Abstract: A complete genetic deficiency of the complement protein C1q results in SLE with nearly 100% penetrance in humans, but the molecular mechanisms responsible for this association have not yet been fully determined. C1q opsonizes ACs for enhanced ingestion by phagocytes, such as Mϕ and iDCs, avoiding the extracellular release of inflammatory DAMPs upon loss of the membrane integrity of the dying cell. We previously showed that human monocyte-derived Mϕ and DCs ingesting autologous, C1q-bound LALs (C1q-polarized Mϕ and C1q-polarized DCs), enhance the production of anti-inflammatory cytokines, and reduce proinflammatory cytokines relative to Mϕ or DC ingesting LAL alone. Here, we show that C1q-polarized Mϕ have elevated PD-L1 and PD-L2 and suppressed surface CD40, and C1q-polarized DCs have higher surface PD-L2 and less CD86 relative to Mϕ or DC ingesting LAL alone, respectively. In an MLR, C1q-polarized Mϕ reduced allogeneic and autologous Th17 and Th1 subset proliferation and demonstrated a trend toward increased Treg proliferation relative to Mϕ ingesting LAL alone. Moreover, relative to DC ingesting AC in the absence of C1q, C1q-polarized DCs decreased autologous Th17 and Th1 proliferation. These data demonstrate that a functional consequence of C1q-polarized Mϕ and DC is the regulation of Teff activation, thereby "sculpting" the adaptive immune system to avoid autoimmunity, while clearing dying cells. It is noteworthy that these studies identify novel target pathways for therapeutic intervention in SLE and other autoimmune diseases.

Abstract: One prerequisite that radiotherapy (RT) and chemotherapy (CT) result in anti-tumor immune responses is triggering of immunogenic cell death forms such as necroptosis. The latter is inducible by inhibition of apoptosis with the pan-caspase inhibitor zVAD-fmk. The design of multimodal therapies that overcome melanoma's resistance to apoptosis is a big challenge of oncoimmunology. As hints exist that immune stimulation by hyperthermia (HT) augments the efficacy of melanoma therapies and that tumors can be sensitized for RT with zVAD-fmk, we asked whether combinations of RT with dacarbazine (DTIC) and/or HT induce immunogenic melanoma cell death and how this is especially influenced by zVAD-fmk. Necroptosis was inducible in poorly immunogenic B16-F10 melanoma cells and zVAD-fmk generally increased melanoma cell necrosis concomitantly with the release of HMGB1. Supernatants (SNs) of melanoma cells whose cell death was modulated with zVAD-fmk induced an upregulation of the activation markers CD86 and MHCII on macrophages. The same was seen on dendritic cells (DCs), but only when zVAD-fmk was added to multimodal tumor treatments including DTIC. DCs of MyD88 KO mice and DCs incubated with SNs containing apyrase did not increase the expression of these activation markers on their surface. The in vivo experiments revealed that zVAD-fmk decreases the tumor growth significantly and results in a significantly reduced tumor infiltration of Tregs when added to multimodal treatment of the tumor with RT, DTIC and HT. Further, a significantly increased DC and CD8+ T-cell infiltration into the tumor and in the draining lymph nodes was induced, as well as an increased expression of IFNγ by CD8+ T cells. However, zVAD-fmk did not further reduce tumor growth in MyD88 KO mice, mice treated with apyrase or RAG KO mice. We conclude that HMGB1, nucleotides and CD8+ T cells mediate zVAD-fmk induced anti-melanoma immune reactions in multimodal therapy settings.

Abstract: Photodynamic therapy (PDT) not only kills tumor cells directly but also rapidly recruits and activates immune cells favoring the development of antitumor adaptive immunity. It is believed that Topical 5-aminolevulinic acid mediated photodynamic therapy (ALA-PDT) can induce anti-tumor immune responses through dangerous signals damage-associated molecular patterns (DAMPs). In this study, we investigated the effect of ALA-PDT induced DAMPs on immune cells. We focused on the stimulation of dendritic cells by major DAMPs, enhanced the expression of calreticulin (CRT), heat shock proteins 70 (HSP70), and high mobility group box 1 (HMGB1), either individually or in combination. We evaluated in vitro and in vivo expressions of DAMPs induced by ALA-PDT using immunohistochemistry, western blot, and ELISA in a squamous cell carcinoma (SCC) mouse model. The role of DAMPs in the maturation of DCs potentiated by ALA-PDT-treated tumor cells was detected by FACS and ELISA. Our results showed that ALA-PDT enhanced the expression of CRT, HSP70, and HMGB1. These induced DAMPs played an important part in activating DCs by PDT-treated tumor cells, including phenotypic maturation (increase of surface expression of MHC-II, CD80, and CD86) and functional maturation (enhanced capability to secrete IFN-γ and IL-12). Furthermore, injecting ALA-PDT-treated tumor cells into naive mice resulted in complete protection against cancer cells of the same origin. Our findings indicate that ALA-PDT can increase DAMPs and enhance tumor immunogenicity, providing a promising strategy for inducing a systemic anticancer immune response.

Abstract: Regulatory T cells (Tregs) have been shown to enhance immune reconstitution and prevent graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation; however, it is unclear how Tregs mediate these effects. Here, we developed a model to examine the mechanism of Treg-dependent regulation of immune reconstitution. Lymphopenic mice were selectively reconstituted with Tregs prior to transfer of conventional CD4+ T cells. Full Treg reconstitution prevented the rapid oligoclonal proliferation that gives rise to pathogenic CD4 effector T cells, while preserving the slow homeostatic form of lymphopenia-induced peripheral expansion that repopulates a diverse peripheral T cell pool. Treg-mediated CTLA-4-dependent downregulation of CD80/CD86 on DCs was critical for inhibition of rapid proliferation and was a function of the Treg/DC ratio achieved by reconstitution. In an allogeneic BM transplant model, selective Treg reconstitution before T cell transfer also normalized DC costimulation and provided complete protection against GVHD. In contrast, cotransfer of Tregs was not protective. Our results indicate that achieving optimal recovery from lymphopenia should aim to improve early Treg reconstitution in order to increase the relative number of Tregs to DCs and thereby inhibit spontaneous oligoclonal T cell proliferation.

Abstract: Circulating monocytes in several mammalian species can be subdivided into functionally distinct subpopulations based on differential expression of surface molecules. We confirm that bovine monocytes express CD172a and MHC class II with two distinct populations of CD14+CD16low/-CD163+ and CD14−CD16++CD163low- cells, and a more diffuse population of CD14+CD16+CD163+ cells. In contrast, ovine monocytes consisted of only a major CD14+CD16+ subset and a very low percentage of CD14−CD16++cells. The bovine subsets expressed similar levels of CD80, CD40 and CD11c molecules and mRNA encoding CD115. However, further mRNA analyses revealed that the CD14−CD16++ monocytes were CX3CR1highCCR2low whereas the major CD14+ subset was CX3CR1lowCCR2high. The former were positive for CD1b and had lower levels of CD11b and CD86 than the CD14+ monocytes. The more diffuse CD14+CD16+ population generally expressed intermediate levels of these molecules. All three populations responded to stimulation with phenol-extracted lipopolysaccharide (LPS) by producing interleukin (IL)-1β, with the CD16++ subset expressing higher levels of IL-12 and lower levels of IL-10. The CD14−CD16++ cells were more endocytic and induced greater allogeneic T cell responses compared to the other monocyte populations. Taken together the data show both similarities and differences between the classical, intermediate and non-classical definitions of monocytes as described for other mammalian species, with additional potential subpopulations. Further functional analyses of these monocyte populations may help explain inter-animal and inter-species variations to infection, inflammation and vaccination in ruminant livestock.

Abstract: Maturation of dendritic cells (DCs) is required to induce T cell immunity, whereas immature DCs can induce immune tolerance. Although the transcription factor STAT5 is suggested to participate in DC maturation, its role in this process remains unclear. In this study, we investigated the effect of STAT5 inhibition on LPS-induced maturation of human monocyte-derived DCs (Mo-DCs). We inhibited STAT5 by treating Mo-DCs with JQ1, a selective inhibitor of BET epigenetic readers, which can suppress STAT5 function. We found that JQ1 inhibits LPS-induced STAT5 phosphorylation and nuclear accumulation, thereby attenuating its transcriptional activity in Mo-DCs. The diminished STAT5 activity results in impaired maturation of Mo-DCs, as indicated by defective upregulation of costimulatory molecules and CD83, as well as reduced secretion of IL-12p70. Expression of constitutively activated STAT5 in JQ1-treated Mo-DCs overcomes the effects of JQ1 and enhances the expression of CD86, CD83, and IL-12. The activation of STAT5 in Mo-DCs is mediated by GM-CSF produced following LPS stimulation. Activated STAT5 then leads to increased expression of both GM-CSF and GM-CSFR, triggering an autocrine loop that further enhances STAT5 signaling and enabling Mo-DCs to acquire a more mature phenotype. JQ1 decreases the ability of Mo-DCs to induce allogeneic CD4(+) and CD8(+) T cell proliferation and production of proinflammatory cytokines. Furthermore, JQ1 leads to a reduced generation of inflammatory CD8(+) T cells and decreased Th1 differentiation. Thus, JQ1 impairs LPS-induced Mo-DC maturation by inhibiting STAT5 activity, thereby generating cells that can only weakly stimulate an adaptive-immune response. Therefore, JQ1 could have beneficial effects in treating T cell-mediated inflammatory diseases.

Abstract: Because dendritic cells (DCs) play critical roles in the pathogenesis of rheumatoid arthritis, modulation of their functions could serve as a novel therapy. In this study, we demonstrated that FTY720 treatment significantly suppressed the incidence and severity of collagen-induced arthritis (CIA) in DBA/1J mice via the modulation of DC functions. In FTY720-treated CIA mice, a decrease in the number of DCs in local draining lymph nodes (LNs) was observed. In vitro, FTY720 inhibited the trafficking of LPS-stimulated bone marrow-derived DCs (BMDCs). Decreased secretion of CCL19 and downregulation of CCR7 on DCs may explain the mechanisms underlying the impairment of DC migration induced by FTY720. In a DC-induced mouse arthritis model, FTY720 treatment also suppressed the incidence and severity of arthritis, which was correlated with a decrease in the migration of injected BMDCs to draining LNs. Although lower levels of costimulatory molecules (CD40, CD80, and CD86) and I-A(q) expressed on LN DCs were observed in FTY720-treated mice, in vitro analysis showed no effect of FTY720 on LPS-stimulated BMDC maturation. Furthermore, LN cells from FTY720-treated CIA mice displayed diminished production of proinflammatory cytokines in response to collagen II and Con A stimulation. In addition, the ratio of Th1/Th2 in the draining LNs of mice with DC-induced arthritis was decreased upon FTY720 treatment. This finding was consistent with the fact that FTY720 suppressed IL-12p70 production in cultured BMDCs. Taken together, these results indicate that inhibition of DC migration by FTY720 may provide a novel approach in treating autoimmune diseases such as rheumatoid arthritis.

Abstract: // Klara Daňova 1,2 , Anna Klapetkova 1,2 , Jana Kayserova 2 , Anna Sediva 2 , Radek Spisek 1,2 and Lenka Palova Jelinkova 1,2 1 Sotio a.s., Prague, Czech Republic 2 Department of Immunology, Charles University, 2nd Medical School, Prague, Czech Republic Correspondence to: Lenka Palova Jelinkova, email: // Keywords : tolerogenic DCs, immunoregulation, stability, activation pathways, glycolysis Received : May 05, 2015 Accepted : May 12, 2015 Published : May 22, 2015 Abstract Tolerogenic dendritic cells (tDCs) may offer an intervention therapy in autoimmune diseases or transplantation. Stable immaturity and tolerogenic function of tDCs after encountering inflammatory environment are prerequisite for positive outcome of immunotherapy. However, the signaling pathways regulating their stable tolerogenic properties are largely unknown. In this study, we demonstrated that human monocyte-derived tDCs established by using paricalcitol (analogue of vitamin D2), dexamethasone and monophosphoryl lipid A exposed for 24h to LPS, cytokine cocktail, polyI:C or CD40L preserved reduced expression of co-stimulatory molecules, increased levels of inhibitory molecules ILT-3, PDL-1 and TIM-3, increased TLR-2, increased secretion of IL-10 and TGF-β, reduced IL-12 and TNF-α secretion and reduced T cell stimulatory capacity. tDCs further induced IL-10-producing T regulatory cells that suppressed the proliferation of responder T cells. In the inflammatory environment, tDCs maintained up-regulated indoleamine 2, 3 dioxygenase but abrogated IκB-α phosphorylation and reduced transcriptional activity of p65/RelA, RelB and c-Rel NF-κB subunits except p50. Mechanistically, p38 MAPK, ERK1/2, mTOR, STAT3 and mTOR-dependent glycolysis regulated expression of ILT-3, PDL-1 and CD86, secretion of IL-10 and T cell stimulatory capacity of tDCs in the inflammatory environment. Stability of tDCs in the inflammatory environment is thus regulated by multiple signaling pathways.

Abstract: Suppressive myeloid cells represent a significant barrier to the generation of productive antitumor immune responses to many solid tumors Eliminating or reprogramming suppressive myeloid cells to abrogate tumor-associated immune suppression is a promising therapeutic approach We asked whether treatment of established aggressive disseminated pancreatic cancer with the immunotherapeutic attenuated Toxoplasma gondii vaccine strain CPS would trigger tumor-associated myeloid cells to generate therapeutic antitumor immune responses CPS treatment significantly decreased tumor-associated macrophages and markedly increased dendritic cell infiltration of the pancreatic tumor microenvironment Tumor-resident macrophages and dendritic cells, particularly cells actively invaded by CPS, increased expression of costimulatory molecules CD80 and CD86 and concomitantly boosted their production of IL12 CPS treatment increased CD4(+) and CD8(+) T-cell infiltration into the tumor microenvironment, activated tumor-resident T cells, and increased IFNγ production by T-cell populations CPS treatment provided a significant therapeutic benefit in pancreatic tumor-bearing mice This therapeutic benefit depended on IL12 and IFNγ production, MyD88 signaling, and CD8(+) T-cell populations Although CD4(+) T cells exhibited activated effector phenotypes and produced IFNγ, CD4(+) T cells as well as natural killer cells were not required for the therapeutic benefit In addition, CD8(+) T cells isolated from CPS-treated tumor-bearing mice produced IFNγ after re-exposure to pancreatic tumor antigen, suggesting this immunotherapeutic treatment stimulated tumor cell antigen-specific CD8(+) T-cell responses This work highlights the potency and immunotherapeutic efficacy of CPS treatment and demonstrates the significance of targeting tumor-associated myeloid cells as a mechanism to stimulate more effective immunity to pancreatic cancer

Abstract: The role and different origin of brain myeloid cells in the brain is central to understanding how the central nervous system (CNS) responds to injury. C-type lectin receptor family 9, member A (DNGR-1/CLEC9A) is a marker of specific DC subsets that share functional similarities, such as CD8α(+) DCs in lymphoid tissues and CD103(+) CD11b(low) DCs in peripheral tissues. Here, we analyzed the presence of DNGR-1 in DCs present in the mouse brain (bDCs). Dngr-1/Clec9a mRNA is expressed mainly in the meningeal membranes and choroid plexus (m/Ch), and its expression is enhanced by fms-like tyrosine kinase 3 ligand (Flt3L), a cytokine involved in DC homeostasis. Using Clec9a(egfp/egfp) mice, we show that Flt3L induces accumulation of DNGR-1-EGFP(+) cells in the brain m/Ch. Most of these cells also express major histocompatibility complex class II (MHCII) molecules. We also observed an increase in specific markers of cDC CD8α+ cells such as Batf-3 and Irf-8, but not of costimulatory molecules such as Cd80 and Cd86, indicating an immature phenotype for these bDCs in the noninjured brain. The presence of DNGR-1 in the brain provides a potential marker for the study of this specific brain cell subset. Knowledge and targeting of brain antigen presenting cells (APCs) has implications for the fight against brain diseases such as neuroinflammation-based neurodegenerative diseases, microbe-induced encephalitis, and brain tumors such as gliomas.

Abstract: Gr-1(+) CD11b(+) myeloid-derived suppressor cells (MDSCs) accumulate in tumor-bearing animals and play a critical negative role during tumor immunotherapy. Strategies for inhibition of MDSCs are expected to improve cancer immunotherapy. Polysaccharide Agaricus blazei Murill (pAbM) has been found to have anti-cancer activity, but the underlying mechanism of this is poorly understood. Here, pAbM directly activated the purified MDSCs through inducing the expression of interleukin-6 (IL-6), IL-12, tumour necrosis factor and inducible nitric oxide synthase (iNOS), CD86, MHC II, and pSTAT1 of it, and only affected natural killer and T cells in the presence of Gr-1(+) CD11b(+) monocytic MDSCs. On further analysis, we demonstrated that pAbM could selectively block the Toll-like receptor 2 (TLR2) signal of Gr-1(+) CD11b(+) MDSCs and increased their M1-type macrophage characteristics, such as producing IL-12, lowering expression of Arginase 1 and increasing expression of iNOS. Extensive study showed that Gr-1(+) CD11b(+) MDSCs by pAbM treatment had less ability to convert the CD4(+) CD25(-) cells into CD4(+) CD25(+) phenotype. Moreover, result from selective depletion of specific cell populations in xenograft mice model suggested that the anti-tumour effect of pAbM was dependent on Gr-1(+ ) CD11b(+) monocytes, nether CD8(+) T cells nor CD4(+) T cells. In addition to, pAbM did not inhibit tumour growth in TLR2(-/-) mice. All together, these results suggested that pAbM, a natural product commonly used for cancer treatment, was a specific TLR2 agonist and had potent anti-tumour effects through the opposite of the suppressive function of Gr-1(+) CD11b(+) MDSCs.

Abstract: The liver maintains a tolerogenic environment to avoid unwarranted activation of its resident immune cells upon continuous exposure to food and bacterially derived Ags. However, in response to hepatotropic viral infection, the liver's ability to switch from a hyporesponsive to a proinflammatory environment is mediated by select sentinels within the parenchyma. To determine the contribution of hepatic dendritic cells (DCs) in the activation of naive CD8(+) T cells, we first characterized resident DC subsets in the murine liver. Liver DCs exhibit unique properties, including the expression of CD8α (traditionally lymphoid tissue specific), CD11b, and CD103 markers. In both the steady-state and following viral infection, liver CD103(+) DCs express high levels of MHC class II, CD80, and CD86 and contribute to the high number of activated CD8(+) T cells. Importantly, viral infection in the Batf3(-/-) mouse, which lacks CD8α(+) and CD103(+) DCs in the liver, results in a 3-fold reduction in the proliferative response of Ag-specific CD8(+) T cells. Limiting DC migration out of the liver does not significantly alter CD8(+) T cell responsiveness, indicating that CD103(+) DCs initiate the induction of CD8(+) T cell responses in situ. Collectively, these data suggest that liver-resident CD103(+) DCs are highly immunogenic in response to hepatotropic viral infection and serve as a major APC to support the local CD8(+) T cell response. It also implies that CD103(+) DCs present a promising cellular target for vaccination strategies to resolve chronic liver infections.

Abstract: In humans, tolerance to renal transplants has been associated with alterations in B-cell gene transcription and maintenance of the numbers of circulating transitional B cells. Here, we use a mouse model of transplantation tolerance to investigate the contribution of B cells to allograft survival. We demonstrate that transfer of B cells from mice rendered tolerant to MHC class I mismatched skin grafts can prolong graft survival in a dose-dependent and antigen-specific manner to a degree similar to that afforded by graft-specific regulatory T (Treg) cells. Tolerance in this model was associated with an increase in transitional-2 (T2) B cells. Only T2 B cells from tolerized mice, not naive T2 nor alloantigen experienced T2, were capable of prolonging skin allograft survival, and suppressing T-cell activation. Tolerized T2 B cells expressed lower levels of CD86, increased TIM-1, and demonstrated a preferential survival in vivo. Furthermore, we demonstrate a synergistic effect between tolerized B cells and graft-specific Treg cells. IL-10 production by T2 B cells did not contribute to tolerance, as shown by transfer of B cells from IL-10(-/-) mice. These results suggest that T2 B cells in tolerant patients may include a population of regulatory B cells that directly inhibit graft rejection.

Abstract: Background:Multiple sclerosis (MS) is an immune-mediated inflammatory disease of the central nervous system. B cells have been strongly implicated in disease pathogenesis based on clinical trials with B-cell ablation. There is a growing body of evidence linking microRNAs with regulation of the immune system. Dicer, a key enzyme involved in microRNA biogenesis, is necessary for normal B-cell function.Objective:We aimed to determine whether Dicer expression is impaired in B cells and is linked to increased expression co-stimulatory molecules in patients with MS.Methods:B cells were separated from blood samples of MS patients and healthy subjects. Expression of Dicer and co-stimulatory molecules CD80 and CD86 was tested. The effect of Dicer modulation on CD80 and CD86 expression in B cells was studied.Results:Dicer expression was decreased in B cells but not in monocytes of patients with MS compared with healthy subjects. CD80 and CD86 expression was increased on B cells of MS patients compared with healthy ...

Abstract: Ginsenoside metabolite compound K (CK; 20-O-d-glucopyranosyl-20(S)-protopanaxadiol), a novel ginsenoside metabolite, belongs to the dammarane-type triterpene saponins, according to its structure. The anti-inflammatory activity of CK has been identified in several studies. Our study demonstrated that CK exerted an anti-inflammatory effect in collagen-induced arthritis (CIA) and adjuvant-induced arthritis animal models, and this effect was due to inhibition of the abnormal activation and differentiation of T cells. However, the mechanism of CK in suppressing T-cell activation remains unclear. In this study, CK had a therapeutic effect in mice with CIA, decreased the percentage of activated T cells and dendritic cells (DCs), and increased the percentage of naive T cells in lymph nodes. The inhibitory effect on T-cell activation of CK was related to suppression of accumulation of DCs in lymph nodes. CK decreased CCL21 levels in lymph nodes and CCR7 expression in DCs and suppressed CCL21/CCR7-mediated migration of DCs, thus reducing accumulation of DCs in lymph nodes. In addition, signals for T-cell activation including major histocompatibility complex class II and costimulatory molecules, such as CD80 and CD86, were suppressed by CK, and the proliferation of T cells induced by DCs was inhibited by CK. In conclusion, this study demonstrated that CK downregulated DC priming of T-cell activation in CIA, and suppression of CCL21/CCR7-mediated DC migration and signaling between T cells and DCs might be the potential mechanism. These results provide an interesting, novel insight into the potential mechanism by which CK contributes to the anti-inflammatory effect in autoimmune conditions.

Abstract: Dendritic cells (DCs) play an important role in the generation of anti-cancer immune responses, however there is evidence that DCs in cancer patients are dysfunctional. Lipid accumulation driven by tumor-derived factors has recently been shown to contribute to DC dysfunction in several human cancers, but has not yet been examined in mesothelioma. This study investigated if mesothelioma tumor cells and/or their secreted factors promote increases in DC lipid content and modulate DC function. Human monocyte-derived DCs (MoDCs) were exposed to human mesothelioma tumor cells and tumor-derived factors in the presence or absence of lipoproteins. The data showed that immature MoDCs exposed to mesothelioma cells or factors contained increased lipid levels relative to control DCs. Lipid accumulation was associated with reduced antigen processing ability (measured using a DQ OVA assay), upregulation of the co-stimulatory molecule, CD86, and production of the tolerogenic cytokine, IL-10. Increases in DC lipid content were further enhanced by co-exposure to mesothelioma-derived factors and triglyceride-rich lipoproteins, but not low-density lipoproteins. In vivo studies using a murine mesothelioma model showed that the lipid content of tumor-infiltrating CD4+CD8α- DCs, CD4-CD8α- DCs DCs and plasmacytoid DCs increased with tumor progression. Moreover, increasing tumor burden was associated with reduced proliferation of tumor-antigen-specific CD8+ T cells in tumor-draining lymph nodes. This study shows that mesothelioma promotes DC lipid acquisition, which is associated with altered activation status and reduced capacity to process and present antigens, which may impair the ability of DCs to generate effective anti mesothelioma T cell responses.

Abstract: Objective:We aimed to clarify whether fingolimod has direct effects on antigen-presenting cells in multiple sclerosis patients.Methods:Frequency and phenotype of directly ex vivo dendritic cells and monocytes were analyzed in 43 individuals, including fingolimod-treated and untreated multiple sclerosis patients as well as healthy subjects. These cells were further stimulated with lipopolysaccharide to determine functional effects of fingolimod treatment.Results:Absolute numbers of CD1c+ dendritic cells and monocytes were not significantly reduced in fingolimod-treated patients indicating that fingolimod did not block the migration of antigen-presenting cells to peripheral blood. CD86 was upregulated on CD1c+ dendritic cells and thus their activation was not impaired under fingolimod treatment. Quantitative analyses of gene transcription in cells and protein content in supernatants from ex vivo CD1c+ dendritic cells and monocytes, however, showed lower secretion of TNFα, IL1-β and IL-6 upon lipopolysacchar...

Abstract: The local immune responses to chronic echinococcal infections in various organs are largely unknown. Since the liver is the most frequently involved organ in such infections in human we aimed to characterize the inflammatory as well as immune cell infiltrate around hydatid cysts in the liver and compared to common inflammatory processes of the liver. Surgical samples from the liver of 21 cystic echinococcosis (CE) patients were studied and the distribution of different types of inflammatory and immune cells were determined by immunohistochemistry. Furthermore, expression levels of costimulatory CTLA4, CD28, CD80 and CD86 molecules were measured at RNA level by PCR. Liver biopsy samples from patients with steatohepatitis (SH, n = 11) and chronic hepatitis (CH, n = 11) were used as non-inflammatory and chronic inflammatory controls, respectively. The composition and density of the inflammatory and immune cell infiltrates have been compared by using morphometry. CD3+ T cells predominated the inflammatory infiltrate in all pathological processes, while in CE samples CD20+ B cells, in CH samples CD68+ macrophages were also frequent. Both myeloperoxidase (MPO) + leukocytes and CD68+ macrophages were found to be significantly decreased in CE as compared to either SH or CH samples. Concerning T cell subtypes, only CD8+ T cells were found to be significantly decreased in SH samples. CD1a + dendritic cells were almost completely missing from CE biopsies unlike in any other sample types. There were no differences detected in the mRNA expression of costimulatory molecules except decreased expression of CD28 in CE samples. In the hydatid lesions of the liver of chronic echinococcal infections T cell-mediated immunity seems to be impaired as compared to other types of chronic inflammatory processes, suggesting an immunosuppressive role for Echinococcus granulosus, which deserve further attentions.

Abstract: Recognition of pathogen-derived nucleic acids by immune cells is critical for the activation of protective innate immune responses. Bacterial cyclic dinucleotides (CDNs) are small nucleic acids that are directly recognized by the cytosolic DNA sensor STING (stimulator of IFN genes), initiating a response characterized by proinflammatory cytokine and type I IFN production. Strategies to improve the immune stimulatory activities of CDNs can further their potential for clinical development. Here, we demonstrate that a simple complex of cylic-di-GMP with a cell-penetrating peptide enhances both cellular delivery and biological activity of the cyclic-di-GMP in murine splenocytes. Furthermore, our findings establish that activation of the TLR-dependent and TLR-independent DNA recognition pathways through combined use of CpG oligonucleotide (ODN) and CDN results in synergistic activity, augmenting cytokine production (IFN-α/β, IL-6, TNF-α, IP-10), costimulatory molecule upregulation (MHC class II, CD86), and antigen-specific humoral and cellular immunity. Results presented herein indicate that 3'3'-cGAMP, a recently identified bacterial CDN, is a superior stimulator of IFN genes ligand than cyclic-di-GMP in human PBMCs. Collectively, these findings suggest that the immune-stimulatory properties of CDNs can be augmented through peptide complexation or synergistic use with CpG oligonucleotide and may be of interest for the development of CDN-based immunotherapeutic agents.

Abstract: Autoimmune peripheral neuropathies such as Guillain-Barre Syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) affect millions of people worldwide. Despite significant advances in understanding the pathology, the molecular and cellular mechanisms of immune-mediated neuropathies remain elusive. T lymphocytes definitely play an important role in disease pathogenesis and CD4(+) T cells have been the main area of research for decades. This is partly due to the fact that the most frequent animal model to study autoimmune peripheral neuropathy is experimental allergic neuritis (EAN). As it is induced commonly by immunization with peripheral nerve proteins, EAN is driven mainly by CD4(+) T cells. However, similarly to what has been reported for patients suffering from multiple sclerosis, a significant body of evidence indicates that CD8(+) T cells may play a pathogenic role in GBS and CIDP disease development and/or progression. Here, we summarize clinical studies pertaining to the presence and potential role of CD8(+) T cells in autoimmune peripheral neuropathies. We also discuss the findings from our most recent studies using a transgenic mouse line (L31 mice) in which the T cell co-stimulator molecule B7.2 (CD86) is constitutively expressed in antigen presenting cells of the nervous tissues. L31 mice spontaneously develop peripheral neuropathy, and CD8(+) T cells are found accumulating in peripheral nerves of symptomatic animals. Interestingly, depletion of CD4(+) T cells accelerates disease onset and increases disease prevalence. Finally, we point out some unanswered questions for future research to dissect the critical roles of CD8(+) T cells in autoimmune peripheral neuropathies.

Abstract: The noninvasive imaging of dendritic cells (DCs) migrated into lymph nodes (LNs) can provide helpful information on designing DCs-based immunotherapeutic strategies. This study is to investigate the influence of transduction of human ferritin heavy chain (FTH) and green fluorescence protein (GFP) genes on inherent properties of DCs, and the feasibility of FTH as a magnetic resonance imaging (MRI) reporter gene to track DCs migration into LNs. FTH-DCs were established by the introduction of FTH and GFP genes into the DC cell line (DC2.4) using lentivirus. The changes in the rate of MRI signal decay (R2*) resulting from FTH transduction were analyzed in cell phantoms as well as popliteal LN of mice after subcutaneous injection of those cells into hind limb foot pad by using a multiple gradient echo sequence on a 9.4 T MR scanner. The transduction of FTH and GFP did not influence the proliferation and migration abilities of DCs. The expression of co-stimulatory molecules (CD40, CD80 and CD86) in FTH-DCs was similar to that of DCs. FTH-DCs exhibited increased iron storage capacity, and displayed a significantly higher transverse relaxation rate (R2*) as compared to DCs in phantom. LNs with FTH-DCs exhibited negative contrast, leading to a high R2* in both in vivo and ex vivo T2*-weighted images compared to DCs. On histological analysis FTH-DCs migrated to the subcapsular sinus and the T cell zone of LN, where they highly expressed CD25 to bind and stimulate T cells. Our study addresses the feasibility of FTH as an MRI reporter gene to track DCs migration into LNs without alteration of their inherent properties. This study suggests that FTH-based MRI could be a useful technique to longitudinally monitor DCs and evaluate the therapeutic efficacy of DC-based vaccines.

Abstract: Microglia activation and neuroinflammation are key events during the progression of neurodegenerative disorders. Microglia exhibits toll-like receptors (TLRs), with predominant expression of TLR4, inducing aberrant neuroinflammation and exacerbated neurotoxicity. Studies suggest that microglia initiate infiltration of T cells into the brain that critically influence disease conditions. We report that LPS-Rs, through TLR4 antagonism, significantly inhibit TLR4 mediated inflammatory molecules like IL-1β, IL-6, TNF-α, COX-2, iNOS, and NO. LPS-Rs regulates JNK/p38 MAPKs and p65-NF-κB signaling pathways, which we report as indispensible for LPS induced neuroinflammation. LPS-Rs mitigates microglial phagocytic activity and we are first to report regulatory role of LPS-Rs which blocked microglia mediated inflammation and apoptotic cell death. LPS-Rs significantly inhibits expression of costimulatory molecules CD80, CD86, and CD40. Chemokine receptor, CCR5, and T cell recruitment chemokines, MIP-1α and CCL5, were negatively regulated by LPS-Rs. Furthermore, LPS-Rs significantly inhibited lymphocyte proliferation with skewed regulatory T (Treg) cell response as evidenced by increased FOXP3, IL-10, and TGF-β. Additionally, LPS-Rs serves to induce coordinated immunosuppressive response and confer tolerogenic potential to activated microglia extending neurosupportive microenvironment. TLR4 antagonism can be a strategy providing neuroprotection through regulation of microglia as well as the T cells.