Plasma membrane Toll‐like receptor activation increases bacterial uptake but abrogates endosomal Lactobacillus acidophilus induction of interferon‐β

Lactobacillus acidophilus induces a potent interferon‐β (IFN‐β) response in dendritic cells (DCs) by a Toll‐like receptor 2 (TLR2) ‐dependent mechanism, in turn leading to strong interleukin‐12 (IL‐12) production. In the present study, we investigated the involvement of different types of endocytosis in the L. acidophilus‐induced IFN‐β and IL‐12 responses and how TLR2 or TLR4 ligation by lipopolysaccharide and Pam3/4CSK4 influenced endocytosis of L. acidophilus and the induced IFN‐β and IL‐12 production. Lactobacillus acidophilus was endocytosed by constitutive macropinocytosis taking place in the immature cells as well as by spleen tyrosine kinase (Syk) ‐dependent phagocytosis but without involvement of plasma membrane TLR2. Stimulation with TLR2 or TLR4 ligands increased macropinocytosis in a Syk‐independent manner. As a consequence, incubation of DCs with TLR ligands before incubation with L. acidophilus enhanced the uptake of the bacteria. However, in these experimental conditions, induction of IFN‐β and IL‐12 was strongly inhibited. As L. acidophilus‐induced IFN‐β depends on endocytosis and endosomal degradation before signalling and as TLR stimulation from the plasma membrane leading to increased macropinocytosis abrogates IFN‐β induction we conclude that plasma membrane TLR stimulation leading to increased macropinocytosis decreases endosomal induction of IFN‐β and speculate that this is due to competition between compartments for molecules involved in the signal pathways. In summary, endosomal signalling by L. acidophilus that leads to IFN‐β and IL‐12 production is inhibited by TLR stimulation from the plasma membrane.     

MD‐2 is involved in the stimulation of matrix metalloproteinase‐1 expression by interferon‐γ and high glucose in mononuclear cells – a potential role of MD‐2 in Toll‐like receptor 4‐independent signalling

We reported recently that treatment of diabetic apolipoprotein E‐deficient mice with the Toll‐like receptor 4 (TLR4) antagonist Rs‐LPS, a lipopolysaccharide isolated from Rhodobacter sphaeroides, inhibited atherosclerosis. Since it is known that Rs‐LPS antagonizes TLR4 by targeting TLR4 co‐receptor MD‐2, this finding indicates that MD‐2 is a potential target for the treatment of atherosclerosis. In this study, we determined if MD‐2 is involved in the gene expression regulated by signalling pathways independent of TLR4. Given that interferon‐γ (IFNγ) and hyperglycaemia play key roles in atherosclerosis, we determined if MD‐2 is involved in IFN‐γ and high‐glucose‐regulated gene expression in mononuclear cells. Results showed that IFN‐γ and high glucose synergistically stimulated matrix metalloproteinase 1 (MMP‐1), a proteinase essential for vascular tissue remodelling and atherosclerosis, in U937 mononuclear cells, but Rs‐LPS inhibited the MMP‐1 stimulation. To provide more evidence for a role of MD‐2 in IFN‐γ‐stimulated MMP‐1, studies using antibodies and small interfering RNA demonstrated that MD‐2 blockade or knockdown attenuated the effect of IFN‐γ on MMP‐1. Furthermore, studies using PCR arrays showed that MD‐2 blockade had a similar effect as IFN‐γ receptor blockade on the inhibition of IFN‐γ‐stimulated pro‐inflammatory molecules. Although these findings indicate the involvement of MD‐2 in IFN‐γ signalling, we also observed that MD‐2 was up‐regulated by IFN‐γ and high glucose. We found that MD‐2 up‐regulation by IFN‐γ played an essential role in the synergistic effect of IFN‐γ and LPS on MMP‐1 expression. Taken together, these findings indicate that MD‐2 is involved in IFN‐γ signalling and IFN‐γ‐augmented MMP‐1 up‐regulation by LPS.     

Murine thymic plasmacytoid dendritic cells

We report herein heterogeneous murine thymic cell subsets expressing CD11c and B220 (CD45R). The CD11c(+)B220(+) subset expresses Ly6C(high) and MHC class II(low) in contrast with previously described thymic DC (CD11c(+)B220(-) cells). Freshly isolated thymic CD11c(+)B220(+) cells show typical plasmacytoid morphology which differentiates to mature DC, in vitro with CpG oligodeoxynucleotides (ODN) 2216; we term this subset thymic plasmacytoid DC (pDC). These thymic pDC are highly sensitive to spontaneous apoptosis in vitro and induce low T cell allo-proliferation activity. Thymic pDC express low TLR2, TLR3 and TLR4 mRNA, normally found on human immature DC, and high TLR7 and TLR9 mRNA, normally found on human pDC. Thymic pDC also produce high amounts of IFN-alpha following culture with CpG ODN 2216 (TLR9 ligands) as compared with the previously defined thymic DC lineage which expresses low TLR9 mRNA and produce high IL-12 (p40) with CpG ODN 2216. These results indicate that thymic pDC are similar to IFN-producing cells as well as human pDC. The TLR and cytokine production profiles are consistent with a nomenclature of pDC. The repertoire of this cell lineage to TLR9 ligands demonstrate that such responses are determined not only by the quantity of expression, but also cell lineage.     

Human β‐defensin 3 increases the TLR9‐dependent response to bacterial DNA

Human β‐defensin 3 (hBD3) is a cationic antimicrobial peptide with potent bactericidal activity in vitro. HBD3 is produced in response to pathogen challenge and can modulate immune responses. The amplified recognition of self‐DNA by human plasmacytoid dendritic cells has been previously reported, but we show here that hBD3 preferentially enhances the response to bacterial DNA in mouse Flt‐3 induced dendritic cells (FLDCs) and in human peripheral blood mononuclear cells. We show the effect is mediated through TLR9 and although hBD3 significantly increases the cellular uptake of both E. coli and self‐DNA in mouse FLDCs, only the response to bacterial DNA is enhanced. Liposome transfection also increases uptake of bacterial DNA and amplifies the TLR9‐dependent response. In contrast to hBD3, lipofection of self‐DNA enhances inflammatory signaling, but the response is predominantly TLR9‐independent. Together, these data show that hBD3 has a role in the innate immune‐mediated response to pathogen DNA, increasing inflammatory signaling and promoting activation of the adaptive immune system via antigen presenting cells including dendritic cells. Therefore, our data identify an additional immunomodulatory role for this copy‐number variable defensin, of relevance to host defence against infection and indicate a potential for the inclusion of HBD3 in pathogen DNA‐based vaccines.     

Bacillus‐derived poly‐γ‐glutamic acid attenuates allergic airway inflammation through a Toll‐like receptor‐4‐dependent pathway in a murine model of asthma

Background Asthma is an inflammatory disease of the airways that is mediated by Th2 responses. Poly‐γ‐glutamic acid (γ‐PGA) is an extracellular polymeric compound that is synthesized by Bacillus cells. Previously, we found that γ‐PGA promoted Th1 cell development in a manner dependent on antigen‐presenting cells, but inhibited Th2 cell development. Objective To investigate the effect of γ‐PGA on dendritic cells (DCs), and its potential for treating Th2‐mediated allergic asthma. Methods Wild‐type, Toll‐like receptor (TLR)‐2 deficient, and TLR‐4‐defective mice were used. DCs derived from the bone marrow and extracted from the lung were stimulated with γ‐PGA and assayed for the expression of signalling molecules, costimulatory molecules, and cytokines. Mice were sensitized and challenged with ovalbumin (OVA) to induce asthma. They were repeatedly injected intranasally with γ‐PGA before and during the challenge period, and inflammation and structural remodelling of the airways were examined. Results γ‐PGA selectively signalled conventional DCs to activate NF‐κB and mitogen‐activated protein kinase, leading to the up‐regulation of CD86, CD40, and IL‐12, but not IL‐10 and IL‐6. These effects of γ‐PGA were dependent on TLR‐4 and independent of TLR‐2. Importantly, the intranasal administration of γ‐PGA to OVA‐sensitized/challenged mice reduced the airway hyperresponsiveness and allergic inflammation such as leucocyte influx, goblet cell hyperplasia, eosinophilia, and Th2 cytokine production. In addition to lowered IgE titres, the treatment of mice with γ‐PGA significantly reduced the multiplication and Th2 polarization of mediastinal lymph node T cells upon allergen‐specific restimulation. These anti‐asthmatic effects of γ‐PGA were also abolished in TLR‐4‐defective mice. Conclusions and Clinical Relevance Our data indicate that γ‐PGA activates DCs to favour Th1 cell induction through a TLR‐4‐dependent pathway and alleviates pathologic symptoms in a Th2‐biased asthmatic model. These findings highlight the potential of γ‐PGA for the treatment of asthma and other allergic disease in which Th2 polarization plays an important role. Cite this as: K. Lee, S.‐H. Kim, H. J. Yoon, D. J. Paik, J. M. Kim and J. Youn, Clinical & Experimental Allergy, 2011 (41) 1143–1156.     

Common variable immunodeficiency revisited: normal generation of naturally occurring dendritic cells that respond to Toll‐like receptors 7 and 9

Patients with common variable immunodeficiency (CVID) have reduced numbers and frequencies of dendritic cells (DCs) in blood, and there is also evidence for defective activation through Toll‐like receptors (TLRs). Collectively, these observations may point to a primary defect in the generation of functional DCs. Here, we measured frequencies of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) in peripheral blood of 26 CVID patients and 16 healthy controls. The results show that the patients have reduced absolute counts of both subsets. However, the decreased numbers in peripheral blood were not reflected in reduced frequencies of CD34⁺ pDC progenitors in the bone marrow. Moreover, studies at the single cell level showed that DCs from CVID patients and healthy controls produced similar amounts of interferon‐α or interleukin‐12 and expressed similar levels of activation markers in response to human cytomegalovirus and ligands for TLR‐7 and TLR‐9. The study represents the most thorough functional characterization to date, and the first to assess bone marrow progenitor output, of naturally occurring DCs in CVID. In conclusion, it seems unlikely that CVID is secondary to insufficient production of naturally occurring DCs or a defect in their signalling through TLR‐7 or TLR‐9.     

Role of A20 in interferon‐α‐mediated functional restoration of myeloid dendritic cells in patients with chronic hepatitis C

Hepatitis C virus (HCV) infection is a global health problem characterized by a high rate of chronic infection, which may in part be due to a defect in myeloid dendritic cells (mDCs). This defect appears to be remedied by treatment with interferon‐α (IFN‐α) ‐based antiviral therapies; however, the molecular mechanisms underlying mDC dysfunction in HCV infection and restoration by IFN‐α treatment are unclear. The ubiquitin‐editing protein A20 plays a crucial role in controlling the maturation, cytokine production and immunostimulatory function of mDCs. We propose that the expression of A20 correlates with the function of mDCs during HCV infection and IFN‐α therapy. In this study, we observed that A20 expression in mDCs isolated from chronically HCV‐infected subjects was significantly higher than healthy subjects or subjects achieving sustained virological responses (SVR) following antiviral treatment. Notably, A20 expression in mDCs from HCV patients during IFN‐α treatment was significantly lower than for untreated patients, SVR patients, or healthy subjects. Besides, A20 expression in mDCs stimulated by polyI:C differed between HCV patients and healthy subjects, and this difference could be abrogated by the treatment with IFN‐α in vitro. Additionally, A20 expression by polyI:C‐activated mDCs, with or without IFN‐α treatment, negatively correlated with the expression of HLA‐DR, CD86 and CCR7, and the secretion of interleukin‐12 (IL‐12), but positively associated with the production of IL‐10. Importantly, silencing A20 expression using small interfering RNAs increased the production of IL‐12 in mDCs of chronically HCV‐infected individuals. These findings suggest that A20 plays a crucial role in negative regulation of innate immune responses during chronic viral infection.     

Pulmonary allergic responses augment interleukin‐13 secretion by circulating basophils yet suppress interferon‐α from plasmacytoid dendritic cells

Background Allergic inflammatory processes may have the capacity to propagate systemically through the actions of circulating leucocytes. Consequently, basophils from allergic individuals are often ‘primed’, as evidenced by their hyperresponsiveness in vitro. IFN‐α secreted predominantly by plasmacytoid dendritic cells (pDCs), suppresses basophil priming for IL‐13 production in vitro. Objective This study sought in vivo correlates arising during experimental allergen challenge that support an ‘axis‐interplay’ between basophils and pDCs. Methods Using segmental allergen challenge (SAC) in the lung, the immune responses of both cell types from the blood were investigated in volunteers (n=10) before and 24 h after allergen exposure. These responses were then correlated with inflammatory parameters measured in bronchoalveolar lavage fluids (BALF). Results In the blood, SAC significantly augmented IL‐13 secretion by basophils induced by IL‐3 (P=0.009), yet reduced IFN‐α secreted by pDCs stimulated with CpG (P=0.018). Both parameters were negatively correlated (P=0.0015), at least among those subjects that secreted the latter. Circulating basophil IL‐13 responses further correlated with post‐SAC bronchoalveolar lavage (BAL) parameters including IL‐13 protein (P=0.04), basophil (P=0.051), eosinophil (P=0.0018), and total cell counts (P<0.003). Basophil and IL‐13 levels in BAL correlated likewise (P=0.0002). Conclusions These results support a mechanism of immune regulation whereby an allergen reduces innate immune responses and IFN‐α production by pDCs, resulting in an enhanced inflammation and basophil cytokine production at sites of allergen exposure. Cite this as: J. T. Schroeder, A. P. Bieneman, K. L. Chichester, L. Breslin, H. Xiao and M. C. Liu, Clinical & Experimental Allergy, 2010 (40) 745–754.     

Origin and filiation of human plasmacytoid dendritic cells

Human plasmacytoid dendritic cells represent a rare population of leukocytes which produce high amounts of type I interferon in response to certain viruses. Although those cells were first described in 1958, there are still unsolved issues related to their origin and function. Recently, a leukemic counterpart of plasmacytoid dendritic cells was identified. Molecular approaches using either normal or leukemic plasmacytoid dendritic cells provide some new insights into the controversial lymphoid origin of those cells. The need for specific markers is still a critical aspect for the identification of plasmacytoid dendritic cells, whatever stage of differentiation, in normal as well as in pathological conditions. Hopefully, novel markers will allow delineation of the relationships between dendritic cells at different stages of differentiation/maturation along the myeloid and lymphoid lineages.     

Reduced expression of oestrogen receptor‐β is associated with tumour invasion and metastasis in oestrogen receptor‐α‐negative human papillary thyroid carcinoma

Oestrogens play an important role in the development and progression of papillary thyroid carcinoma (PTC) through oestrogen receptor (ER)‐α and ‐β, which may exert different or even opposing actions in PTC. The roles of ERβ in ERα‐negative PTC are still not clear. This study investigated the expression dynamics of ERβ1 (wild‐type ERβ) and its clinical significance in female ERα‐negative PTC patients. ERβ1 expression was detected in thyroid tissues of 136 female patients diagnosed with PTC. The relationships between ERβ1 expression and clinicopathological/biological factors were also analysed in female ERα‐negative PTC patients. The total score for ERβ1 was significantly lower in female ERα‐negative PTC patients with LNM or ETE when compared to those without LNM or ETE (Z = ?2.923, P = 0.003 and Z = ?3.441, P = 0.001). Accordingly, the total score for ERβ1 was significantly higher in ERα‐negative PTC patients expressing E‐cadherin compared to patients negative for E‐cadherin expression (Z = ?2.636, P = 0.008). The total score was lower in ERα‐negative PTC patients positive for VEGF expression compared to those negative for VEGF expression (Z = ?1.914, P = 0.056). This preliminary study indicates that reduced expression of ERβ1 in female ERα‐negative PTC patients is associated with greater progression of the disease. This may provide insights into the underlying molecular mechanisms of ERβ1 and could help design targeted approaches for treating or even preventing this disease.     

Interleukin‐27 suppresses osteoclastogenesis via induction of interferon‐γ

Interleukin (IL)‐27 is a heterodimeric cytokine that is known to have both stimulatory and inhibitory functions during immune responses. We investigated the effects of IL‐27 on arthritis and bone erosion in the murine collagen‐induced arthritis (CIA) model. We demonstrate that the inhibitory effect of IL‐27 on osteoclastogenesis is associated with interferon‐γ (IFN‐γ) production by using an IFN‐γ knockout mouse model. The IL‐27‐Fc was injected into both CIA and IFN‐γ‐deficient mice. The effects of IL‐27‐Fc on osteoclast differentiation were evaluated both in vitro and in vivo. The IL‐27‐Fc‐injected mice showed significantly lower arthritis indices and fewer tartrate‐resistant acid‐phosphatase‐positive osteoclasts in their joint tissues than untreated mice. Interleukin‐27 inhibited osteoclastogenesis from bone marrow‐derived mononuclear cells in vitro, which was counteracted by the addition of anti‐IFN‐γ antibody. The IL‐27‐Fc did not affect arthritis in IFN‐γ knockout mice. Interleukin‐27 also suppressed osteoclast differentiation in human and intriguingly, it could promote the expression of IFN‐γ on priming osteoclasts. These results imply that IL‐27 suppressed the generation of CIA and osteoclastogenesis, which were mediated by the induction of IFN‐γ.     

Role of natural interferon-producing cells and T lymphocytes in porcine monocyte-derived dendritic cell maturation

Maturation of dendritic cells (DC) is a key immunological process regulating immune responses to pathogens and vaccines, as well as tolerance and autoimmune processes. Consequently, the regulation of DC maturation should reflect these multifaceted immunological processes. In the present study, we have defined the role of particular cytokines, Toll-like receptor (TLR) ligands and T lymphocytes in the porcine monocyte-derived DC (MoDC). Interferon-alpha (IFN-alpha) alone was a poor inducer of MoDC maturation, but in association with tumour necrosis factor-alpha (TNF-alpha), or TLR ligands such as lipopolysaccharide and polyinosinic-polycytidylic acid I:C, an up-regulation of major histocompatibility complex II and CD80/86 expression was noted, along with reduced endocytic activity. In contrast, TNF-alpha alone or in combination with the TLR ligands was a poor inducer of DC maturation, but co-operated with T-lymphocytes in the presence of antigen to induce DC maturation. Natural interferon producing cells (NIPC, or plasmacytoid DCs) represent a danger-recognition system of the immune defences, and can respond to viruses not otherwise recognized as posing a danger. Indeed, MoDC did not respond to transmissible gastroenteritis virus (TGEV), whereas NIPC produced high levels of IFN-alpha and TNF-alpha after TGEV stimulation. Moreover, supernatants from the stimulated NIPC induced maturation in MoDCs. These matured MoDCs displayed an enhanced ability to present antigen to and thus stimulate T cells. Taken together, the present work demonstrates that maturation of MoDC not only results from TLR signalling, but can require co-operation with various cell types--principally NIPC and activated T cells--which would reflect the particular immunological situation.     

Activation of invariant natural killer T cells in regional lymph nodes as new antigen‐specific immunotherapy via induction of interleukin‐21 and interferon‐γ

Invariant natural killer T (iNKT) cells play important immunoregulatory functions in allergen‐induced airway hyperresponsiveness and inflammation. To clarify the role of iNKT cells in allergic rhinitis (AR), we generated bone marrow‐derived dendritic cells (BMDCs), which were pulsed by ovalbumin (OVA) and α‐galactosylceramide (OVA/α‐GalCer‐BMDCs) and administered into the oral submucosa of OVA‐sensitized mice before nasal challenge. Nasal symptoms, level of OVA‐specific immunoglobulin (IgE), and T helper type 2 (Th2) cytokine production in cervical lymph nodes (CLNs) were significantly ameliorated in wild‐type (WT) mice treated with OVA/α‐GalCer‐BMDCs, but not in WT mice treated with OVA‐BMDCs. These anti‐allergic effects were not observed in Jα18^–/– recipients that lack iNKT cells, even after similar treatment with OVA/α‐GalCer‐BMDCs in an adoptive transfer study with CD4⁺ T cells and B cells from OVA‐sensitized WT mice. In WT recipients of OVA/α‐GalCer‐BMDCs, the number of interleukin (IL)‐21‐producing iNKT cells increased significantly and the Th1/Th2 balance shifted towards the Th1 dominant state. Treatment with anti‐IL‐21 and anti‐interferon (IFN)‐γ antibodies abrogated these anti‐allergic effects in mice treated with α‐GalCer/OVA‐BMDCs. These results suggest that activation of iNKT cells in regional lymph nodes induces anti‐allergic effects through production of IL‐21 or IFN‐γ, and that these effects are enhanced by simultaneous stimulation with antigen. Thus, iNKT cells might be a useful target in development of new treatment strategies for AR.     

The atypical IκB protein IκBNS is important for Toll‐like receptor‐induced interleukin‐10 production in B cells

Although a major function of B cells is to mediate humoral immunity by producing antigen‐specific antibodies, a specific subset of B cells is important for immune suppression, which is mainly mediated by the secretion of the anti‐inflammatory cytokine interleukin‐10 (IL‐10). However, the mechanism by which IL‐10 is induced in B cells has not been fully elucidated. Here, we report that IκB_NS, an inducible nuclear IκB protein, is important for Toll‐like receptor (TLR)‐mediated IL‐10 production in B cells. Studies using IκB_NS knockout mice revealed that the number of IL‐10‐producing B cells is reduced in IκB_NS^?/? spleens and that the TLR‐mediated induction of cytoplasmic IL‐10‐positive cells and IL‐10 secretion in B cells are impaired in the absence of IκB_NS. The impairment of IL‐10 production by a lack of IκB_NS was not observed in TLR‐triggered macrophages or T‐cell‐receptor‐stimulated CD4⁺ CD25⁺ T cells. In addition, IκB_NS‐deficient B cells showed reduced expression of Prdm1 and Irf4 and failed to generate IL‐10⁺ CD138⁺ plasmablasts. These results suggest that IκB_NS is selectively required for IL‐10 production in B cells responding to TLR signals, so defining an additional role for IκB_NS in the control of the B‐cell‐mediated immune responses.