To Toll-like Receptor Agonists Research Articles

SARS-CoV-2 suppresses TLR4-induced immunity by dendritic cells via C-type lectin receptor DC-SIGN.

SARS-CoV-2 causes COVID-19, an infectious disease with symptoms ranging from a mild cold to severe pneumonia, inflammation, and even death. Although strong inflammatory responses are a major factor in causing morbidity and mortality, superinfections with bacteria during severe COVID-19 often cause pneumonia, bacteremia and sepsis. Aberrant immune responses might underlie increased sensitivity to bacteria during COVID-19 but the mechanisms remain unclear. Here we investigated whether SARS-CoV-2 directly suppresses immune responses to bacteria. We studied the functionality of human dendritic cells (DCs) towards a variety of bacterial triggers after exposure to SARS-CoV-2 Spike (S) protein and SARS-CoV-2 primary isolate (hCoV-19/Italy). Notably, pre-exposure of DCs to either SARS-CoV-2 S protein or a SARS-CoV-2 isolate led to reduced type I interferon (IFN) and cytokine responses in response to Toll-like receptor (TLR)4 agonist lipopolysaccharide (LPS), whereas other TLR agonists were not affected. SARS-CoV-2 S protein interacted with the C-type lectin receptor DC-SIGN and, notably, blocking DC-SIGN with antibodies restored type I IFN and cytokine responses to LPS. Moreover, blocking the kinase Raf-1 by a small molecule inhibitor restored immune responses to LPS. These results suggest that SARS-CoV-2 modulates DC function upon TLR4 triggering via DC-SIGN-induced Raf-1 pathway. These data imply that SARS-CoV-2 actively suppresses DC function via DC-SIGN, which might account for the higher mortality rates observed in patients with COVID-19 and bacterial superinfections.

Impaired functionality of antigen presenting cells in HIV- exposed uninfected infants in the first six months of life.

HIV-exposed uninfected infants (HEU) have increased morbidity and mortality due to infections in the first 6 months of life that tapers down to 2 years of life. The underlying immunologic defects remain undefined. We investigated antigen-presenting cells (APC) by comparing the phenotype of unstimulated APC, responses to toll-like receptor (TLR) stimulation, and ability to activate natural killer (NK) cells in 24 HEU and 64 HIV-unexposed infants (HUU) at 1-2 days of life (birth) and 28 HEU and 45 HUU at 6 months of life. At birth, unstimulated APC showed higher levels of activation and cytokine production in HEU than HUU and stimulation with TLR agonists revealed lower expression of inflammatory cytokines and activation markers, but similar expression of IL10 regulatory cytokine, in APC from HEU compared to HUU. Differences were still present at 6 months of life. From birth to 6 months, APC underwent extensive phenotypic and functional changes in HUU and minimal changes in HEU. TLR stimulation also generated lower NK cell expression of CD69 and/or IFNγ in HEU compared with HUU at birth and 6 months. In vitro experiments showed that NK IFNγ expression depended on APC cytokine secretion in response to TLR stimulation. Ex vivo IL10 supplementation decreased APC-mediated NK cell activation measured by IFNγ expression. We conclude that APC maturation was stunted or delayed in the first 6 months of life in HEU compared with HUU. Deficient inflammatory APC responses and/or the imbalance between inflammatory and regulatory responses in HEU may play an important role in their increased susceptibility to severe infections.

Development of an Enzyme-Mediated, Site-Specific Method to Conjugate Toll-Like Receptor 2 Agonists onto Protein Antigens: Toward a Broadly Protective, Four Component, Group A Streptococcal Self-Adjuvanting Lipoprotein-Fusion Combination Vaccine.

Subunit vaccines composed of protein antigens covalently attached to Toll-like receptor (TLR) agonists elicit superior immune responses compared to mixtures of antigens and TLR agonists. Among different conjugation approaches, enzyme-mediated ligation is one of the few that provides an opportunity for the generation of homogeneous, molecularly defined products in which protein antigens are maintained with native structures, which is most critical to elicit protective immune responses upon vaccination. Four highly conserved protein antigens from Group A Streptococcus (GAS) have the potential to be safe and efficacious vaccine candidates. After a TLR2 agonist fibroblast-stimulating lipopeptide-1 (FSL-1) was successfully attached onto each antigen using sortase A and techniques for their purification were developed, a combination vaccine containing interleukin 8 (IL-8) protease (Streptococcus pyogenes cell envelope proteinase [SpyCEP]), Group A Streptococcal C5a peptidase (SCPA), anchorless virulence factor arginine deiminase (ADI), and trigger factor (TF)-TLR2 conjugates was produced. This combination was assessed for immunity in mice and compared with mixtures of the four antigens with FSL-1 or alum. High titer antigen-specific IgG antibodies were detected from all vaccine groups, with antibodies elicited from FSL-1 conjugates around 10-fold higher compared to the FSL-1 mixture group. Furthermore, the FSL-1 conjugates afforded a more balanced TH1/TH2 immune response than the alum-adjuvanted group, suggesting that this combination vaccine represents a promising candidate for the prevention of GAS diseases. Thus, we established a conjugation platform that allows for the production of defined, site-specific antigen-adjuvant conjugates, which maintain the native three-dimensional structure of antigens and can be potentially applied to a variety of protein antigens.

PSX-41 Effect of Polyinosinic-polycytidylic acid on gene expression in goat blood

Abstract The objective of this study was to investigate the effect of Polyinosinic-polycytidylic [poly(I:C)] acid on gene activation in goat blood. Polyinosinic-polycytidylic acid is a synthetic dsRNA analogue that binds to Toll-like receptor (TLR) 3. Synthetic TLR agonists are promising immune modulators. Blood samples were collected from the jugular vein of BoerXSpanish goats (n = 3) into tubes containing an anticoagulant. Whole blood was treated with the 12.5 µg/ml of poly I:C or 200µl of PBS which served as control. Cells were incubated at 37°C with 5% CO2 and 85% humidity for 30 minutes. Total RNA was isolated from the pellet using Trizol and then converted to cDNA using RETROscript kit (Qiagen). The expression of 84 genes in the human TLR signaling pathway RT2 PCR Array was evaluated using real-time PCR. Fold change in gene expression was calculated using the 2−ΔΔCt method. The housekeeping gene GAPDH, ACTB, HPRT1, TBP, and YWHAZ was used to normalize the data. Fold change was set at a cutoff of 2. Following treatment with poly I:C, 24 genes were up-regulated, 15 genes were down-regulated. The gene MAPK8 was induced by poly I:C treatment. Only 74 genes were expressed in the control. Thirty-nine genes were expressed in both the control group and poly I:C treatment group. Treatment with poly I:C also down-regulated some of the genes tested. Our results show that treatment poly I:C modulated the expression of genes in the TLR signaling pathway and provides insights into how goats respond to viral pathogens for the design of adjuvants to enhance the immune response.

PlGF enhances TLR-dependent inflammatory responses in human mononuclear phagocytes.

Levels of placental growth factor (PlGF) peak during third trimester of pregnancy, a time when women are at increased risk of virus-induced morbidity. We hypothesized PlGF might contribute to an exaggerated inflammatory response to Toll-like receptor (TLR) activation. Primary human adult and cord blood CD14+ cells were cultured in the presence of TLR ligands and/or PlGF. PlGF significantly enhanced the magnitude and duration of TNF messenger RNA and protein production by TLR-7/8-activated monocytes, and increased subsequent production of TNF-independent inflammatory cytokines. This PlGF/TLR effect involved multiple inflammatory cytokines/chemokines and was seen with the majority of TLR agonists. PlGF enhanced phosphorylation of IkappaB kinase (IKK) in monocytes stimulated with the TLR-7/8 agonist R848, and IKK inhibition completely suppressed the PlGF effect. PlGF enhances TLR-signaling upstream of IKK and contributes to an exaggerated pathologic pro-inflammatory state in response to activation of maternal and fetal mononuclear phagocytes by specific TLR agonists.

The Differential Response of Human Epithelial Derived Colonic Organoids to TLR Agonists

The intestinal epithelium expresses a range of pattern recognition receptors and is thought to contribute to the innate immune response in the intestine. However, as the cytokines produced by the epithelial cells are the same as those produced by the intestinal immune system it is difficult to define the epithelial response. Here we have used epithelial derived colonic organoids from healthy individuals to characterise the response of the colonic epithelium to toll like receptor (TLR) agonists. Colonoids were grown for 15 days from biopsies from the transverse colon of healthy control patients. The expression of TLRs in the organoids was compared with that of the native colonic epithelium with qPCR and Western blotting, and the response of the organoids to TLR agonists was quantified with qPCR. The response of SW480 cells to agonists was used as a positive control. n = number of patients from which organoids were grown. Two distinct types of organoids developed, colonospheres, which have a poorly developed epithelial layer consisting of thin fusiform cells, and colonoids, which have a well‐developed columnar epithelium. Both colonospheres and colonoids had transcript levels of TLR 1, 2, 4, 5 & 6 comparable to that seen in the native colonic epithelium. Furthermore, transcript for the accessory proteins associated with TLR4 ‐ CD14, lipopolysaccharide binding protein (LBP) and myeloid differentiation factor 2 (MD2) ‐ were also present in the organoids and the expression of TLR 4 & 5 protein in the organoids was comparable to that of the native epithelium (n=5). Despite this, treatment of the organoids with either the TLR 2 agonists [Pam 2CSK4 & Pam3CSK4 (200ng ml−1) or lipoteichoic acid (LTA, 10μg ml−1)] or the TLR4 agonist (lipopolysaccharide (LPS, 200ng ml−1)] failed to stimulate an increase in either IL‐8 or TNF‐α transcript at 3, 6 or 24 h in either colonospheres or colonoids (n=5). In contrast, flagellin (1 μg ml−1) stimulated an increase in both IL‐8 and TNF‐α at all time points (n=5). To further investigate the differential response to the TLR agonists, the effect of increasing concentrations of LPS and flagellin (1, 10, 100, 1000 ng ml−1) on IL‐8 and TNF‐α transcript levels in colonospheres and colonoids was investigated (n=5). Flagellin stimulated a dose dependent increase in both cytokines, whereas LPS failed to stimulate an increase in either IL‐8 or TNF‐α at any dose, although comparable measurements with LPS in SW480 cells resulted in a robust response. Furthermore, LPS (1 μg ml−1) did not stimulate an increase in transcript levels of a range of cytokines associated with intestinal epithelial cells (TSLP, IL33, IL‐1β, or APRIL), whereas flagellin (1 μg ml−1) significantly increased expression of these cytokines (n=5). These data indicate that colonic epithelial cells are unresponsive to common components of the commensal bacteria (LTA, LPS) but flagellin, a virulence factor associated with both gram positive and negative bacteria, induces a robust response. It remains to be determined what the roles of the TLR2 and TLR4 receptors are in the colonic epithelial cells.Support or Funding InformationSupported by a University of Otago Research Grant, a University of Otago postgraduate scholarship and grants from and the Department of Physiology and the Dean's Funds, Otago School of Biomedical Sciences and Dunedin School of Medicine.

Activation of NK cell cytotoxicity by aerosolized CpG-ODN/poly(I:C) against lung melanoma metastases is mediated by alveolar macrophages

Controversies remain about NK cells direct responsiveness to Toll-like receptor (TLR) agonists or dependence on macrophages. In a melanoma lung metastasis model, aerosolized TLR9 and TLR3 agonists have been reported to induce antitumor immunity through NK cells activation. In the current study, we demonstrated that in vitro TLR9/TLR3 stimulation induced IFN-γ secretion by NK cells, but an increase in their cytotoxicity was detected only after NK cells co-culture with in vitro TLR9/TLR3 agonists pretreated alveolar macrophages. Alveolar macrophages from melanoma lung metastases-bearing mice, treated with aerosolized TLR agonists, also promoted NK cell cytotoxicity. Activated NK cells from lungs of melanoma metastases-bearing mice that were given aerosolized TLR9/TLR3 agonists were able to polarize naive alveolar macrophages toward a M1-like phenotype. Our results demonstrate that activation of NK cells in the lung after TLR engagement is mediated by alveolar macrophages and that activated NK cells shape macrophage behavior.

Deficiency of the c-JUN Inhibitor DACH1 in FA Macrophages Accounts for the Cytokine Overproduction Phenotype

▪The inflammatory cytokines TNFα and IL-1β contribute to the bone marrow failure phenotype in Fanconi anemia (FA) as well as to clonal evolution to MDS and AML. Mononuclear phagocytes deficient in FANCA or FANCC overproduce these cytokines in response to toll-like receptor agonists (TLRa), but the precise molecular mechanisms by which FA proteins suppress cytokine production remains enigmatic. We show here that pretreatment of control macrophages with interferon (IFN) α enhances (by 3-fold) TLRa-induced production of both TNFα and IL-1β. FANCC-deficient cells produce more of these proteins (by 3-fold) after exposure to TLRa alone and IFNα does not enhance production of these cytokines. We therefore hypothesized that TLR stimulation of FANCC-deficient cells activates an IFNα-like pathway, one constrained by FANCC in normal cells. To test this notion, we performed gene expression microarray analysis (Affymetrix HTA 2.0) using RNA from FANCC-deficient (T-shFC) and control (T-shNT) THP-1 human mononuclear phagocytes treated with IFNα, the TLR7/8 agonist R848, or a combination of IFNα plus R848. We found that treatment of T-shFC cells with R848 alone was sufficient to enhance expression of 49 genes that were activated by IFNα in control cells. Thirteen of these genes were not induced by R848 in control cells but were induced by R848 in T-shFC cells (one of which, IFNgamma-inducible protein 30 (IFI30), is known to be overexpressed in Fancd2 -/- progenitor cells). These results support our hypothesis that FANCC functions normally to constrain IFNα pathway activation in TLRa-activated macrophages. Of greater functional importance vis-à-vis cytokine production, DACH1 mRNA was suppressed by 11-fold and DACH1 protein was barely detectable in FANCC-deficient cells under all conditions when compared to control cells. DACH1 is known to bind to and suppress the activities of a variety of transcription factors, notably c-JUN in fibroblasts. We tested the hypothesis that DACH1 deficiency is sufficient to account for the TLRa hypersensitivity of FA macrophages using gain- and loss-of-function studies. Knockdown of DACH1 in control (T-shNT) macrophages resulted in a 7-fold enhancement of R848-induced TNFα production, increased c-JUN protein (1.6-fold) and c-JUN phosphorylation (2.6-fold). Reporter gene expression (secreted embryonic alkaline phosphatase [SEAP]) from constructs containing both AP-1 and NF-κB sites was activated by DACH1 knockdown but deletion of the AP-1 site completely abrogated activation. Results of gain-of-function experiments with wild-type and mutant DACH1 cDNA sequences now taking place will be reported. Taken together, these results suggest that FANCC directly or indirectly enhances ground-state expression of DACH1 and thereby suppresses the TLR pathway in normal cells specifically by inhibiting R848-dependent activation of c-JUN. Conversely, loss of DACH1 expression in FANCC-deficient cells leads to unconstrained c-JUN activity, resulting in overproduction inflammatory cytokines. In light of the contribution of such cytokines to both bone marrow failure and clonal evolution, activation of DACH1 gene expression is a rational therapeutic objective in the management of patients with Fanconi anemia and is worthy of investigating in preclinical (animal) models. DisclosuresNo relevant conflicts of interest to declare.

Phenotype and Polyfunctional Deregulation Involving Interleukin 6 (IL-6)- and IL-10-Producing Monocytes in HIV-Infected Patients Receiving Combination Antiretroviral Therapy Differ From Those in Healthy Older Individuals.

Despite the relevance of monocytes as promoters of the inflammatory response, whether human immunodeficiency virus (HIV) infection induces premature age-related changes to the phenotype and function of monocytes or whether these alterations are different and/or specifically driven by HIV remains to be mechanistically determined. We assayed the activation phenotype and the responsiveness in vitro to Toll-like receptor (TLR) agonists in classical, intermediate, and nonclassical subsets of monocytes by assessing intracellular interleukin 1α (IL-1α), IL-1β, interleukin 6 (IL-6), interleukin 8, tumor necrosis factor α, and interleukin 10 (IL-10) production in 20 HIV-infected patients receiving combination antiretroviral therapy (cART) and 2 groups of uninfected controls (20 age-matched young individuals and 20 older individuals aged >65 years). HIV-infected patients showed a more activated phenotype of monocytes than older controls. Regarding functionality, under unstimulated conditions HIV-infected patients showed a higher percentage of classical monocytes producing IL-6 and IL-10 than control subjects. The percentage of cells with production of multiple cytokines (polyfunctionality), including IL-10, in response to TLR agonists was greater among HIV-infected patients than among control subjects. Inflammatory alterations associated with monocytes during HIV infection are different from those in aging individuals. This monocyte dysfunction, mainly characterized by high levels of IL-6- and IL-10-producing monocytes, may have clinical implications in HIV-infected patients that are different from those in aging individuals.

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.

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.

An in vitro screen employing human neonatal dendritic cells identifies distinct synergy between Toll-like receptor and C-type lectin receptor agonists (VAC10P.966)

Abstract Due to impairments in cell-mediated immunity, newborns are markedly susceptible to infection with intracellular pathogens. Impaired newborn immunity includes a reduced response to Toll-like Receptor agonists (TLRAs) by dendritic cells (DCs), placing them at risk for infection and limiting Th1-responses to many vaccines. Our hypothesis was that dual stimulation with TLRAs and C-type Lectin Receptor agonists (CLRAs) may overcome the reduced response of newborn DCs to common vaccine formulations. We screened TLRAs, CLRAs and their combinations for their ability to induce Th1-polarizing cytokine production from neonatal dendritic cells. Human adult- and cord blood monocyte-derived DCs (MoDCs) were generated in the presence of autologous plasma and stimulated for 18 hours. Cytokines and markers for cellular toxicity were measured to assess correlates of immunogenicity and reactogenicity. Dual activation of newborn DCs with a TLR8 agonist (R848) and a Mincle agonist (TDB) or with a TLR4 agonist (MPLA or GLA) and a Dectin-1 agonist (alkali-treated Zymosan) synergistically enhanced the production of the T-cell activating and -polarizing cytokines TNF-α, IL-12p70 and IL-1β. Our data suggest that targeting vaccinal antigens to the endocytic receptors Mincle or Dectin-1 is a powerful approach to induce Th1-mediated immunity in newborns. This study has the potential to inform development of novel adjuvanted neonatal vaccines and reduce infections in infants.

1-Dehydro-[10]-gingerdione from ginger inhibits IKKβ activity for NF-κB activation and suppresses NF-κB-regulated expression of inflammatory genes.

Pungent constituents of ginger (Zingiber officinale) have beneficial effects on inflammatory pain and arthritic swelling. However, the molecular basis for these pharmacological properties is only partially understood. Here, we investigated the molecular target of 1-dehydro-[10]-gingerdione (D10G), one of the pungent constituents of ginger, that mediates its suppression of NF-κB-regulated expression of inflammatory genes linked to toll-like receptor (TLR)-mediated innate immunity. RAW 264.7 macrophages or primary macrophages-derived from bone marrows of C57BL/6 or C3H/HeJ mice were stimulated with the TLR4 agonist LPS in the presence of D10G. Catalytic activity of inhibitory κB (IκB) kinase β (IKKβ) was determined by a kinase assay and immunoblot analysis, and the expression of inflammatory genes by RT-PCR analysis and a promoter-dependent reporter assay. D10G directly inhibited the catalytic activity of cell-free IKKβ. Moreover, D10G irreversibly inhibited cytoplasmic IKKβ-catalysed IκBα phosphorylation in macrophages activated by TLR agonists or TNF-α, and also IKKβ vector-elicited NF-κB transcriptional activity in these cells. These effects of D10G were abolished by substitution of the Cys(179) with Ala in the activation loop of IKKβ, indicating a direct interacting site of D10G. This mechanism was shown to mediate D10G-induced disruption of NF-κB activation in LPS-stimulated macrophages and the suppression of NF-κB-regulated gene expression of inducible NOS, COX-2 and IL-6. This study demonstrates that IKKβ is a molecular target of D10G involved in the suppression of NF-κB-regulated gene expression in LPS-activated macrophages; this suggests D10G has therapeutic potential in NF-κB-associated inflammation and autoimmune disorders.

Cyclophosphamide induces bone marrow to yield higher numbers of precursor dendritic cells in vitro capable of functional antigen presentation to T cells in vivo

We have shown recently that cyclophosphamide (CTX) treatment induced a marked increase in the numbers of immature dendritic cells (DCs) in blood, coinciding with enhanced antigen-specific responses of the adoptively transferred CD8 + T cells. Because this DC expansion was preceded by DC proliferation in bone marrow (BM), we tested whether BM post CTX treatment can generate higher numbers of functional DCs. BM was harvested three days after treatment of C57BL/6 mice with PBS or CTX and cultured with GM-CSF/IL-4 in vitro. Compared with control, BM from CTX-treated mice showed faster generation and yielded higher numbers of DCs with superior activation in response to toll-like receptor (TLR) agonists. Vaccination with peptide-pulsed DCs generated from BM from CTX-treated mice induced comparable adjuvant effects to those induced by control DCs. Taken together, post CTX BM harbors higher numbers of DC precursors capable of differentiating into functional DCs, which be targeted to create host microenvironment riches in activated DCs upon treatment with TLR agonists.

A central role for monocytes in Toll‐like receptor‐mediated activation of the vasculature

There is increasing evidence that activation of inflammatory responses in a variety of tissues is mediated co-operatively by the actions of more than one cell type. In particular, the monocyte has been implicated as a potentially important cell in the initiation of inflammatory responses to Toll-like receptor (TLR)-activating signals. To determine the potential for monocyte-regulated activation of tissue cells to underpin inflammatory responses in the vasculature, we established cocultures of primary human endothelial cells and monocytes and dissected the inflammatory responses of these systems following activation with TLR agonists. We observed that effective activation of inflammatory responses required bidirectional signalling between the monocyte and the tissue cell. Activation of cocultures was dependent on interleukin-1 (IL-1). Although monocyte-mediated IL-1beta production was crucial to the activation of cocultures, TLR specificity to these responses was also provided by the endothelial cells, which served to regulate the signalling of the monocytes. TLR4-induced IL-1beta production by monocytes was increased by TLR4-dependent endothelial activation in coculture, and was associated with increased monocyte CD14 expression. Activation of this inflammatory network also supported the potential for downstream monocyte-dependent T helper type 17 activation. These data define co-operative networks regulating inflammatory responses to TLR agonists, identify points amenable to targeting for the amelioration of vascular inflammation, and offer the potential to modify atherosclerotic plaque instability after a severe infection.

Intra-amniotic LPS modulation of TLR signaling in lung and blood monocytes of fetal sheep

Epidemiological studies suggest that intra-uterine exposure to inflammation may prime postnatal immune responses. In fetal sheep, intra-amniotic injection of lipopolysaccharide (LPS) induced chorioamnionitis, lung inflammation and maturation, matured lung monocytes to macrophages and initiated systemic tolerance of fetal monocytes to subsequent challenge with LPS. We hypothesized that LPS-mediated chorioamnionitis altered the response of lung and blood monocytes to Toll-like receptor (TLR) ligands such as PamCysK4 (TLR2), flagellin (TLR5), and human CpG-DNA (TLR9). Time-mated ewes were given intra-amniotic injections of LPS or saline. Blood and lung monocytes were assessed after 2 days, 7 days and 2 days and 7 days repetitive LPS injections before delivery at 124 days gestational age (term 150 days). Responsiveness of blood and lung monocytes to TLR-ligands in vitro was assessed by interleukin (IL)-6, tumor necrosis factor-alpha (TNF-alpha) and hydrogen peroxide. Monocytes from preterm controls had minimal responses. Lipopolysaccharide-mediated chorioamnionitis increased IL-6, TNF- alpha and hydrogen peroxide to all TLR agonists in blood and lung monocytes. Repetitive exposure to antenatal LPS reduced IL-6, TNF- alpha and hydrogen peroxide to TLR-ligands suggesting tolerance. Tolerance to TLR-ligands reduced IL-1 receptor associated kinase-4 expression. Thus, repeated fetal exposure to LPS induced tolerance to other TLR-ligands. These modulations of fetal innate immunity have implications for host defense and injury responses in preterm infants.

Hyper innate responses in neonates lead to increased morbidity and mortality after infection

Neonates suffer high morbidity and mortality in infection, presumably because of the lack of a fully developed adaptive and innate immune system. Evidence of poor innate responses in neonates has been shown by using a model that sensitizes the host to Toll-like receptor (TLR)-mediated inflammation with d-galactosamine (d-GalN). However, we show that neonatal mice demonstrate much stronger inflammatory responses than adult mice in response to LPS stimulation, and such hypersensitivity extends to other TLR agonists including actual viral infection. Our study reveals that the ensuing inflammatory reaction after d-GalN sensitization reflects preferential toxicity of d-GalN to adult liver cells, rather than accurately reflecting the TLR response to LPS. We show further that an uncontrolled proinflammatory innate response due to inadequate T cells makes neonates more vulnerable to TLR agonists or viral infection. Remarkably, through transfer of T cells into neonates or depletion of T cells in adult mice, we show that T cells are sufficient and necessary to control the early inflammatory response to LPS. Therefore, neonates might suffer from the unleashed innate responses caused by an insufficient number of T cells, which leads to increased morbidity and mortality.

Successful Correction of Immune Defects in a Patient with NEMO Mutation by Allogeneic Transplantation Uncovers Intrinsic Epithelial Basis for Colitis

RationaleMutations in critical components of the nuclear factor-κB (NF-κB) signaling pathway, such as NF-κB essential modulator (NEMO), result in primary immunodeficiency. Whether both the immune defects and other manifestations, such as colitis, seen in boys with NEMO mutation are correctable by allogeneic transplantation is not known.MethodsWe report a patient with NEMO mutation, immunodeficiency and corticosteroid-dependent colitis. An HLA-matched unaffected sibling was born after in vitro fertilization and preimplantation genetic diagnosis. The patient underwent sibling matched hematopoietic stem cell transplantation (HSCT) after myeloablative conditioning. Responses to Toll-like receptor (TLR) agonists, natural killer cell cytotoxicity and CD40-mediated responses were assessed pre and post-HSCT. The clinical course of colitis and corticosteroid use post-HSCT were also examined.ResultsThe patient tolerated conditioning and had 98-100% donor chimerism in all immune cells tested. Defects in responses to TLR agonists, natural killer cell cytotoxicity, and CD40-mediated proliferation were all corrected following allogeneic HSCT. However, the patient continued to have flares of colitis, often precipitated by bacterial infection, requiring treatment with corticosteroids.ConclusionsPreimplantation genetic diagnosis was used successfully in conjunction with allogeneic HSCT to correct the immune defects in a patient with NEMO mutation. However, the patient's colitis persisted. Our data strongly suggest that lack of NEMO function and NF-κB signaling in intestinal epithelium is sufficient to drive susceptibility to colitis. RationaleMutations in critical components of the nuclear factor-κB (NF-κB) signaling pathway, such as NF-κB essential modulator (NEMO), result in primary immunodeficiency. Whether both the immune defects and other manifestations, such as colitis, seen in boys with NEMO mutation are correctable by allogeneic transplantation is not known. Mutations in critical components of the nuclear factor-κB (NF-κB) signaling pathway, such as NF-κB essential modulator (NEMO), result in primary immunodeficiency. Whether both the immune defects and other manifestations, such as colitis, seen in boys with NEMO mutation are correctable by allogeneic transplantation is not known. MethodsWe report a patient with NEMO mutation, immunodeficiency and corticosteroid-dependent colitis. An HLA-matched unaffected sibling was born after in vitro fertilization and preimplantation genetic diagnosis. The patient underwent sibling matched hematopoietic stem cell transplantation (HSCT) after myeloablative conditioning. Responses to Toll-like receptor (TLR) agonists, natural killer cell cytotoxicity and CD40-mediated responses were assessed pre and post-HSCT. The clinical course of colitis and corticosteroid use post-HSCT were also examined. We report a patient with NEMO mutation, immunodeficiency and corticosteroid-dependent colitis. An HLA-matched unaffected sibling was born after in vitro fertilization and preimplantation genetic diagnosis. The patient underwent sibling matched hematopoietic stem cell transplantation (HSCT) after myeloablative conditioning. Responses to Toll-like receptor (TLR) agonists, natural killer cell cytotoxicity and CD40-mediated responses were assessed pre and post-HSCT. The clinical course of colitis and corticosteroid use post-HSCT were also examined. ResultsThe patient tolerated conditioning and had 98-100% donor chimerism in all immune cells tested. Defects in responses to TLR agonists, natural killer cell cytotoxicity, and CD40-mediated proliferation were all corrected following allogeneic HSCT. However, the patient continued to have flares of colitis, often precipitated by bacterial infection, requiring treatment with corticosteroids. The patient tolerated conditioning and had 98-100% donor chimerism in all immune cells tested. Defects in responses to TLR agonists, natural killer cell cytotoxicity, and CD40-mediated proliferation were all corrected following allogeneic HSCT. However, the patient continued to have flares of colitis, often precipitated by bacterial infection, requiring treatment with corticosteroids. ConclusionsPreimplantation genetic diagnosis was used successfully in conjunction with allogeneic HSCT to correct the immune defects in a patient with NEMO mutation. However, the patient's colitis persisted. Our data strongly suggest that lack of NEMO function and NF-κB signaling in intestinal epithelium is sufficient to drive susceptibility to colitis. Preimplantation genetic diagnosis was used successfully in conjunction with allogeneic HSCT to correct the immune defects in a patient with NEMO mutation. However, the patient's colitis persisted. Our data strongly suggest that lack of NEMO function and NF-κB signaling in intestinal epithelium is sufficient to drive susceptibility to colitis.

Dendritic cells from HIV-1 infected individuals are less responsive to toll-like receptor (TLR) ligands

We compared TLR responsiveness in PBMC from HIV-1-infected and uninfected individuals using the TLR agonists: TLR7 (3M-001), TLR8 (3M-002), and TLR7/8 (3M-011). Activation and maturation of plasmacytoid dendritic cells (pDC) were measured by evaluating CD86, CD40, and CD83 expression and myeloid dendritic cell (mDC) activation was measured by evaluating CD40 expression. All agonists tested induced activation and maturation of pDC in PBMC cultures of cells from HIV + and HIV − individuals. The TLR7 agonist induced significantly less pDC maturation in cells from HIV + individuals. Quantitative assessment of secreted IFN-α and pro-inflammatory cytokines at the single cell level showed that pDC from HIV + individuals stimulated with TLR7 and TLR7/8 induced IFN-α. TLR8 and TLR7/8 agonists induced IL-12 and COX-2 expression in mDC from HIV + and HIV − individuals. Understanding pDC and mDC activation and maturation in HIV-1 infection could lead to more rational development of immunotherapeutic strategies to stimulate the adaptive immune response to HIV-1.