CpG oligodeoxynucleotide induction of antiviral effector molecules in sheep

Immunostimulatory CpG oligodeoxynucleotide (ODN) can protect mice against infection by many pathogens but the mechanisms mediating disease protection are not well defined. Furthermore, the mechanisms of CpG ODN induced disease protection in vivo have not been investigated in other species. We investigated the induction of antiviral effector molecules in sheep treated with a class B CpG ODN (2007). Subcutaneous injection of ODN 2007 induced a dose-dependent increase in serum levels of the antiviral effector molecule, 2'5'-A synthetase. Peak levels of enzyme were observed 4 days following ODN injection and enzyme levels remained elevated for the following 3-5 days. Repeated ODN injections induced a more sustained elevation of serum 2'5'-A synthetase activity. Finally, formulation of ODN 2007 in emulsigen increased the level of serum 2'5'-A synthetase activity and this response was CpG-specific. Elevated serum 2'5'-A synthetase activity suggested that CpG ODN acted through the induction of either interferon (IFN)-alpha or IFN-gamma. ODN 2007 did not induce detectable levels of IFN-alpha or IFN-gamma when incubated with peripheral blood mononuclear cells, but both IFN-alpha and IFN-gamma were detected following stimulation of lymph node cells with ODN 2007. CpG ODN induction of 2'5'-A synthetase in vitro correlated with the secretion of both IFN-alpha and IFN-gamma. Furthermore, immunohistochemical staining of skin revealed a marked cellular infiltration at the site of ODN 2007 injection. This cellular infiltration was CpG-specific and consisted of primarily CD172(+) myeloid cells. Many of the cells recruited to the site of ODN 2007 injection expressed IFN-alpha and some IFN-gamma. These observations support the conclusion that localized cell recruitment and activation contribute to CpG ODN induction of antiviral effector molecules, such as interferon and 2'5'-A synthetase.     

Stimulation of innate immune responses by CpG oligodeoxynucleotide in newborn lambs can reduce bovine herpesvirus-1 shedding

Stimulation of the innate immune system is potentially very important in neonates who have an immature adaptive immune system and vaccination cannot be used to reduce the risk of infection. CpG oligodeoxynucleotide (ODN) can stimulate innate immune responses in newborn chickens and mice, but similar studies are lacking in other mammalian species. We have shown previously that CpG ODN can both stimulate an acute-phase immune response and induce the antiviral effector molecule, 2'5'-A synthetase, in adult sheep. Therefore, the immunostimulatory activity of A class and B class CpG ODN was evaluated in newborn lambs, and the capacity of CpG ODN-induced responses to reduce viral shedding was evaluated following aerosol challenge with the respiratory pathogen, bovine herpesvirus-1 (BHV-1). In vitro CpG ODN stimulation of peripheral blood mononuclear cells (PBMC) isolated from newborn lambs (3-5 days old) and adult sheep induced equivalent CpG-specific proliferative responses and interferon-alpha (IFN-alpha) secretion. CpG ODN-induced IFN-alpha secretion by neonatal PBMCs was, however, significantly (p < 0.01) enhanced 6 days after subcutaneous (s.c.) injection of 100 microg/kg CpG ODN 2007. Newborn lambs injected s.c. with B class CpG ODN 2007 or the inverted GpC control ODN formulated in 30% Emulsigen (MVP Laboratories, Ralston, NE) displayed CpG ODN-specific increases in body temperature (p < 0.0001), serum 2'5'-A synthetase activity (p = 0.0015), and serum haptoglobin (p = 0.07). CpG ODN-treated lambs also displayed a transient reduction in viral shedding on day 2 postinfection (p < 0.05), which correlated (p < 0.03) with serum 2'5'-A synthetase levels on the day of viral challenge. These observations confirmed that CpG ODNs effectively activate innate immune responses in newborn lambs and CpG ODN-induced antiviral responses correlated with a reduction in viral shedding.     

Oligodeoxynucleotides containing CpG motifs induce low levels of TNF-alpha in human B lymphocytes: possible adjuvants for Th1 responses

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) represent potential adjuvants for specific immunotherapy of type I allergies because they foster Th1-like immune responses. However, previous work has shown that CpG-ODN induce systemically active levels of TNF-alpha in murine macrophages. The goal of the present study was to evaluate the release of TNF-alpha in human cells by a CpG-ODN proven to induce Th1 immune responses in cells from atopic individuals and in mice. CpG-ODN induced TNF-alpha in cells from atopic and healthy individuals. However, the amounts were low, as determined by comparison with commonly used Ags. Intracellular cytokine staining of PBMC revealed that CpG-ODN-induced TNF-alpha derived exclusively from B lymphocytes. TNF-alpha contributed to the CpG-ODN-augmented proliferation and Ig synthesis in PBMC, but was not involved in IFN-gamma synthesis. In conclusion, our findings indicate that certain CpG-ODN induce low amounts of TNF-alpha in human B lymphocytes and may therefore be used to modulate Th2-biased immune responses in allergic patients.     

CpG-A-induced monocyte IFN-gamma-inducible protein-10 production is regulated by plasmacytoid dendritic cell-derived IFN-alpha

Unmethylated CpG motifs in bacterial DNA or synthetic oligodeoxynucleotides (ODN) are known for inducing a Th1 cytokine/chemokine environment, but the mechanisms regulating this have been unclear. Recent studies have defined two classes of CpG ODN, CpG-A ODN that induce plasmacytoid dendritic cells (pDC) to secrete very high levels of IFN-alpha, and CpG-B ODN that induce only low levels of IFN-alpha production, but strongly activate B cells. We now demonstrate that a CpG-A ODN directly activates pDC secretion of IFN-alpha and other soluble factors that secondarily induce purified monocytes to secrete high levels of the Th1-promoting chemokine IFN-gamma-inducible protein-10 (IP-10). Cell contact between the monocytes and pDC is not required for this interaction. IFN-alpha is necessary, but only partially sufficient, for this indirect CpG-induced monocyte IP-10 production. Although CpG ODN induce human PBMC to make only very slight amounts of IFN-gamma, we find that these low concentrations synergize with IFN-alpha for inducing monocyte production of IP-10. These studies provide a better understanding of the mechanisms through which CpG ODN create a Th1-like environment.     

Identification of cytosine-phosphorothioate-guanine oligodeoxynucleotide sequences that induce interferon-γ production in feline immune cells

Unmethylated CpG-ODN are known to enhance Th1-type immune response. However, optimal sequences of CpG-ODN for activating Th1-type immune cells vary among species. It is necessary to identify the effective CpG-ODN sequences in each species. In the present study, in order to identify the sequences of CpG-ODN that produce fIFN-γ in cats, 14 kinds of ODN were synthesized and examined regarding their ability to induce fIFN-γ in feline PBMC and splenocytes. It was shown that some CpG-ODN significantly induced fIFN-γ production in splenocytes, but not in PBMC. We found that three kinds of CpG-ODN (no. 2, 5'-ggTGCATCGATGCAGggggG-3'; no. 5, 5'-ggTGCGTCGACGCAGggggG-3'; no. 10, 5'-ggTGCTACGTAGCAGggggG-3') specifically and significantly induced fIFN-γ production in feline splenocytes. The reverse sequences, GpC-ODN, do not cause significant fIFN-γ production. The fIFN-γ production inductivity of a mixture of CpG-ODN nos. 2, 5 and 10 was higher than those of individual CpG-ODN. When the CpG-ODN mixture was encapsulated in an MCL and administrated to cats, the number of fIFN-γ(+) cells in PBMC significantly increased. CpG-ODN nos. 2, 5 and 10 should be useful to elicit a Th1-type immune response as a vaccine adjuvant in cats.     

Transfection of human monocyte-derived dendritic cells with CpG oligonucleotides

Monocyte-derived dendritic cells (mDC), the most frequently applied DC subset in clinical studies, which can be obtained easily from peripheral blood monocytes after incubation with GM-CSF and IL-4, have not been clearly demonstrated to be activated by CpG oligodeoxynucleotides (ODN). The development of novel molecular strategies - such as the use of CpG-ODN - to increase immunological functions and thus improve the therapeutic efficiency of mDC vaccines in the treatment of malignant diseases is highly desirable. CpG-ODN need to be internalized into specific intracellular compartments to be active. Therefore, we applied electroporation and lipofection and compared these techniques with incubation to overcome possible defects in localization. Conditions of CpG-ODN transfection of these cells were optimized using fluorescein-marked ODN 2216. We were able to achieve high transfection efficiencies with various methods of delivery. However, we did not observe increased expression of maturation-associated and functionally relevant surface antigens (CD14, HLA-DR, CD40, CD83, CD80 and CD86), significant secretion of IL-12 and IFN-alpha in culture supernatant, or enhanced antitumour activation of cytokine-induced killer cells. In conclusion, our results show that non-viral transfection of CpG-ODN is not sufficient to overcome resistance of mDC to CpG activation.     

Effective DNA vaccination against listeriosis by prime/boost inoculation with the gene gun.

Protective immunity against Listeria monocytogenes strongly depends on CD8+ T lymphocytes, and both IFN-gamma secretion and target cell killing are considered relevant to protection. We analyzed whether we could induce a protective type 1 immune response by DNA vaccination with the gene gun using plasmids encoding for two immunodominant listerial Ags, listeriolysin and p60. To induce a Th1 response, we 1) coprecipitated a plasmid encoding for GM-CSF, 2) employed a prime/boost vaccination schedule with a 45-day interval, and 3) coinjected oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs. DNA immunization of BALB/c mice with plasmids encoding for listeriolysin (pChly) and p60 (pCiap) efficiently induced MHC class I-restricted, Ag-specific CD8+ T cells that produced IFN-gamma. Coinjection of CpG-ODN significantly increased the frequency of specific IFN-gamma-secreting T cells. Although pChly induced specific CD8+ T cells expressing CTL activity, it failed to stimulate CD4+ T cells. Only pCiap induced significant CD4+ T cell and humoral responses, which were predominantly of Th2 type. Vaccination with either plasmid induced protective immunity against listerial challenge, and coinjection of CpG ODN improved vaccine efficacy in some situations. This study demonstrates the feasibility of gene gun administration of plasmid DNA for inducing immunity against an intracellular pathogen for which protection primarily depends on type 1 CD8+ T cells.     

Impaired monocyte maturation in response to CpG oligodeoxynucleotide is related to viral RNA levels in human immunodeficiency virus disease and is at least partially mediated by deficiencies in alpha/beta interferon responsiveness and production

The biological activity of CpG oligodeoxynucleotide 2216 (ODN2216), a Toll-like receptor 9 agonist, was investigated with monocytes from human immunodeficiency virus (HIV)-negative and HIV-positive (HIV+) donors. Exposure of peripheral blood mononuclear cells to CpG ODN2216 led to decreased expression of the monocyte marker CD14 and increased expression of the dendritic cell marker CD83, as well as increased expression of HLA-DR, CD40, CD80, and CD86 among the monocytes. Several features of the CpG ODN-induced maturation were diminished in monocytes from HIV+ donors, and these deficiencies were related to increased viremia but not to CD4 cell counts. Alpha interferon (IFN-alpha) was implicated as at least a partial mediator of the CpG ODN-induced monocyte maturation. Reduced production of IFN-alpha in response to CpG ODN and reduced frequencies of plasmacytoid dendritic cells, the principal IFN-alpha-producing cell type in peripheral blood, were observed in peripheral blood mononuclear cells from HIV+ donors. These deficiencies also were related to levels of plasma HIV RNA. Responses of monocytes from HIV+ donors to direct stimulation with IFN-alpha also were partially impaired. Thus, reduced production of IFN-alpha and reduced IFN-alpha responsiveness may contribute to diminished functional responses to CpG ODN in HIV disease. Application of CpG ODNs in HIV disease for adjuvant or immunoregulatory purposes may be particularly useful for HIV+ donors without high-level viremia.     

Differential effects of interferon-alpha and interferon-gamma on interleukin 1 secretion by monocytes

We examined the effect of interferon (IFN)-alpha and IFN-gamma on the ability of human monocytes to secrete interleukin 1 (IL 1). IFN-alpha directly induced IL 1 secretion by monocytes. IFN-gamma did not induce any IL 1. IFN-gamma-stimulated monocyte supernatants were also negative for pyrogenic activity. However, IFN-gamma greatly enhanced the amount of IL 1 secreted when monocytes were stimulated by lipopolysaccharide or Staphylococcus aureus, even at concentrations which by themselves did not induce IL 1. IFN-alpha did not enhance IL 1 secretion induced by other stimuli. IFN-gamma enhanced IL 1 secretion by priming monocytes to be more sensitive to an IL 1-inducing stimulus. However, IFN-gamma does not enhance IL 1 induced by all stimuli, because there was no enhancement of IL 1 induced by PMA. Thus, IFN-alpha and IFN-gamma have very distinct roles in the induction and enhancement of IL 1 by monocytes.     

TLR9- and FcepsilonRI-mediated responses oppose one another in plasmacytoid dendritic cells by down-regulating receptor expression

Plasmacytoid dendritic cells (pDC) express not only TLR9 molecules through which ligation with CpG DNA favors Th1 responses but also possess IgE receptors (FcepsilonRI) implicated in allergen presentation and induction of Th2 responses. This dichotomy prompted an investigation to determine whether TLR9- and IgE receptor-mediated responses oppose one another in pDC by affecting receptor expression and associated functional responses. Results showed that IgE cross-linking reduced TLR9 in pDC and inhibited the capacity of these cells to secrete IFN-alpha when stimulated with the CpG oligodeoxynucleotide (ODN)-2216. In contrast, an approximately 15-fold reduction in FcepsilonRIalpha mRNA and a loss in surface protein were seen in pDC first exposed to TLR9 ligation with ODN-2216. Results indicated that type I IFNs partly mediated this effect, as rIFN-alpha also caused a significant approximately 4-fold reduction in FcepsilonRIalpha mRNA. Finally, this reduction in FcepsilonRIalpha mediated by ODN-2216 correlated with a selective suppression of allergen-induced CD4+ T cell proliferation, but not of responses resulting from tetanus toxoid. Overall, these results imply mechanisms by which specific innate and IgE-dependent immune responses counterregulate one another at the dendritic cell level and may have significant impact on whether an ensuing response is either of Th1 or Th2 in nature.     

Human B cells express functional TRAIL/Apo-2 ligand after CpG-containing oligodeoxynucleotide stimulation

CpG-containing oligodeoxynucleotides (CpG ODN) have broad-ranging immunostimulatory effects, including the generation of antitumor immune responses. Analysis of different CpG ODN have identified two classes: CpG-A ODN, which stimulate high levels of IFN-alpha production from plasmacytoid dendritic cells and weakly activate B cells, and CpG-B ODN, which strongly activate B cells but stimulate low production of IFN-alpha from plasmacytoid dendritic cells. Previously, we observed that CpG-B ODN (2006) induces TRAIL/Apo-2 ligand (Apo-2L)-mediated killing of tumor cells by CD14(+) PBMC. In this study, we extend our investigation of CpG ODN-induced TRAIL/Apo-2L expression and activity in PBMC to include CpG-A ODN. Of the two classes, IFN-alpha production and TRAIL/Apo-2L-mediated killing of tumor cells was greatest with CpG-A ODN. Surprisingly, CD3(+), CD14(+), CD19(+), and CD56(+) PBMC expressed high levels of TRAIL/Apo-2L following CpG-A ODN stimulation. When isolated, the CD19(+) PBMC (B cells) were able to kill tumor cells in a TRAIL/Apo-2L-dependent manner. As with CD14(+) PBMC, CD19(+) sorted B cells were capable of up-regulating TRAIL/Apo-2L expression when stimulated with IFN-alpha alone. Interestingly, agonist anti-CD40 mAb further enhanced the IFN-alpha-induced TRAIL/Apo-2L expression on CD19(+) B cells. These results are the first to demonstrate human B cell-mediated killing of tumor cells in a TRAIL/Apo-2L-dependent fashion.     

Plasmacytoid dendritic cell-derived IFN-alpha induces TNF-related apoptosis-inducing ligand/Apo-2L-mediated antitumor activity by human monocytes following CpG oligodeoxynucleotide stimulation

Immunostimulatory oligodeoxynucleotides (ODN) containing the CpG motif are being tested as immune adjuvants in many disease settings. Of the human PBMC examined, plasmacytoid dendritic cells (pDC) are a major source of type I IFN upon stimulation with CpG ODN. IFNs have numerous immunostimulatory effects, including the induction of TNF-related apoptosis-inducing ligand (TRAIL)/Apo-2L on monocytes, NK cells, and T cells. Importantly, IFN has also been linked to antitumor responses. Thus, we tested whether CpG ODN stimulation of PBMC led to TRAIL/Apo-2L-induced tumor cell death. When PBMC were stimulated with CpG ODN, TRAIL/Apo-2L-dependent tumor cell death was observed. Further examination of CpG ODN-stimulated PBMC revealed that TRAIL/Apo-2L expression was limited to CD14(+) cells, which, when depleted, led to a loss of the TRAIL/Apo-2L-mediated tumor cell killing. Moreover, pDC depletion also abolished the TRAIL/Apo-2L-mediated killing of tumor cell targets. Analysis of the pDC showed IFN-alpha production after CpG ODN stimulation. Finally, inclusion of neutralizing IFN-alpha antiserum with the PBMC during CpG ODN stimulation abrogated TRAIL/Apo-2L-mediated tumor cell killing. These results define a mechanism by which CpG ODN induces TRAIL/Apo-2L-dependent killing of tumor cells by CD14(+) PBMC, in which CpG ODN-activated pDC produce IFN-alpha that stimulates CD14(+) PBMC to express functional TRAIL/Apo-2L.     

Vaccination with Leishmania soluble antigen and immunostimulatory oligodeoxynucleotides induces specific immunity and protection against Leishmania donovani infection

In this report, we investigated the effect of ODN containing immunostimulatory CG motifs as adjuvant with soluble antigen (SA) from Leishmania donovani. BALB/c mice were vaccinated with the soluble antigen with or without CpG-ODN as adjuvant and then challenged with L. donovani metacyclic promastigotes. CpG-ODN alone resulted in partial protection against challenge with L. donovani. Immunization of mice with SA and CpG-ODN showed enhanced reduction in parasite load ( approximately 60%) when compared to SA ( approximately 40%) immunized mice. Immunization with SA by itself resulted in a mixed Th1/Th2 response whereas co-administration of SA with CpG-ODN resulted in a strong Th1 promoting isotype as they together promoted production of immunoglobulin G2a. Leishmania-specific Th1 cytokine response was induced by co-administering CpG-ODN and SA as they together promoted production of IFN-gamma and IL-12. In the present study, we demonstrate that immunostimulatory phosphorothioate-modified ODN are promising immune enhancers for vaccination against visceral leishmaniaisis.     

Release of lymphokines after infection with Epstein Barr virus in vitro. II. A monocyte-dependent inhibitor of interleukin 1 downregulates the production of interleukin 2 and interferon-gamma in rheumatoid arthritis

Epstein Barr virus (EBV)-infection of normal peripheral blood mononuclear cells (PBMC) in vitro induces IFN-alpha secretion from B cell and natural killer (NK) cell populations, and IFN-gamma secretion from T cells. IFN-gamma depends on prior elaboration of IL 2 and IL 1 that originates from monocytes and NK cells. PBMC from rheumatoid arthritis (RA) patients released moderately elevated levels of IFN-alpha (236 +/- 62 U/ml vs 168 +/- 34 in normals). In contrast, IFN-gamma was significantly lower in RA (88 +/- 34 U/ml vs 209 +/- 32) with an associated deficit in IL 2. A monocyte-dependent factor was shown to be responsible for this deficit, since monocyte depletion of RA cultures normalized the levels of IL 2 and IFN-gamma. Significantly lower levels of IL 1 activity were present in the supernatants of RA PBMC cultures as compared with normal cultures, and this was shown to be associated with presence of a nondialyzable IL 1 inhibitor. This inhibitor was capable of preventing the IL 1-dependent synthesis of IL 2 and IFN-gamma by normal PBMC. Exogenous IL 1 or IL 2 restored the deficient IFN-gamma secretion in RA PBMC. Thus, the deficient ability of RA lymphocytes to control EBV infection may be secondary to impairment of a monocyte-T cell interaction at the level of IL 1.     

Expansion of bone marrow IFN-alpha-producing dendritic cells in New Zealand Black (NZB) mice: high level expression of TLR9 and secretion of IFN-alpha in NZB bone marrow

Patients with systemic lupus erythematosus have elevated IFN-alpha production. Furthermore, sera IFN-alpha levels correlate with disease activity. We have focused our attention on whether this phenotype is also seen in the New Zealand Black (NZB) mice and simultaneously addressed the underlying mechanisms. Specifically, we analyzed: 1) levels of sera IFN-alpha after type A CpG ODN 2216 injection in autoimmunity-prone NZB and control mice, and 2) levels of IFN-alpha synthesized by IFN-alpha-producing dendritic cells (IPDCs) using highly enriched populations of CD11c+B220+ IPDCs derived from NZB and control mice; IPDCs are divided into two subpopulations (CD4+CD11c+B220+ and CD4-CD11c+B220+). Our data demonstrate that NZB mice produced higher levels of sera IFN-alpha after type A CpG ODN 2216 injection when compared with control mice (p < 0.01). In addition, the cell numbers, frequency, and TLR9 mRNA levels of CD4+ and CD4- IPDC were markedly increased in the bone marrow (BM) of NZB mice. Upon in vitro stimulation with TLR9 ligand-CpG ODN 2216, higher levels of IFN-alpha were synthesized by IPDCs from the BM of NZB. The major contributor of IFN-alpha was the CD4-CD11c+B220+ IPDC subpopulation. Furthermore, NZB BM IPDCs manifest impaired expression of homing chemokine CCR7 and CD62L, and IL-12 production. These data on the functional characteristics of the IPDC lineages explain in part the mechanism of hyper-IFN-alpha production and help clarify the mechanism for the expansion of NZB BM IPDCs.     

Activation with CpG-A and CpG-B oligonucleotides reveals two distinct regulatory pathways of type I IFN synthesis in human plasmacytoid dendritic cells

Two different CpG oligonucleotides (ODN) were used to study the regulation of type I IFN in human plasmacytoid dendritic cells (PDC): ODN 2216, a CpG-A ODN, known to induce high amounts of IFN-alpha in PDC, and ODN 2006, a CpG-B ODN, which is potent at stimulating B cells. CpG-A ODN showed higher and prolonged kinetics of type I IFN production compared with that of CpG-B ODN. In contrast, CpG-B ODN was more active than CpG-A ODN in stimulating IL-8 production and increasing costimulatory and Ag-presenting molecules, suggesting that CpG-A and CpG-B trigger distinct regulatory pathways in PDC. Indeed, CpG-A ODN, but not CpG-B ODN, activated the type I IFNR-mediated autocrine feedback loop. PDC were found to express high constitutive levels of IFN regulatory factor (IRF)7. IRF7 and STAT1, but not IRF3, were equally up-regulated by both CpG-A and CpG-B. CD40 ligand synergistically increased CpG-B-induced IFN-alpha independent of the IFNR but did not affect CpG-B-induced IFN-beta. In conclusion, our studies provide evidence for the existence of two distinct regulatory pathways of type I IFN synthesis in human PDC, one dependent on and one independent of the IFNR-mediated feedback loop. The alternate use of these pathways is based on the type of stimulus rather than the quantity of IFN-alphabeta available to trigger the IFNR. Constitutive expression of IRF7 and the ability to produce considerable amounts of IFN-alpha independent of the IFNR seem to represent characteristic features of PDC.     

Induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded siRNAs is sequence-dependent and requires endosomal localization

The potential induction of inflammatory cytokines and interferon responses by small-interfering RNAs (siRNAs) represents a major obstacle for their use as inhibitors of gene expression. Therapeutic applications of siRNAs will require a better understanding of the mechanisms that trigger such unwanted effects, especially in freshly isolated human cells. Surprisingly, the induction of tumor necrosis factor (TNF-alpha) and interleukin-6 (IL-6) in adherent peripheral blood mononuclear cells (PBMC) was not restricted to double-stranded siRNAs, because induction was also obtained with single-stranded siRNAs (sense or antisense strands). The immunostimulatory effects were sequence-dependent, since only certain sequences are prone to induce inflammatory responses while others are not. The induction of TNF-alpha, IL-6 and interferon alpha (IFN-alpha) was chloroquine-sensitive and dependent more likely on endosomal Toll-like receptor signaling in particular TLR8. Indeed, no significant immunostimulatory effects were detected when either double or single-stranded siRNAs were delivered directly to cytoplasm via electroporation. Both RNA types activated a NF-kappaB promoter-driven luciferase gene in transiently transfected human adherent PBMC. Moreover, culture of immature dendritic cells with either double or single-stranded siRNAs stimulated interleukin-12 production and induced the expression of CD83, an activation marker. Interestingly, several double-stranded siRNAs did not induce TNF-alpha, IL-6 and IFN-alpha production, however, their single-stranded sense or antisense did. Taken together, the present data indicate for the first time that the induction of inflammatory cytokines and IFN-alpha responses by either double-stranded or single-stranded siRNAs in adherent PBMC is sequence-dependent and requires endosomal intracellular signaling. The finding that endosomal localization of self-RNAs (sense strands) can trigger Toll-like receptor signaling in adherent human PBMC is intriguing because it indicates that endosomal self-RNAs can display a molecular pattern capable for activating innate immunity.     

A human 15-kDa IFN-induced protein induces the secretion of IFN-gamma.

A 15,000 molecular weight protein (15-kDa), induced and secreted by human PBMC after treatment with IFN-alpha or -beta, was assessed for its ability to modulate cellular function. Although it had no effect on growth or 2'5'-A synthetase activity in Daudi, U-937, or HL-60 cells, when incubated with fresh human PBMC, LPS-induced monocyte cytotoxicity against WEHI-164 target cells was augmented. This stimulation was inhibited by both an antibody against TNF-alpha and a rabbit polyclonal antiserum to the 15-kDa protein. Furthermore, when the 15-kDa protein was added to PBMC an increase in GTP cyclohydrolase I activity, as assessed by neopterin secretion, resulted. Neopterin secretion by PBMC in response to the 15-kDa was increased in a dose-responsive manner up to more than sixfold over baseline, with a 15-kDa concentration of less than 10 ng/ml effective. The 15-kDa protein also stimulated indoleamine 2,3-dioxygenase (IDO) activity in fresh, human PBMC. Induction of neopterin secretion and IDO activity was inhibited by a polyclonal antiserum to 15-kDa. LPS-induced cytotoxic activity was not augmented by 15-kDa pretreatment of purified monocytes, indicating the need for the presence of a second cell population and the indirect action of the 15-kDa on the induction of monocyte activities. When PBMC or purified CD3+ cells, but not purified CD14+ cells, were incubated with the 15-kDa protein, secretion of a factor was induced that resulted in the induction of IDO activity in PMA-differentiated THP-1 cells. An antibody to IFN-gamma, but not IFN-alpha, inhibited the induction of IDO activity by this secreted factor. In addition, antiserum to the 15-kDa blocked the secretion of IFN-gamma from the CD3+ cells. Thus, a 15-kDa product of IFN-alpha- and IFN-beta-treated monocytes and lymphocytes can stimulate secretion of IFN-gamma from CD3+ cells.     

The Toll-like receptor 7 (TLR7) agonist, imiquimod, and the TLR9 agonist, CpG ODN, induce antiviral cytokines and chemokines but do not prevent vaginal transmission of simian immunodeficiency virus when applied intravaginally to rhesus macaques

The initial host response to viral infection occurs after Toll-like receptors (TLRs) on dendritic cells (DC) are stimulated by viral nucleic acids (double-stranded RNA, single-stranded RNA) and alpha interferon (IFN-alpha) and IFN-beta are produced. We hypothesized that pharmacologic induction of innate antiviral responses in the cervicovaginal mucosa by topical application of TLR agonists prior to viral exposure could prevent or blunt vaginal transmission of simian immunodeficiency virus (SIV). To test this hypothesis, we treated rhesus monkeys intravaginally with either the TLR9 agonist, CpG oligodeoxynucleotides (ODN), or the TLR7 agonist, imiquimod. Both immune modifiers rapidly induced IFN-alpha and other antiviral effector molecules in the cervicovaginal mucosa of treated animals. However, both CpG ODN and imiquimod also induced proinflammatory cytokine expression in the cervicovaginal mucosa. In the vaginal mucosa of imiquimod-treated monkeys, we documented a massive mononuclear cell infiltrate consisting of activated CD4(+) T cells, DC, and beta-chemokine-secreting cells. After vaginal SIV inoculation, all TLR agonist-treated animals became infected and had plasma vRNA levels that were higher than those of control monkeys. We conclude that induction of mucosal innate immunity including an IFN-alpha response is not sufficient to prevent sexual transmission of human immunodeficiency virus.