Enlarged spleens without enlarged lymph nodes in tlr3-/- pkr-/- mice.

Initial phenotypic studies in a mouse containing mutations in both toll-like receptor 3 (TLR3) and RNA-de-pendent protein kinase R (PKR) revealed comparable spleen and bone marrow cell populations in tlr3(-/)-, pkr(-/-), and tlr3(-/-)pkr(-/-) mice to wild-type controls. Splenomegaly developing between 8 and 10 weeks of age was observed in tlr3(-/-) and tlr3(-/-)pkr(-/-) mice but not in wild-type or pkr(-/-) mice. Palpably enlarged cervical, axillary, and inguinal lymph nodes accompanied by enlarged spleens were observed in 12-18-week-old tlr3(-/-) mice at a higher frequency compared with other genotypes. The enlarged spleens and lymph nodes observed in tlr3(-/-) mice were accompanied by destruction of organ architecture and lymphocyte infiltration. However, the enlargement of these organs was not the result of clonal proliferation of one lymphocyte subset. It is likely this phenotype is a result of TLR3 deficiency in combination with an additional, uncharacterized genetic defect or the presence of an infectious agent. These data also suggest that PKR may have a role in preventing progression from splenomegaly to lymphadenopathy in these mice.

Synergistic activation of innate immunity by double-stranded RNA and CpG DNA promotes enhanced antitumor activity.

Double-stranded RNA (dsRNA) and unmethylated CpG sequences in DNA are pathogen-associated molecular patterns of viruses and bacteria that activate innate immunity. To examine whether dsRNA and CpG DNA could combine to provide enhanced stimulation of innate immune cells, murine macrophages were stimulated with poly-rI:rC (pIC), a dsRNA analog, and CpG-containing oligodeoxynucleotides (CpG-ODN). Combined treatments demonstrated synergy in nitric oxide, interleukin (IL)-12, tumor necrosis factor alpha, and IL-6 production. Studies using neutralizing antibodies for type I interferons (IFNs), IFN-alpha and IFN-beta, indicated that nitric oxide synthase synergism is mediated by paracrine/autocrine effects of IFN-beta. In contrast, enhanced cytokine production occurred independent of type I IFN and was maintained in macrophages from IFN-alpha/beta receptor knockout mice. Cotransfection of human Toll-like receptors 3 and 9 (receptors for dsRNA and CpG DNA, respectively) into 293T cells supported synergistic activation of an IL-8 promoter reporter construct by pIC, indicating interaction of the signaling pathways in driving the synergy response. In vivo stimulation of mice with pIC and CpG-ODN demonstrated synergy for serum IL-6 and IL-12p40 levels that correlated with an enhanced antitumor effect against established B16-F10 experimental pulmonary metastases. Treatment of tumor-bearing mice with pIC and CpG-ODN in combination resulted in enhanced nitric oxide synthase expression in lung tissue and enhanced up-regulation of class I major histocompatibility complex on splenic dendritic cells relative to treatments with either agent alone. In conclusion, the combined detection of viral pathogen-associated molecular patterns, i.e., dsRNA and CpG DNA, may mimic definitive viral recognition, resulting in an enhanced innate immune response that could be used for tumor vaccination or immunotherapy.

Negative regulation of TLR-signaling pathways by activating transcription factor-3.

Activating transcription factor-3 (ATF3) is rapidly induced by LPS in mouse macrophages and regulates TLR4 responses. We show that ATF3 is rapidly induced by various TLRs in mouse macrophages and plasmacytoid dendritic cells (DCs), as well as plasmacytoid and myeloid subsets of human DCs. In primary macrophages from mice with a targeted deletion of the atf3 gene (ATF3-knockout (KO)), TLR-stimulated levels of IL-12 and IL-6 were elevated relative to responses in wild-type macrophages. Similarly, targeted deletion of atf3 correlated with enhanced responsiveness of myeloid DCs to TLR activation as measured by IL-12 secretion. Ectopic expression of ATF3 antagonized TLR-stimulated IL-12p40 activation in a reporter assay. In vivo, CpG-oligodeoxynucleotide, a TLR9 agonist, given i.p. to ATF3-KO mice resulted in enhanced cytokine production from splenocytes. Furthermore, while ATF3-KO mice challenged with a sublethal dose of PR8 influenza virus were delayed in body weight recovery in comparison to wild type, the ATF3-KO mice showed higher titers of serum neutralizing Ab against PR8 5 mo postinfection. Thus, ATF3 behaves as a negative regulatory transcription factor in TLR pathways and, accordingly, deficiency in atf3 alters responses to immunological challenges in vivo. ATF3 dysregulation merits further exploration in diseases such as type I diabetes and cancer, where altered innate immunity has been implicated in their pathogenesis.