To Toll-like Receptor Research Articles

Abstract 1096: Murine mammary carcinoma produce more CCL2 in response to TLR agonist treatment than dendritic cells because of differences in NF-kB levels

Abstract Previously we reported that murine mammary carcinoma and dendritic cells elicit distinct responses to Toll-like receptor (TLR) agonists. Here we used a combination of computational biology and molecular analysis to identify the reason for the distinct responses. For this purpose we created a Netlogo model of the TLR4-Myd88 signaling pathway from TLR4 through nuclear translocation of NF-kB culminating in CCL2 transcription. Assessing CCL2 allowed us to validate our model by comparing how much CCL2 was generated by the model versus how much CCL2 was produced in our lab experiments. CCL2 was also of interest because previous studies have shown that CCL2 expression correlates with breast cancer progression. The 4T1 murine mammary carcinoma was used for these studies because it represents a model for stage IV breast cancer and also produces CCL2. Bone marrow derived CD11c+ dendritic cells (DC), which elicit a prototypical TLR agonist response, were used as a control. The model was developed by incorporating qRT-PCR data of the relative mRNA concentrations from each cell type. The experimental values were then used as the starting concentration of proteins in the model. The model of the TLR4-Myd88 signaling cascade allowed us to make predictions by running computational experiments that generate CCL2 expression data over a set amount of time. For instance, the model could be used to predict what happens in response to a TLR agonist if any of the TLR signaling proteins are up- or down-regulated. So far, the model predicted that the tumor cells would produce significantly more CCL2 than DC, results that were validated with experimental data. These results were surprising because most of the TLR signaling proteins were expressed at greater levels by the DC. Since only Tab3 and NF-kB were expressed at greater levels by the tumor cells we used the model to address whether the increased amount of NF-kB in the tumor cells was responsible for the greater level of CCL2 production by the tumor cells. The model showed that when the level of NF-kB in the tumor cells was reduced to the level found in the DC the tumor cells produced lower levels of CCL2. These data suggest that the level of NF-kB in the tumor cells was responsible for the greater production of CCL2 by the tumor cells relative to the DC. Thus, our computational model successfully predicted the experimental data and revealed that NF-kB may be a critical protein responsible for tumor-derived CCL2 expression. Collectively, a more thorough understanding of the differences in TLR signaling between the tumor cells and DC may reveal how targeting the TLR4 signaling cascade can be used to slow tumor progression. Citation Format: Tiffany Phuong, Carli B. Jones, Robert A. Kurt, Chun Wai Liew. Murine mammary carcinoma produce more CCL2 in response to TLR agonist treatment than dendritic cells because of differences in NF-kB levels. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1096. doi:10.1158/1538-7445.AM2015-1096

Immune composition of peripheral blood and responsiveness to Toll-like receptor (TLR) ligands at Day 29 of induction chemotherapy for pediatric acute lymphoblastic leukemia (ALL)

Introduction: Although ALL has long been viewed as a poor target for immune therapy, peripheral blood (PB) absolute lymphocyte count (ALC) at end of induction chemotherapy (Day 29) seems to be prognostic for outcome. However, whether ALC is simply a marker of bone marrow recovery or whether certain immune cell subsets confer a beneficial effect is unknown.

MEK1 dependent and independent ERK activation regulates IL-10 and IL-12 production in bone marrow derived macrophages

The mitogen activated protein kinases ERK1/2 play an important role in response to toll like receptor (TLR) activation and cytokine production, including IL-10 and IL-12. Here, we examined the role of MEK1 in ERK1/2 activation in response to TLR4 agonist by using bone marrow-derived macrophages (BMDMs) from wild type (WT) and Mek1d/dSox2Cre mice. Our data demonstrates that MEK1 is essential for ERK1/2 activation in response to LPS. Furthermore, stimulation of the TLR4 receptor of BMDMs derived from Mek1d/dSox2Cre mice showed enhanced STAT4 phosphorylation and increased IL-12 secretion, but exhibited a significantly lower IL-10 production as compared to WT macrophages. Most interestingly, TLR ligation in the presence of recombinant IL-10 (rIL-10) or retinoic acid (RA) led to ERK1/2 activation independent of MEK1 in BMDMs derived from Mek1d/dSox2Cre mice and led to inhibition of STAT4 and decreased IL-12 levels. Collectively, these data suggest that MEK1 is required for TLR4 mediated ERK activation and in turn regulates the production of IL-10 and IL-12. It also indicates that ERK1/2 can be activated independent of MEK1 in the presence of IL-10 and RA and this activation negatively regulates IL-12, but positively regulates IL-10 production. These findings may have significant implications for the development of drugs that modulate MEK1 activity in the treatment of inflammatory, autoimmune and proliferative diseases such as cancer.

Tanshinone IIA Attenuates Chronic PancreatitisInduced Pain in Rats via Downregulation of HMGB1 and TRL4 Expression in the Spinal Cord

Background: Chronic pancreatitis (CP) is a long-standing inflammation of the exocrine pancreas, which typically results in severe and constant abdominal pain. Previous studies on the mechanisms underlying CP-induced pain have primarily focused on the peripheral nociceptive system. A role for a central mechanism in the mediation or modulation of abdominal pain is largely unknown. Tanshinone IIA (TSN IIA), an active component of the traditional Chinese medicine Danshen, exhibits anti-inflammatory properties via downregulation of the expression of high-mobility group protein B1 (HMGB1), a late proinflammatory cytokine. HMGB1 binds and activates toll-like receptor 4 (TLR4) to induce spinal astrocyte activation and proinflammatory cytokine release in neuropathic pain. Objective: In this study, we investigated the effect of TSN IIA on pain responses in rats with trinitrobenzene sulfonic acid (TNBS)-induced CP. The roles of central mechanisms in the mediation or modulation of CP were also investigated. Study Design: A randomized, double-blind, placebo-controlled animal trial. Methods: CP was induced in rats by intrapancreatic infusion of trinitrobenzene sulfonic acid (TNBS). Pancreatic histopathological changes were characterized with semi-quantitative scores. The abdomen nociceptive behaviors were assessed with von Frey filaments. The effects of intraperitoneally administered TSN IIA on CP-induced mechanical allodynia were tested. The spinal protein expression of HMGB1 was determined by western blot. The spinal mRNA and protein expression of proinflammatory cytokines IL-1β, TNF-α, and IL-6 were determined by RT-PCR and western blot, respectively. The spinal expression of the HMGB1 receptor TRL4 and the astrocyte activation marker glial fibrillary acidic protein (GFAP) were determined by western blot or immunohistological staining after intraperitoneal injection of TSN IIA or intrathecal administration of a neutralizing anti-HMGB1 antibody. Results: TNBS infusion resulted in pancreatic histopathological changes of chronic pancreatitis and mechanical allodynia in rats. TSN IIA significantly attenuated TNBS-induced mechanical allodynia in a dose-dependent manner. TNBS significantly increased the spinal expression of HMGB1 and proinflammatory cytokines IL-1β, TNF-α, and IL-6. These TNBS-induced changes were significantly inhibited by TSN IIA in a dose-dependent manner. Furthermore, TSN IIA, but not the neutralizing anti-HMGB1 antibody, significantly inhibited TNBS-induced spinal TLR4 and GFAP expression. Limitations: In addition to TLR4, HMGB1 can also bind to toll-like receptor-2 (TLR2) and the receptor for advanced glycation end products (RAGE). Additional studies are warranted to ascertain whether HMGB1 contributes to CP-induced pain through activation of these receptors. Conclusions: Our results suggest that spinal HMGB1 contributes to the development of CPinduced pain and can potentially be a therapeutic target. TSN IIA attenuates CP-induced pain via downregulation of spinal HMGB1 and TRL4 expression. Therefore, TSN IIA may be a potential anti-nociceptive drug for the treatment of CP-induced pain. Key words: Chronic pancreatitis, HMGB1, proinflammatory cytokine, Tanshinone IIA, spinal cord, astrocyte, TLR4

Src family kinase members Fyn and Lyn mediate type I interferon production by plasmacytoid dendritic cells. (INM7P.345)

Abstract Plasmacytoid dendritic cells (pDCs) are the most powerful type I interferon (IFN-I) producing cells and are therefore important for antiviral defense and for autoimmune diseases such as systemic lupus erythematosus. However, the signaling factors that regulate pDC IFN-I production are not completely understood. Here we investigate the role of Src Family Kinases (SFKs), a family of non-receptor tyrosine kinases, in pDC responses to toll like receptor (TLR) stimulation. We first found that while human pDCs expressed mostly Lyn, mouse pDCs expressed both Lyn and Fyn, two members of SFKs. Importantly, we observed that pDCs from either Fyn or Lyn deficient mice exhibited impaired IFN-I and pro-inflammatory cytokine production after TLR-9 and TLR-7 stimulation, whereas no difference was observed in conventional DCs. Consistently, incubation of murine or human pDCs with a pan-SFKs inhibitor resulted in a dramatic ablation of pDC IFN-I and cytokine production in a dose dependent manner. Finally, treatment of a human pDC cell line (CAL-1) or pDCs from human peripheral blood with Bafetinib (a selective dual Bcr-Abl/Lyn inhibitor) profoundly ablated their IFN-I production after TLR stimulation. Taken together, our data indicate that the SFKs members Fyn and Lyn promote pDC IFN-I and pro-inflammatory cytokine production and suggest that Bafetinib could be considered as a therapeutic candidate for attenuating pDC responses in autoimmune diseases.

Cytosolic phospholipase A2 modulates TLR2 signaling in synoviocytes.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovitis leading to destruction of cartilage and bone. PLA2 enzymes are key players in inflammation regulating the release of unsaturated fatty acids such as arachidonic acid (AA), a precursor of pro-inflammatory eicosanoids. Several lines of evidence point to toll-like receptors (TLRs) as drivers of synovitis and joint destruction in RA. However, few studies have addressed the implication of PLA2 activity downstream TLR activation in the synovium. Here, we aimed to characterize PLA2 enzyme involvement in TLR2-induced signaling in synovial fibroblast-like cells. TLRs1-7 and a range of sPLA2, iPLA2 and cPLA2 enzymes were found to be transcriptionally expressed in cultured synoviocytes. Activation of TLR2/1 and TLR2/6 led to phosphorylation of cPLA2α at Ser505, and induced AA release and PGE2 production; effects that were attenuated by cPLA2α inhibitors. In contrast, sPLA2 inhibitors did not affect AA or PGE2 release. cPLA2α inhibitors furthermore attenuated TLR-induced expression of IL-6, IL-8 and COX2. COX1/2 inhibitors attenuated TLR2/6-induced IL-6 transcription and protein production comparable to cPLA2α inhibition. Moreover, exogenously PGE2 added alone induced IL-6 production and completely rescued IL-6 transcription when added simultaneously with FSL-1 in the presence of a cPLA2α inhibitor. Our results demonstrate for the first time that cPLA2α is involved in TLR2/1- and TLR2/6-induced AA release, PGE2 production and pro-inflammatory cytokine expression in synoviocytes, possibly through COX/PGE2-dependent pathways. These findings expand our understanding of cPLA2α as a modulator of inflammatory molecular mechanisms in chronic diseases such as RA.

Enterococcus faecalislipoteichoic acid suppressesAggregatibacter actinomycetemcomitanslipopolysaccharide-induced IL-8 expression in human periodontal ligament cells

Periodontitis is caused by multi-bacterial infection and Aggregatibacter actinomycetemcomitans and Enterococcus faecalis are closely associated with inflammatory periodontal diseases. Although lipopolysaccharide (LPS) of A. actinomycetemcomitans (Aa.LPS) and lipoteichoic acid of E. faecalis (Ef.LTA) are considered to be major virulence factors evoking inflammatory responses, their combinatorial effect on the induction of chemokines has not been investigated. In this study, we investigated the interaction between Aa.LPS and Ef.LTA on IL-8 expression in human periodontal ligament (PDL) cells. Aa.LPS, but not Ef.LTA, substantially induced IL-8 expression at the protein and mRNA levels. Interestingly, Ef.LTA suppressed Aa.LPS-induced IL-8 expression without affecting the binding of Aa.LPS to Toll-like receptor (TLR) 4. Ef.LTA reduced Aa.LPS-induced phosphorylation of mitogen-activated protein kinases, including ERK, JNK and p38 kinase. Furthermore, Ef.LTA inhibited the Aa.LPS-induced transcriptional activities of the activating protein 1, CCAAT/enhancer-binding protein and nuclear factor-kappa B transcription factors, all of which are known to regulate IL-8 gene expression. Ef.LTA augmented the expression of IL-1 receptor-associated kinase-M (IRAK-M), a negative regulator of TLR intracellular signaling pathways, in the presence of Aa.LPS at both the mRNA and protein levels. Small interfering RNA silencing IRAK-M reversed the attenuation of Aa.LPS-induced IL-8 expression by Ef.LTA. Collectively, these results suggest that Ef.LTA down-regulates Aa.LPS-induced IL-8 expression in human PDL cells through up-regulation of the negative regulator IRAK-M.

Targeting Toll-like receptor 2 inhibits growth of head and neck squamous cell carcinoma.

Infection-driven inflammation has been proposed to be involved in the tumorigenesis of head and neck squamous cell carcinoma (HNSCC). Oral HNSCC is often colonized with microbes such as gram-positive bacteria and yeast, where ligands derived from their wall components have been shown to specifically bind to Toll-like receptor 2 (TLR2). Although TLR2 has been described to be expressed in oral HNSCC, its function has not been well characterized. Here, we show the expression of TLR2 in both HNSCC cell lines and primary patient-derived HNSCC xenograft tumors. Activation of TLR2 with a yeast-derived ligand of TLR2, zymosan, promoted organoid formation in an ex vivo model of tumor growth, while blockade with anti-TLR2 antibodies inhibited organoid formation. Zymosan also induced phosphorylation of ERK and the p65 subunit of NF-κB, which was inhibited in the presence of anti-TLR2 antibodies, indicating that this receptor is functional in HNSCC and that the signaling through these pathways is intact. TLR2 blockade also inhibited growth of human xenografted tumors in immunodeficient mice. In summary, our data show that TLR2 is a functional receptor expressed in human HNSCC that plays a direct pro-tumorigenic role, and that it can be therapeutically targeted with blocking antibodies to reduce tumor growth.

Heat-Shock Proteins 70 Induce Pro-Inflammatory Maturation Program in Decidual CD1a(+) Dendritic Cells.

The aim of the study was to assess possible binding of a mixture of constitutive Hsc70 and inducible Hsp70 forms (HSP70) to Toll-like receptor (TLR) 4 and CD91 receptors on decidual CD1a(+) dendritic cells (DCs) and their influence on DCs maturation status. Immunohistology and immunofluorescence of paraffin-embedded first trimetester and term pregnancy decidua were performed together with flow cytometry detection of antigens in DCs after stimulation of decidual mononuclear cells with HSP70. Hsc70 and Hsp70 labeling revealed intracellular and nuclear staining in trophoblast cells. The numbers of Hsc70(+) and Hsp70(+) cells of decidual tissue were higher in early pregnancy decidua than in decidua at term. HSP70 binds CD91 and TLR4 receptors on CD1a(+) DCs and increased the expression of CD83, HLA-DR, CD80, and CD86, but decreased CC receptor (CCR) 5. HSP70 increased CC ligand (CCL) 3 and CCL22. HSP70 in the concentration of 1 μg/mL increased the percentage of interferon-γ and interleukin (IL)-15-expressing cells over the cells expressing IL-4. HSP70 binds CD91 and TLR4 on decidual CD1a(+) DCs, causes their maturation, and increases IL-15 in the context of Th1 cytokine/chemokine domination, which could support immune response harmful for ongoing pregnancy.

Influence of histamine on the expression of CCL20 in human gingival fibroblasts.

Histamine plays an important role during allergic and inflammatory reactions, and it has been suggested to influence periodontal inflammation. The aim of this study was to investigate the effects of histamine on the expression of the antimicrobial peptide C-C chemokine ligand 20 (CCL20) in human gingival fibroblasts (HGFs) when exposed to toll-like receptor (TLR) agonists. Monolayers of HGFs from three different donors were exposed to histamine, alone, and in combination with Pam3CSK4 (a TLR2 agonist) or lipopolysaccharide (LPS) from Escherichia coli (a TLR4 agonist), for 2, 4, 6 or 12 h. In another experimental group, cells were pretreated with a specific histamine-1 receptor antagonist (H1R) antagonist, cetirizine. Real-time PCR analysis was performed to detect expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), CCL20 and interleukin-8 (IL8) genes. The levels of CCL20 and IL-8 protein were determined by ELISA. In HGFs, histamine induced expression of CCL20 and IL8 genes in a time-dependent manner (p < 0.05). Combined stimulation with histamine and Pam3CSK4 or LPS led to a significant amplification in expression of CCL20 and IL-8 when compared with treatment with each stimulant alone (p < 0.05), and this effect was mediated via pathways involving the H1R (p < 0.05). The results of this study suggest a sensitizing effect of histamine on early innate immune responses of HGFs when simultaneously exposed to bacterial virulence factors.

The clinically approved drugs dasatinib and bosutinib induce anti-inflammatory macrophages by inhibiting the salt-inducible kinases.

Macrophages switch to an anti-inflammatory, ‘regulatory’-like phenotype characterized by the production of high levels of interleukin (IL)-10 and low levels of pro-inflammatory cytokines to promote the resolution of inflammation. A potential therapeutic strategy for the treatment of chronic inflammatory diseases would be to administer drugs that could induce the formation of ‘regulatory’-like macrophages at sites of inflammation. In the present study, we demonstrate that the clinically approved cancer drugs bosutinib and dasatinib induce several hallmark features of ‘regulatory’-like macrophages. Treatment of macrophages with bosutinib or dasatinib elevates the production of IL-10 while suppressing the production of IL-6, IL-12p40 and tumour necrosis factor α (TNFα) in response to Toll-like receptor (TLR) stimulation. Moreover, macrophages treated with bosutinib or dasatinib express higher levels of markers of ‘regulatory’-like macrophages including LIGHT, SPHK1 and arginase 1. Bosutinib and dasatinib were originally developed as inhibitors of the protein tyrosine kinases Bcr-Abl and Src but we show that, surprisingly, the effects of bosutinib and dasatinib on macrophage polarization are the result of the inhibition of the salt-inducible kinases. Consistent with the present finding, bosutinib and dasatinib induce the dephosphorylation of CREB-regulated transcription co-activator 3 (CRTC3) and its nuclear translocation where it induces a cAMP-response-element-binding protein (CREB)-dependent gene transcription programme including that of IL-10. Importantly, these effects of bosutinib and dasatinib on IL-10 gene expression are lost in macrophages expressing a drug-resistant mutant of salt-inducible kinase 2 (SIK2). In conclusion, our study identifies the salt-inducible kinases as major targets of bosutinib and dasatinib that mediate the effects of these drugs on the innate immune system and provides novel mechanistic insights into the anti-inflammatory properties of these drugs.

Innate and adaptive immune responses to transfused alloantigens

Over 300 different variant alloantigens on red blood cells (RBCs) have been described in humans. Thus, anytime a transfusion of non-autologous RBCs is given, the recipient is exposed to myriad RBC alloantigens. Despite such exposures, on average only 3–6% of transfused humans develop a measurable RBC alloantibody. Once a recipient is alloimmunised to at least one antigen, they are much more likely to become alloimmunised to additional specificities; in contrast, patients who are not alloimmunised tend not to become alloimmunised, despite multiple transfusions. Using murine models, we have reported that exposure of recipients to Toll-like receptor (TLR) agonists, which are known to induce inflammation and activate innate immunity, significantly increase both rates and magnitude of alloimmunisation. Moreover, transfusion of uninflamed mice not only produces no measurable alloimmunisation, but then makes mice non-responsive to a subsequent transfusion in the presence of TLR agonists, which would otherwise result in alloimmunisation. In aggregate, these studies implicate innate immune activation as a regulatory switch of responder/non-responder biology, in a mouse model. Both published and new data will be presented, in both murine models and also limited human studies, exploring the role of innate immune activation in adaptive immunisation to RBC transfusion. Over 300 different variant alloantigens on red blood cells (RBCs) have been described in humans. Thus, anytime a transfusion of non-autologous RBCs is given, the recipient is exposed to myriad RBC alloantigens. Despite such exposures, on average only 3–6% of transfused humans develop a measurable RBC alloantibody. Once a recipient is alloimmunised to at least one antigen, they are much more likely to become alloimmunised to additional specificities; in contrast, patients who are not alloimmunised tend not to become alloimmunised, despite multiple transfusions. Using murine models, we have reported that exposure of recipients to Toll-like receptor (TLR) agonists, which are known to induce inflammation and activate innate immunity, significantly increase both rates and magnitude of alloimmunisation. Moreover, transfusion of uninflamed mice not only produces no measurable alloimmunisation, but then makes mice non-responsive to a subsequent transfusion in the presence of TLR agonists, which would otherwise result in alloimmunisation. In aggregate, these studies implicate innate immune activation as a regulatory switch of responder/non-responder biology, in a mouse model. Both published and new data will be presented, in both murine models and also limited human studies, exploring the role of innate immune activation in adaptive immunisation to RBC transfusion.

Innate and adaptive immune responses to transfused alloantigens

Over 300 different variant alloantigens on red blood cells (RBCs) have been described in humans. Thus, anytime a transfusion of non-autologous RBCs is given, the recipient is exposed to myriad RBC alloantigens. Despite such exposures, on average only 3–6% of transfused humans develop a measurable RBC alloantibody. Once a recipient is alloimmunised to at least one antigen, they are much more likely to become alloimmunised to additional specificities; in contrast, patients who are not alloimmunised tend not to become alloimmunised, despite multiple transfusions. Using murine models, we have reported that exposure of recipients to Toll-like receptor (TLR) agonists, which are known to induce inflammation and activate innate immunity, significantly increase both rates and magnitude of alloimmunisation. Moreover, transfusion of uninflamed mice not only produces no measurable alloimmunisation, but then makes mice non-responsive to a subsequent transfusion in the presence of TLR agonists, which would otherwise result in alloimmunisation. In aggregate, these studies implicate innate immune activation as a regulatory switch of responder/non-responder biology, in a mouse model. Both published and new data will be presented, in both murine models and also limited human studies, exploring the role of innate immune activation in adaptive immunisation to RBC transfusion.

In silico approach to identify the role of a putative protein MAP1138c in the virulence of Johne’s disease

The complete sequencing of Mycobacterium avium subspecies paratuberculosis (MAP) strain K-10 genome has shown the existence of over 4,000 genes. This opens up many opportunities to study the interaction of MAP with its hosts and the environment. Understanding the immune response directed at MAP antigens at different stages of infection, would be enhanced by the characterization of putative antigens. In this context, our comprehensive in silico analysis of MAP1138c, a putative protein demonstrates its sequential, physicochemical, structural and functional homology with Rv1411c (LprG) lipoprotein. The InterPro Scan studies have also shown that MAP1138c protein is a member of LppX/LprAFG family of lipoprotein involved in cell wall biogenesis and pathogenesis of Mycobacterium species. The structure assessment tools reveal that the theoretical structure of MAP1138c protein generated by SWISS-MODEL server shows homology with the crystal structure of Rv1411c (LprG) lipoprotein with respect to the global, local and stereochemical properties. Additionally, the structure-based ligand interaction studies using AutoDock Vina 1.1.2 shows that the triacylated glycoprotein (Ac1PIM2) also interacts with the hydrophobic pockets in the 3D theoretical structure of MAP1138c protein. Similar interactions of Rv1411c (LprG)-Ac1PIM2 leads to Toll-like Receptor 2 (TLR-2) mediated evasion of immune responses within host macrophages in tuberculosis infection. Hence, these results support our hypothesis that the MAP1138c protein is probably involved in immune evasion within host macrophages leading to virulence and infection in ruminants and human, respectively.

Bone marrow-imprinted gut-homing of plasmacytoid dendritic cells (pDCs) in acute simian immunodeficiency virus infection results in massive accumulation of hyperfunctional CD4+ pDCs in the mucosae.

Plasmacytoid dendritic cells (pDCs), a primary source of interferon α (IFN-α), provide a first line of innate immune defense against human immunodeficiency virus infection. However, their kinetics and functions during acute infection are poorly understood. In mucosal tissues of normal rhesus macaques, we found CD4(+) pDCs to be the subset responsible for most IFN-α and tumor necrosis factor α (TNF-α) production in response to Toll-like receptor (TLR) 7/8 stimulation, compared with relatively anergic CD4(-) pDCs. During acute simian immunodeficiency virus (SIV) infection, gut homing was imprinted on pDCs in the bone marrow, resulting in a decline in pDCs from circulation and secondary lymphoid tissues. Although the accumulated pDCs in the gut mucosae had robust cytokine responses to TLR7/8 stimulation in vitro, pDC gut migration occurred after infection and detection of SIV in plasma. Our data suggest that innate pDC responses do not control initial SIV seeding and dissemination but instead may contribute to ongoing immune activation in the gut.

Nanovaccine loaded with poly I:C and STAT3 siRNA robustly elicits anti-tumor immune responses through modulating tumor-associated dendritic cells in vivo

Although cancer vaccine-based immunotherapy holds great potential for cancer treatment, tumor-induced dendritic cell (DC) dysfunction remains to be the major obstacle for developing effective vaccines. Compared with normal DCs, tumor-associated DCs (TADCs) are less matured with poor responsiveness to Toll-like receptor (TLR) stimulation, which has been related with STAT3 hyperactivity. In the present study, Poly I:C (PIC, a TLR3 agonist), STAT3 siRNA and OVA antigen were co-encapsulated by poly (ethylene glycol)-b-poly (l-lysine)-b-poly (l-leucine) (PEG-PLL-PLLeu) polypeptide micelles to generate PMP/OVA/siRNA nanovaccine, which was aimed to effectively overcome DC dysfunction in vivo by deleting STAT3 gene in situ. The results showed that PMP/OVA/siRNA simultaneously facilitated the cellular uptake of OVA antigen and siRNA about 3–200 folds, and decreased STAT3 expression in TADCs over 50% both in vitro and in vivo. PMP/OVA/siRNA also elevated CD86 and CD40 expression as well as IL-12 production by TADCs more effectively than PMP/OVA did, indicating its strong potency of inducing TADC maturation and activation. Moreover, the immunization of PMP/OVA/siRNA rather than PMP/OVA effectively abrogated immunosuppression in the tumor microenvironment by increasing mature DCs and decreasing immunosuppressive cells in tumor-draining lymph nodes, which thereby led to potent anti-tumor immune responses and dramatic tumor regression with prolonged survival. Hence, in vivo co-delivery of immunopotentiator (PIC) and immunosuppressive gene silencer (STAT3 siRNA) by nanovaccines are expected to be a promising strategy to improve the therapeutic efficacy of cancer vaccines by modulating TADCs and overcoming tumor immunosupression.

Cutaneous Innate Immune Sensing of Toll-like Receptor 2-6 Ligands Suppresses T Cell Immunity by Inducing Myeloid-Derived Suppressor Cells

Skin is constantly exposed to bacteria and antigens, and cutaneous innate immune sensing orchestrates adaptive immune responses. In its absence, skin pathogens can expand, entering deeper tissues and leading to life-threatening infectious diseases. To characterize skin-driven immunity better, we applied living bacteria, defined lipopeptides, and antigens cutaneously. We found suppression of immune responses due to cutaneous infection with Gram-positive S.aureus, which was based on bacterial lipopeptides. Skin exposure to Toll-like receptor (TLR)2-6-binding lipopeptides, but not TLR2-1-binding lipopeptides, potently suppressed immune responses through induction of Gr1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs). Investigating human atopic dermatitis, in which Gram-positive bacteria accumulate, we detected high MDSC amounts in blood and skin. TLR2 activation in skin resident cells triggered interleukin-6 (IL-6), which induced suppressive MDSCs, which are then recruited to the skin suppressing Tcell-mediated recall responses such as dermatitis. Thus, cutaneous bacteria can negatively regulate skin-driven immune responses by inducing MDSCs via TLR2-6 activation.

Rapid fucosylation of intestinal epithelium sustains host-commensal symbiosis in sickness.

Systemic infection induces conserved physiological responses that include both resistance and ‘tolerance of infection’ mechanisms1. Temporary anorexia associated with an infection is often beneficial2,3 reallocating energy from food foraging towards resistance to infection4 or depriving pathogens of nutrients 5. It imposes, however, a stress on intestinal commensals, as they also experience reduced substrate availability and impacting host fitness due to the loss of caloric intake and colonization resistance (protection from additional infections)6. We hypothesized that the host might utilize internal resources to support the gut microbiota during the acute phase of the disease. Here we show that systemic exposure to Toll-like receptor (TLR) ligands causes rapid α1,2-fucosylation of the small intestine epithelial cells (IEC), which requires sensing of TLR agonists and production of IL-23 by dendritic cells, activation of innate lymphoid cells and expression of α1,2-Fucosyltransferase-2 (Fut2) by IL-22-stimulated IECs. Fucosylated proteins are shed into the lumen and fucose is liberated and metabolized by the gut microbiota, as shown by reporter bacteria and community-wide analysis of microbial gene expression. Fucose affects the expression of microbial metabolic pathways and reduces the expression of bacterial virulence genes. It also improves host tolerance of the mild pathogen Citrobacter rodentium. Thus, rapid IEC fucosylation appears to be a protective mechanism that utilizes the host's resources to maintain host-microbial interactions during pathogen-induced stress.

Immunology: Starve a fever, feed the microbiota.

A study finds that the cells lining the gut are modified in response to systemic infection, increasing the host's tolerance to infection in a manner that is dependent on the microorganisms that inhabit the gut. See Letter p.638 A healthy gut microbiota is important for the host's well being, so it would make sense for host resources to be expended to protect 'good' microbes during a systemic response to infection. L-fucosylation could be such a resource: L-fucose attached to glycoproteins and glycolipids is accessible for microbial, but not for host energy harvest. Joseph Pickard et al. show that systemic exposure to Toll-like receptor (TLR) ligands induces fucosylation of the small intestine epithelium via a dendritic cell and interleukin-22 (IL-22) dependent mechanism. Some of the fucose is shed into the intestinal lumen where it provides nourishment for the microbiota, and contributes to the production of short-chain fatty acids, which are beneficial for the host.

Standard of hygiene and immune adaptation in newborn infants

The prevalence of immune-mediated diseases, such as allergies and type 1 diabetes, is on the rise in the developed world. In order to explore differences in the gene expression patterns induced in utero in infants born in contrasting standards of living and hygiene, we collected umbilical cord blood RNA samples from infants born in Finland (modern society), Estonia (rapidly developing society) and the Republic of Karelia, Russia (poor economic conditions). The whole blood transcriptome of Finnish and Estonian neonates differed from their Karelian counterparts, suggesting exposure to toll-like receptor (TLR) ligands and a more matured immune response in infants born in Karelia. These results further support the concept of a conspicuous plasticity in the developing immune system: the environmental factors that play a role in the susceptibility/protection towards immune-mediated diseases begin to shape the neonatal immunity already in utero and direct the maturation in accordance with the surrounding microbial milieu.