Maternal host responses to poly(I:C) during pregnancy leads to both dysfunctional immune profiles and altered behaviour in the offspring.

PROBLEM: Autism spectrum disorder (ASD)-like phenotypes in murine models are linked to elevated pro-inflammatory cytokine profiles caused by maternal immune activation (MIA), but whether MIA alters the immune response in the offspring remains unclear. METHOD OF STUDY: Polyinosinic:polycytidylic acid (poly:[IC]) was used to induce MIA in immunocompetent and control TLR3-deficient pregnant mice, and cytokine levels were measured in maternal and foetal organs. Furthermore, cytokines and behaviour responses were tested after challenge with lipopolysaccharide in 7-day-old and adult mice. RESULTS: MIA induced on E12 resulted in changes in the cytokine expression profile in maternal and foetal organs and correlated with TNFα and IL-18 dysregulation in immune organs and brains from neonatal mice born to MIA-induced dams. Such changes further correlated with altered behavioural responses in adulthood. CONCLUSION: MIA induced by pathogens during pregnancy can interfere with the development of the foetal immune and nervous systems leading to dysfunctional immune responses and behaviour in offspring.

The majority of murine γδ T cells at the maternal-fetal interface in pregnancy produce IL-17.

Compared with lymphoid tissues, the immune cell compartment at mucosal sites is enriched with T cells bearing the γδ T-cell receptor (TCR). The female reproductive tract, along with the placenta and uterine decidua during pregnancy, are populated by γδ T cells predominantly expressing the invariant Vγ6(+)Vδ1(+) receptor. Surprisingly little is understood about the function of these cells. We found that the majority of γδ T cells in the non-pregnant uterus, pregnant uterus, decidua and placenta of mice express the transcription factor RORγt and produce interleukin-17 (IL-17). In contrast, IFNγ-producing γδ T cells were markedly reduced in gestational tissues compared with uterine-draining lymph nodes and spleen. Both uterine-resident invariant Vγ6(+) and Vγ4(+) γδ T cells which are more typically found in lymphoid tissues and circulating blood, were found to express IL-17. Vγ4(+) γδ T cells were particularly enriched in the placenta, suggesting a pregnancy-specific recruitment or expansion of these cells. A small increase in IL-17-producing γδ T cells was observed in allogeneic compared with syngeneic pregnancy, suggesting a contribution to regulating the maternal response to paternally-derived alloantigens. However, their high proportions also in non-pregnant uteri and gestational tissues of syngeneic pregnancy imply a role in the prevention of intrauterine infection or quality control of fetal development. These data suggest the need for a more rigorous evaluation of the role of IL-17 in sustaining normal pregnancy, particularly as emerging data points to a pathogenic role for IL-17 in pre-eclampsia, pre-term birth, miscarriage and maternal immune activation-induced behavioral abnormalities in offspring.

Multi-parameter flow cytometric analysis of uterine immune cell fluctuations over the murine estrous cycle.

Investigating immune cell populations within various reproductive tissues commonly utilises flow cytometric methods. With advances in fluorophore technology and equipment capabilities, multiple cell types from a single tissue sample can be identified by using different combinations of cell surface markers to distinguish specific cell populations. Here a protocol optimized for mouse uterine tissue was used to show the proportional changes in dendritic cells, monocyte/macrophages, T and B cells, NK and NK T cells, and the granulocytes, neutrophils and eosinophils at each of the four stages of the estrous cycle. Importantly, we demonstrate that use of anti-SiglecF or assessment of FSC/SSC plots could be used to differentiate monocyte/macrophage and eosinophil populations that otherwise cannot be distinguished by use of the common combination of antibodies against F4/80 and CD11b. Our results clearly indicate that within the uterus a dynamic population of immune cells resides, with many cell types reaching peak abundance at estrus and metestrus phases of the cycle, consistent with their importance in the response to paternal antigens and/or pathogens encountered after insemination.

The multifunctional alarmin HMGB1 with roles in the pathophysiology of sepsis and cancer.

Although originally described as a highly conserved nuclear protein involved in DNA replication, transcription and repair, high-mobility group box-1 protein (HMGB1) has emerged as a key mediator in the regulation of immune responses to infection and sterile injury by exhibiting all the properties of a prototypic 'alarmin'. These include rapid passive release in response to pathogenic infection and/or traumatic injury, active secretion providing for chemotactic and cytokine-like function and an ability to resolve inflammation, including tissue repair and remodelling. In this review, we will give an overview of the post-translational modifications necessary for such diversity in biological activity, concentrating particularly on how differences in oxidation of highly conserved redox-sensitive cysteine residues can potentiate inflammatory responses and dictate cellular fate. We will also review the most recent literature on HMGB1 and its involvement in the pathophysiology of sepsis and cancer, as well as cancer therapy-induced mucositis.

Inhibiting the TLR4-MyD88 signalling cascade by genetic or pharmacological strategies reduces acute alcohol-induced sedation and motor impairment in mice.

BACKGROUND AND PURPOSE: Emerging evidence implicates a role for toll-like receptor 4 (TLR4) in the CNS effects of alcohol. The aim of the current study was to determine whether TLR4-MyD88-dependent signalling is involved in the acute behavioural actions of alcohol and if alcohol can activate TLR4-downstream MAPK and NF-κB pathways. EXPERIMENTAL APPROACH: The TLR4 pathway was evaluated using the TLR4 antagonist (+)-naloxone (µ-opioid receptor-inactive isomer) and mice with null mutations in the TLR4 and MyD88 genes. Sedation and motor impairment induced by a single dose of alcohol were assessed by loss of righting reflex (LORR) and rotarod tests, separately. The phosphorylation of JNK, ERK and p38, and levels of IκBα were measured to determine the effects of acute alcohol exposure on MAPK and NF-κB signalling. KEY RESULTS: After a single dose of alcohol, both pharmacological inhibition of TLR4 signalling with (+)-naloxone and genetic deficiency of TLR4 or MyD88 significantly (P < 0.0001) reduced the duration of LORR by 45-78% and significantly decreased motor impairment recovery time to 62-88% of controls. These behavioural actions were not due to changes in the peripheral or central alcohol pharmacokinetics. IκBα levels responded to alcohol by 30 min in mixed hippocampal cell samples, from wild-type mice, but not in cells from TLR4- or MyD88-deficient mice. CONCLUSIONS AND IMPLICATIONS: These data provide new evidence that TLR4-MyD88 signalling is involved in the acute behavioural actions of alcohol in mice.

Innate immune recognition of poxviral vaccine vectors.

The study of poxviruses pioneered the field of vaccinology after Jenner's remarkable discovery that 'vaccination' with the phylogenetically related cowpox virus conferred immunity to the devastating disease of smallpox. The study of poxviruses continues to enrich the field of virology because the global eradication of smallpox provides a unique example of the potency of effective immunization. Other poxviruses have since been developed as vaccine vectors for clinical and veterinary applications and include modified vaccinia virus strains such as modified vaccinia Ankara and NYVAC as well as the avipox viruses, fowlpox virus and canarypox virus. Despite the empirical development of poxvirus-based vectored vaccines, it is only now becoming apparent that we need to better understand how the innate arm of the immune system drives adaptive immunity to poxviruses, and how this information is relevant to vaccine design strategies, which are the topics addressed in this article.

Attenuation of microglial and IL-1 signaling protects mice from acute alcohol-induced sedation and/or motor impairment.

Alcohol-induced proinflammatory central immune signaling has been implicated in the chronic neurotoxic actions of alcohol, although little work has examined if these non-neuronal actions contribute to the acute behavioral responses elicited by alcohol administration. The present study examined if acute alcohol-induced sedation (loss of righting reflex, sleep time test) and motor impairment (rotarod test) were influenced by acute alcohol-induced microglial-dependent central immune signaling. Inhibition of acute alcohol-induced central immune signaling, through the reduction of proinflammatory microglial activation with minocycline, or by blocking interleukin-1 (IL-1) receptor signaling using IL-1 receptor antagonist (IL-1ra), reduced acute alcohol-induced sedation in mice. Mice treated with IL-1ra recovered faster from acute alcohol-induced motor impairment than control animals. However, minocycline led to greater motor impairment induced by alcohol, implicating different mechanisms in alcohol-induced sedation and motor impairment. At a cellular level, IκBα protein levels in mixed hippocampal cells responded rapidly to alcohol in a time-dependent manner, and both minocycline and IL-1ra attenuated the elevated levels of IκBα protein by alcohol. Collectively these data suggest that alcohol is capable of rapid modification of proinflammatory immune signaling in the brain and this contributes significantly to the pharmacology of alcohol.

Antigen-specific T-cell responses to a recombinant fowlpox virus are dependent on MyD88 and interleukin-18 and independent of Toll-like receptor 7 (TLR7)- and TLR9-mediated innate immune recognition.

Fowlpox virus (FWPV) is a double-stranded DNA virus long used as a live-attenuated vaccine against poultry diseases, but more recent interest has focused on its use as a mammalian vaccine vector. Here, in a mouse model system using FWPV encoding the nominal target antigen chicken ovalbumin (OVA) (FWPV(OVA)), we describe for the first time some of the fundamental processes by which FWPV engages both the innate and adaptive immune systems. We show that Toll-like receptor 7 (TLR7) and TLR9 are important for type I interferon secretion by dendritic cells, while TLR9 is solely required for proinflammatory cytokine secretion. Despite this functional role for TLR7 and TLR9 in vitro, only the adapter protein myeloid differentiation primary response gene 88 (MyD88) was shown to be essential for the formation of adaptive immunity to FWPV(OVA) in vivo. The dependence on MyD88 was confined only to the T-cell compartment and was not related to its contribution to TLR signaling, dendritic cell maturation, or the capture and presentation of FWPV-derived OVA antigen. We demonstrate that this is not by means of mediating T-cell-dependent interleukin-1 (IL-1) signaling, but rather, we suggest that MyD88 functions to support T-cell-specific IL-18 receptor signaling, which in turn is essential for the formation of adaptive immunity to FWPV-encoded OVA.

Induction of both cellular and humoral immunity following a rational prime-boost immunization regimen that incorporates recombinant ovine atadenovirus and fowlpox virus.

Recombinant fowlpox viruses (rFPV) and ovine atadenoviruses (rOAdV) are being developed as safe, nonpathogenic, prophylactic and therapeutic vaccine vectors. There is scope, however, to improve the limited immune responses elicited by each of these vaccine vectors. Using previously determined and optimized routes of administration and viral doses, we characterized the primary adaptive immune responses elicited by recombinant variants of each virus. We demonstrate the contrasting nature of the response elicited by each recombinant virus. Whereas rFPV generates predominately cell-mediated immunity to our nominal target antigen, ovalbumin (OVA), rOAdV drives strong humoral responses. By defining the time taken to achieve maximal cytotoxic T cell responses and by studying the different patterns and kinetics of major histocompatibility complex class I-restricted OVA antigen expression postimmunization, we proposed a heterologous prime-boost regimen of immunization with rOAdV followed by rFPV. The subsequent experimental results showed that this approach produced robust cell-mediated and humoral immune responses against OVA that, importantly, were accompanied by weak anti-viral vector antibody responses. These results, therefore, represent a novel and potentially clinically applicable way to achieve broadly based and effective immunity to the antigens encoded by vectored vaccines.

Dasatinib alters the metastatic phenotype of B16-OVA melanoma in vivo.

The Src/Abl tyrosine kinase inhibitor dasatinib is an approved chronic myeloid leukemia treatment and is under investigation for solid tumor therapy. Members of the Src family of kinases (SFKs) are involved in the process of metastasis and dasatinib inhibits the migration and invasiveness of human melanoma cell lines in vitro. SFKs are also involved in immune function and angiogenesis, which both contribute to As active and passive immunotherapies continue to be investigated in metastatic melanoma, we investigated possible interactions between kinase inhibitors and immunotherapies. A murine syngenic model of metastatic melanoma in which B16F10 cells expressed ovalbumin (B16-OVA) was employed and the active immunotherapy comprised immunization with an OVA-expressing recombinant fowlpox virus (FPVOVA).Dasatinib did not affect B16-OVA viability, proliferation, migration or soft agar colony formation. However, depending on drug dose and schedule, differences in the metastatic behavior of B16-OVA were observed in vivo after dasatinib therapy. At a dose of 5 mg/kg/day given before tumor challenge, dasatinib therapy reduced the number of pulmonary metastases. Conversely, a higher dose (25 mg/kg/day), did not affect the number of pulmonary metastases and increased the number of extra-pulmonary metastases. Finally, immunization of B16-OVA-bearing mice with FPVOVA reduced the number of lung metastases. Prior treatment of these mice with dasatinib 5 mg/kg/day did not affect the incidence of lung metastases. Although the mechanisms by which dasatinib alters the metastatic behavior of B16-OVA cells in vivo remain to be determined, we hypothesize that dasatinib acts via multiple tumor-extrinsic processes that include immune function and neoangiogenesis.

Type I interferons mediate the innate cytokine response to recombinant fowlpox virus but not the induction of plasmacytoid dendritic cell-dependent adaptive immunity.

Type I interferons (IFNs) are considered to be important mediators of innate immunity due to their inherent antiviral activity, ability to drive the transcription of a number of genes involved in viral clearance, and their role in the initiation of innate and adaptive immune responses. Due to the central role of type I IFNs, we sought to determine their importance in the generation of immunity to a recombinant vaccine vector fowlpox virus (FPV). In analyzing the role of type I IFNs in immunity to FPV, we show that they are critical to the secretion of a number of innate and proinflammatory cytokines, including type I IFNs themselves as well as interleukin-12 (IL-12), tumor necrosis factor-alpha (TNF-alpha), IL-6, and IL-1beta, and that deficiency leads to enhanced virus-mediated antigen expression. Interestingly, however, type I IFNs were not required for adaptive immune responses to recombinant FPV even though plasmacytoid dendritic cells (pDCs), the primary producers of type I IFNs, have been shown to be requisite for this to occur. Furthermore, we provide evidence that the importance of pDCs may lie in their ability to capture and present virally derived antigen to T cells rather than in their capacity as professional type I IFN-producing cells.

Dasatinib inhibits the secretion of TNF-alpha following TLR stimulation in vitro and in vivo.

OBJECTIVE: Dasatinib (SPRYCEL, BMS-354825) is a small molecule Src/Abl tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. However, kinases inhibited by dasatinib are also involved in the induction and regulation of innate immunity. The purpose of this study was to evaluate the effect of dasatinib on cytokine secretion in response to toll-like receptor (TLR) stimulation. MATERIALS AND METHODS: Dasatinib-treated mice were administered intraperitoneally with lipopolysaccharide (LPS) and serum cytokine (tumor necrosis factor-alpha [TNF-alpha], interleukin [IL]-10, and IL-6) levels and neutrophil accumulation in the lungs were analyzed. Cytokine secretions (TNF-alpha and IL-6) from TLR3-, TLR4-, and TLR9-stimulated RAW264.7, as well as TLR4- and TLR9-stimulated bone marrow-derived macrophages (BMDM) were also evaluated. RESULTS: Dasatinib-treated mice had reduced serum levels of TNF-alpha in response to LPS administration; however, other inflammatory hallmarks of systemic LPS administration, such as secretion of IL-6 and accumulation of neutrophils in the lung, were unaffected. In contrast to the reduced TNF-alpha levels, dasatinib treatment increased serum levels of IL-10 following LPS administration. The production of TNF-alpha was also impaired in vitro in response to TLR3, TLR4, and TLR9 stimulation of the mouse macrophage cell line RAW264.7, as well as TLR4 and TLR9 stimulation of BMDM; IL-6 production was also impaired in dasatinib-treated BMDM. CONCLUSIONS: These findings further support the ability of dasatinib to modulate the host immune response and highlights scope for off-target applications of dasatinib for the control of TNF-alpha-mediated inflammatory disorders.

Recombinant fowlpox virus elicits transient cytotoxic T cell responses due to suboptimal innate recognition and recruitment of T cell help.

Recombinant fowlpox viruses (FPVs) have been used in a variety of vaccine strategies; however strong data clearly demonstrating the characteristics of the strength and nature of the resultant immune response elicited by these vectors are lacking. By utilising a recombinant variant of FPV which expresses the nominal antigen chicken ovalbumin (OVA), and assessing innate FPV- and OVA-specific adaptive immune responses, we show that recombinant FPV induces a rapid type I interferon (IFN) response, mediated primarily by plasmacytoid dendritic cells (pDCs). These cells are necessary for the development of a strong but transient CD8(+) T cell effector response directed against OVA-expressing target cells. We propose that a combination of suboptimal type I IFN production, poor CD4(+) T cell helper function and inefficient DC licensing likely contribute to this transient response. These findings now provide a sound basis for rational modifications to be made to recombinant FPV, designed to improve subsequent vaccine responses.