a history of atopic disease is associated with impaired TLR‐4‐mediated innate immune function compared with non‐atopic children. Cite this as: D. Préfontaine, A.‐A. Banville‐Langelier, P.‐O. Fiset, J. Guay, J. An, M. Mazer, Q. Hamid and B. D. Mazer, Clinical & Experimental Allergy, 2010 (40) 1648–1657.     

Species‐dependent role of type I IFNs and IL‐12 in the CTL response induced by humanized CpG complexed with β‐glucan

CpG oligodeoxynucleotide (ODN) is one of promising nucleic acid‐based adjuvants. We recently improved its ability to enhance CD8⁺ T‐cell responses to coadministered protein antigen without conjugation or emulsion, by forming a nanoparticulate complex between CpG ODN (K3) and mushroom‐derived β‐glucan schizophyllan (SPG), namely K3‐SPG. Here, we sought to elucidate the cellular immunological mechanisms by which K3‐SPG induce such potent CD8⁺ T‐cell responses to coadministered antigen. By focusing on two DC subsets, plasmacytoid DCs and CD8α⁺ DCs, as well as the secreted cytokines, IFN‐α and IL‐12, we found that K3‐SPG strongly activates mouse plasmacytoid DCs to secrete IFN‐α and CD8α⁺ DCs to secrete IL‐12, respectively. Although a single cytokine deficiency had no impact on adjuvant effects, the lack of both type I IFN and IL‐12 in mice resulted in a significant reduction of Th1 type immune responses and CD8⁺ T‐cell responses elicited by protein vaccine model. By sharp contrast, type I IFN, but not IL‐12, was required for the production of IFN‐γ by human PBMCs as well as antigen‐specific CD8⁺ T‐cell proliferation. Taken together, K3‐SPG may overcome the species barrier for CpG ODN to enhance antigen‐specific CD8⁺ T‐cell responses despite the differential role of IL‐12 between human and mice.     

Antigen-activated T cells induce IL-12p75 production from dendritic cells in an IFN-γ-independent manner

The addition of IL‐12p75 to naïve CD4⁺ T cells promotes their differentiation towards a T_H1‐type cytokine pattern. Dendritic cells stimulated by LPS generate IL‐12p75, but only if the environment also contains IFN‐γ. Thus, it appears that IFN‐γ is needed to start the response that will result in further production of IFN‐γ. We previously reported that paradoxically DCs produce IL‐12p75 only after engaging primed, but not naïve T cells. This study examines the mechanism by which primed T cells trigger IL‐12p75 secretion and asks whether this induction is also dependent on the presence of IFN‐γ. Here, we show that, in contrast to LPS, primed T cells induce IL‐12p75 in an IFN‐γ‐independent manner. Addition of rIFN‐γ to cocultures of naïve T cells with DCs did not induce IL‐12p75. Moreover, antigen‐activated CD4⁺ T cells from wild type or IFN‐γ‐deficient mice both initiated IL‐12p75 production from DCs. Surprisingly, we found that synergies between three T‐cell‐derived factors – CD40 Ligand, IL‐4 and GM‐CSF – were necessary and sufficient for IL‐12p75 production. These results suggest that there are at least two distinct pathways for IL‐12p75 production in vivo. Furthermore, the T‐cell‐dependent pathway of IL‐12p75 production employs molecules that are not classically associated with a T_H1‐type response.     

α‐Galactosylceramide‐activated murine NK1.1+ invariant‐NKT cells in the myometrium induce miscarriages in mice

Innate immunity, which is unable to discriminate self from allo‐antigens, is thought to be important players in the induction of miscarriages. Here, we show that the administration of IL‐12 to syngeneic‐mated C57BL/6 mice on gestation day 7.5 (Gd 7.5), drives significant miscarriages in pregnant females. Furthermore, the administration on Gd 7.5 of α‐galactosylceramide (α‐GalCer), which is known to activate invariant natural killer T (iNKT) cells, induced miscarriages in both syngeneic‐mated C57BL/6 mice and allogeneic‐mated mice (C57BL/6 (♀) × BALB/c (♂)). Surprisingly, the percentages of both DEC‐205⁺ DCs and CD1d‐restricted NK1.1⁺ iNKT cells were higher in the myometrium of pregnant mice treated i.p. with α‐GalCer than in the decidua. IL‐12 secreted from α‐GalCer‐activated DEC‐205⁺ DCs stimulated the secretion of cytokines, including IL‐2, IL‐4, IFN‐γ, TNF‐α, perforin, and granzyme B, from the NK1.1⁺ iNKT cells in the myometrium, leading to fetal loss in pregnant mice. Finally, the i.p. administration of IL‐12 and/or α‐GalCer in iNKT‐deficient Jα18(‐/‐) (Jα18 KO) mice did not induce miscarriages. This study provides a new perspective on the importance of the myometrium, rather than the decidua, in regulating pregnancy and a mechanism of miscarriage mediated by activated DEC‐205⁺ DCs and NK1.1⁺ iNKT cells in the myometrium of pregnant mice.     

Mycobacterium tuberculosis PstS1 amplifies IFN‐γ and induces IL‐17/IL‐22 responses by unrelated memory CD4+ T cells via dendritic cell activation

The immunological mechanisms that modulate protection during Mycobacterium tuberculosis (Mtb) infection or vaccination are not fully understood. Secretion of IFN‐γ and, to a lesser extent, of IL‐17 by CD4⁺ T cells plays a major role both in protection and immunopathology. Few Mtb Ags interacting with DCs affect priming, activation, and regulation of Ag‐unrelated CD4⁺ T‐cell responses. Here we demonstrate that PstS1, a 38 kDa‐lipoprotein of Mtb, promotes Ag‐independent activation of memory T lymphocytes specific for Ag85B or Ag85A, two immunodominant protective Ags of Mtb. PstS1 expands CD4⁺ and CD8⁺ memory T cells, amplifies secretion of IFN‐γ and IL‐22 and induces IL‐17 production by effector memory cells in an Ag‐unrelated manner in vitro and in vivo. These effects were mediated through the stimulation of DCs, particularly of the CD8α^? subtype, which respond to PstS1 by undergoing phenotypic maturation and by secreting IL‐6, IL‐1β and, to a lower extent, IL‐23. IL‐6 secretion by PstS1‐stimulated DCs was required for IFN‐γ, and to a lesser extent for IL‐22 responses by Ag85B‐specific memory T cells. These results may open new perspectives for immunotherapeutic strategies to control Th1/Th17 immune responses in Mtb infections and in vaccinations against tuberculosis.     

Autologous tolerogenic dendritic cells derived from monocytes of systemic lupus erythematosus patients and healthy donors show a stable and immunosuppressive phenotype

Systemic lupus erythematosus (SLE) is an autoimmune disease with unrestrained T‐cell and B‐cell activity towards self‐antigens. Evidence shows that apoptotic cells (ApoCells) trigger an autoreactive response against nuclear antigens in susceptible individuals. In this study, we focus on generating and characterizing tolerogenic dendritic cells (tolDCs) to restore tolerance to ApoCells. Monocyte‐derived dendritic cells (DCs) from healthy controls and patients with SLE were treated with dexamethasone and rosiglitazone to induce tolDCs. Autologous apoptotic lymphocytes generated by UV irradiation were given to tolDCs as a source of self‐antigens. Lipopolysaccharide (LPS) was used as a maturation stimulus to induce the expression of co‐stimulatory molecules and secretion of cytokines. TolDCs generated from patients with SLE showed a reduced expression of co‐stimulatory molecules after LPS stimulation compared with mature DCs. The same phenomenon was observed in tolDCs treated with ApoCells and LPS. In addition, ApoCell‐loaded tolDCs stimulated with LPS secreted lower levels of interleukin‐6 (IL‐6) and IL‐12p70 than mature DCs without differences in IL‐10 secretion. The functionality of tolDCs was assessed by their capacity to prime allogeneic T cells. TolDCs displayed suppressor properties as demonstrated by a significantly reduced capacity to induce allogeneic T‐cell proliferation and activation. ApoCell‐loaded tolDCs generated from SLE monocytes have a stable immature/tolerogenic phenotype that can modulate CD4⁺ T‐cell activation. These properties make them suitable for an antigen‐specific immunotherapy for SLE.     

Dendritic cells from human mesenteric lymph nodes in inflammatory and non‐inflammatory bowel diseases: subsets and function of plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDC) in mesenteric lymph nodes (MLN) may be important regulators of both inflammatory and non‐inflammatory mucosal immune responses but human studies are rare. Here we compare pDC from human MLN and peripheral blood (PB) by phenotype and function. MLN from patients with or without inflammatory bowel disease (IBD) undergoing colon surgery and PB from patients with IBD and from controls were used to isolate mononuclear cells. The pDC were analysed by flow cytometry for the expression of CD40, CD80, CD83, CD86, CCR6, CCR7, CX3CR1, CD103 and HLA‐DR. Purified pDC from MLN and PB were stimulated with staphylococcus enterotoxin B (SEB), CpG‐A, interleukin‐3 (IL‐3), SEB + IL‐3, CpG‐A + IL‐3 or left unstimulated, and cultured alone or with purified allogeneic CD4⁺ CD45RA⁺ HLA‐DR‐ T cells. Subsequently, concentrations of IL‐1β, IL‐2, IL‐4, IL‐6, IL‐8, IL‐10, IL‐12, IL‐17, interferon‐α (IFN‐α), IFN‐γ and tumour necrosis factor‐α (TNF‐α) in culture supernatants were determined by multiplex bead array. The PB pDC from IBD patients exhibited an activated and matured phenotype whereas MLN pDC and control PB pDC were less activated. CpG‐A and CpG‐A + IL‐3‐stimulated MLN pDC secreted less IL‐6 and TNF‐α compared with PB pDC from controls. Compared with co‐cultures of naive CD4 T cells with PB pDC, co‐cultures with MLN pDC contained more IL‐2, IL‐10 and IFN‐γ when stimulated with SEB and SEB + IL‐3, and less IFN‐α when stimulated with CpG‐A. MLN pDC differ phenotypically from PB pDC and their pattern of cytokine secretion and may contribute to specific outcomes of mucosal immune reactions.     

Inhibitory effects of suplatast tosilate on the differentiation and function of monocyte-derived dendritic cells from patients with asthma

Background Dendritic cells (DCs) are antigen‐presenting cells that efficiently activate T cells. Objective We examined the effects of suplatast tosilate, which prevents T‐helper type 2 responses, on the differentiation and function of monocyte‐derived DCs (moDCs). Methods DCs were differentiated in vitro from peripheral monocytes from patients with asthma by the addition of granulocyte macrophage colony‐stimulating factor and IL‐4 in the presence or absence of suplatast tosilate. Cell surface molecules (CD1a, CD14, CD80, CD83, CD86, HLA‐DR) on immature and mature DCs were analysed with flow cytometry, and the secretion of CC chemokine ligand (CCL)17 (thymus and activation‐regulated chemokine), IL‐12p70, IL‐12p40, and IL‐10 was measured with an ELISA. We also studied the proliferative responses of allogeneic CD4⁺ T cells from healthy subjects to DCs differentiated in the presence of suplatast tosilate. In addition, the production of IFN‐γ and IL‐5 by CD4⁺ T cells after coculture with untreated DCs or suplatast tosilate‐treated DCs was measured with ELISA. Results Suplatast tosilate significantly inhibited the expression of CD1a, CD80, and CD86 on immature DCs and of CD1a, CD80, CD83, and CD86 on mature DCs. Suplatast tosilate also significantly inhibited the secretion of CCL17, IL‐12p70, and IL‐12p40; however, the secretion of IL‐10 was not affected. The proliferative responses of allogeneic CD4⁺ T cells to suplatast tosilate‐treated DCs were suppressed. Moreover, suplatast tosilate‐treated DCs had an impaired capacity to stimulate CD4⁺ T cells to produce IFN‐γ and IL‐5. Conclusion Suplatast tosilate inhibits the differentiation, maturation, and function of moDCs.     

Human milk oligosaccharides promote immune tolerance via direct interactions with human dendritic cells

Human milk oligosaccharides (HMOS) are a complex mixture of bioactive components supporting the immune development of breastfed‐infants. Dendritic cells (DCs) play a central role in the regulation of immune responses, being specialized in antigen presentation and driving T‐cell priming as well as differentiation. However, little is known about the direct effects of HMOS on human DC phenotypes and functions. Here, we report that HMOS mixture isolated from pooled human milk, induced semi‐maturation of human monocytes‐derived DCs (moDCs), and elevated levels of IL‐10, IL‐27 and IL‐6 but not IL‐12p70 and TNF‐α. Consistently, HMOS‐conditioned human moDCs promoted Treg generation from naïve CD4⁺ T cells. Interestingly, HMOS limited LPS‐induced maturation of human moDCs, while maintained IL‐10 and IL‐27 secretion and reduced LPS‐induced production of IL‐12p70, IL‐6 and TNF‐α. Furthermore, HMOS+LPS‐stimulated DCs induced a higher frequency of Tregs and increased IL‐10 production, while a reduction in Tbet+Th1 frequency and IFN‐γ production was detected as compared to LPS‐DCs. The regulatory effects of HMOS seemed to be mediated by interactions of HMOS with receptors, including but not limited to TLR4 and DC‐SIGN on human moDCs. In conclusion, HMOS contain tolerogenic factors influencing human moDCs and thereby modulating the development of the neonatal immune system.     

Blockade of PD‐1 or p38 MAP kinase signaling enhances senescent human CD8+ T‐cell proliferation by distinct pathways

Immune enhancement is desirable in situations where decreased immunity results in increased morbidity. We investigated whether blocking the surface inhibitory receptor PD‐1 and/or p38 MAP kinase could enhance the proliferation of the effector memory CD8⁺ T‐cell subset that re‐expresses CD45RA (EMRA) and exhibits characteristics of senescence, which include decreased proliferation and telomerase activity but increased expression of the DNA damage response related protein γH2AX. Blocking of both PD‐1 and p38 MAPK signaling in these cells enhanced proliferation and the increase was additive when both pathways were inhibited simultaneously in both young and old human subjects. In contrast, telomerase activity in EMRA CD8⁺ T cells was only enhanced by blocking the p38 but not the PD‐1 signaling pathway, further indicating that nonoverlapping signaling pathways were involved. Although blocking p38 MAPK inhibits TNF‐α secretion in the EMRA population, this decrease was counteracted by the simultaneous inhibition of PD‐1 signaling in these cells. Therefore, end‐stage characteristics of EMRA CD8⁺ T cells are stringently controlled by distinct and reversible cell signaling events. In addition, the inhibition of PD‐1 and p38 signaling pathways together may enable the enhancement of proliferation of EMRA CD8⁺ T cells without compromising their capacity for cytokine secretion.