Pilot Clinical Trial to Evaluate In Situ Calcium Phosphate Cement Injection for Conservative Surgical Management of Appendicular Osteosarcoma in Dogs.

Cementoplasty is a minimally invasive procedure that consists of injecting a bone substitute into the tumor lesion to provide bone reinforcement and alleviate pain. This study aimed to demonstrate the feasibility, safety, and efficacy of cementoplasty with a calcium phosphate cement in osteosarcoma to reduce pain and preserve limb function. Throughout the 6-month study, dogs received no adjuvant therapy, and dogs' evaluations included a clinical examination, monitoring of postoperative complications, radiographic follow-up, and assessment of limb function and pain scores. Out of 12 dogs enrolled, 10 were withdrawn before study completion due to deterioration in their general condition. Nine (9) dogs were followed until D28, six until D56, and two until D183. Compared to D0, more than 50% of the dogs showed improvement in both veterinarian and owner scores at their final visit. Throughout the study, 10 major and 4 minor complications were reported, all unrelated to the procedure. This open non-controlled study provides first evidence of the feasibility, safety, and efficacy of cementoplasty procedure using a calcium phosphate bone cement to relieve pain and preserve limb function in dogs suffering from appendicular osteosarcoma.

Activation of the endoplasmic reticulum stress sensor IRE1α by the vaccine adjuvant AS03 contributes to its immunostimulatory properties.

The oil-in-water emulsion Adjuvant System 03 (AS03) is one of the few adjuvants used in licensed vaccines. Previous work indicates that AS03 induces a local and transient inflammatory response that contributes to its adjuvant effect. However, the molecular mechanisms involved in its immunostimulatory properties are ill-defined. Upon intramuscular injection in mice, AS03 elicited a rapid and transient downregulation of lipid metabolism-related genes in the draining lymph node. In vitro, these modifications were associated with profound changes in lipid composition, alteration of endoplasmic reticulum (ER) morphology and activation of the unfolded protein response pathway. In vivo, treatment with a chemical chaperone or deletion of the ER stress sensor kinase IRE1α in myeloid cells decreased AS03-induced cytokine production and its capacity to elicit high affinity antigen-specific antibodies. In summary, our results indicate that IRE1α is a sensor for the metabolic changes induced by AS03 in monocytic cells and may constitute a canonical pathway that could be exploited for the design of novel vaccine adjuvants.

Tristetraprolin regulation of interleukin 23 mRNA stability prevents a spontaneous inflammatory disease.

Interleukin (IL) 12 and IL23 are two related heterodimeric cytokines produced by antigen-presenting cells. The balance between these two cytokines plays a crucial role in the control of Th1/Th17 responses and autoimmune inflammation. Most studies focused on their transcriptional regulation. Herein, we explored the role of the adenine and uridine-rich element (ARE)-binding protein tristetraprolin (TTP) in influencing mRNA stability of IL12p35, IL12/23p40, and IL23p19 subunits. LPS-stimulated bone marrow-derived dendritic cells (BMDCs) from TTP(-/-) mice produced normal levels of IL12/23p40. Production of IL12p70 was modestly increased in these conditions. In contrast, we observed a strong impact of TTP on IL23 production and IL23p19 mRNA stability through several AREs in the 3' untranslated region. TTP(-/-) mice spontaneously develop an inflammatory syndrome characterized by cachexia, myeloid hyperplasia, dermatitis, and erosive arthritis. We observed IL23p19 expression within skin lesions associated with exacerbated IL17A and IL22 production by infiltrating γδ T cells and draining lymph node CD4 T cells. We demonstrate that the clinical and immunological parameters associated with TTP deficiency were completely dependent on the IL23-IL17A axis. We conclude that tight control of IL23 mRNA stability by TTP is critical to avoid severe inflammation.

Interferon regulatory factor 3 controls interleukin-17 expression in CD8 T lymphocytes.

IFN regulatory factor (IRF) 3 plays a key role in innate responses against viruses. Herein we assessed its contribution to T-cell activation. We observed that poly(I:C)-induced IRF3 activation in CD8 T cells represses IL-17 expression in a type I IFN-independent fashion. Even in the absence of poly(I:C), polyclonally activated naïve IRF3(-/-) CD8 T cells expressed high levels of IL-17 and IL-23R in comparison with wild-type cells. Furthermore, IRF3(-/-) OT1 cells adoptively transferred into wild-type hosts also produced higher IL-17 levels upon immunization than their wild-type counterparts. This phenotype could be reversed by ectopic expression of IRF3, confirming that this effect is intrinsic to T cells. We show that IRF3 directly interacts with RORγt in the cytoplasm through its IRF interaction domain and limits its ability to bind and transactivate the IL-17 promoter. These observations uncover an unexpected role of IRF3 in the control of CD8 T-cell polarization.

A conventional protein kinase C inhibitor targeting IRF-3-dependent genes differentially regulates IL-12 family members.

Protein kinase C (PKC) isoforms play a critical role in the regulation of innate immune responses. We have previously demonstrated that conventional PKC (cPKC) α is involved in interferon regulatory factor 3 (IRF-3) activation and IFN-ß synthesis. Herein, we investigated the role of cPKCs in the regulation of IL-12 family members expression mediated by the Toll-like receptor 3 (TLR3) and TLR4. First, inhibition of cPKCs activity in human DCs by a cPKC-specific inhibitor, Gö6976 downregulated the expression of IL-12p70 and IL-27p28 but not IL-12/IL-23p40, IL-23, IL-27EBI3 induced by LPS or poly(I:C). Furthermore, reporter gene assays in RAW 264.7 macrophages showed that cPKCs regulate IL-12p35 and IL-27p28 promoter activities since Gö6976 repressed LPS and poly(I:C)-mediated transcriptional activities of IL-12p35 and IL-27p28. In contrast, no effect was observed with IL-12/IL-23p40 and IL-23p19 reporter constructs. These results prompted us to study the role of IRF-3 on IL-23 expression. Bone marrow-derived DC (BMDCs) from IRF-3(-/-) mice produced comparable levels of IL-23 induced by both LPS and poly(I:C) as compared to wild type BMDCs, indicating that IRF-3 is not involved in IL-23 production. Finally, BMDCs from PKCα(-/-) mice displayed a reduced synthesis of IL-27 induced by poly(I:C). Collectively, these data identify cPKCs as critical components that control IRF-3-dependent IL-12p35 and IL-27p28 gene expression downstream of TLR3 and TLR4.

Critical role of the IFN-stimulated gene factor 3 complex in TLR-mediated IL-27p28 gene expression revealing a two-step activation process.

In myeloid dendritic cells, activation of the IL-27p28 gene is selectively induced by ligands of TLR4 or TLR3, both coupled to the Toll/IL-1R-related domain-containing adaptor-inducing IFN/IFN regulatory factor (IRF)3 pathway. In response to both ligands, autocrine type 1 IFN production was required for optimal IL-27p28 expression. Type I IFN signaling was necessary for sustained IRF1 activation and formation of the IRF9-containing IFN-stimulated gene factor 3 complex. Indeed, we demonstrated that IRF1 and IRF9 are sequentially activated and recruited to the IL-27p28 IFN-stimulated regulatory element site. Involvement of IRF1 and IRF9 in the induction of IL-27p28 was confirmed in vitro and upon in vivo exposure to TLR ligands. Thus, in response to TLR4 or TLR3 ligation, the initial induction of the IL-27p28 gene depends on the recruitment of IRF1 and IRF3, whereas transcriptional amplification requires recruitment of the IFN-stimulated gene factor 3 complex. These results highlight the complex molecular interplay between TLRs and type I IFNs for the control of IL-27 synthesis.

IL-27 synthesis induced by TLR ligation critically depends on IFN regulatory factor 3.

IL-27 is a heterodimeric cytokine composed of EBV-induced gene 3 and p28. Produced by dendritic cells (DCs) in response to TLR ligands, IL-27 recently emerged as a key regulator of inflammatory responses. In this study, we first demonstrate that Toll/IL-1R-containing adaptor inducing IFN-beta and its associated IFN regulatory factor (IRF) 3 transcription factor are critically involved in IL-27p28 expression in mouse DCs stimulated by TLR ligands. We then show that IL-27 serum levels are dramatically reduced in IRF3(-/-) upon LPS injection, indicating a critical role for IRF3 in TLR4-mediated IL-27 production in vivo. We identified an IRF3-binding site within the IL-27p28 promoter region which is required for IL-27p28 gene activation in reporter gene assays. In human DCs, IL-27p28 mRNA was preferentially induced by Toll/IL-1R-containing adaptor inducing IFN-beta-coupled TLR ligands and following CMV infection. Furthermore, chromatin immunoprecipitation studies demonstrate that IRF3 is recruited to the endogenous p28 promoter in TLR4-stimulated human DCs. We conclude that IRF3 activation is a master switch for IL-27 synthesis.

Interferon regulatory factor 3 is involved in Toll-like receptor 4 (TLR4)- and TLR3-induced IL-12p35 gene activation.

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by dendritic cells (DCs) in response to Toll-like receptor (TLR) ligation. While the mechanisms regulating IL-12p40 chain gene expression are well characterized, molecular events involved in IL-12p35 chain gene activation remain to be clarified. Since IL-12p35 mRNA was induced in human DCs activated through TLR3 or TLR4 but not TLR2, we investigated the potential role of interferon regulatory factor 3 (IRF-3) in IL-12p35 gene transactivation. First, a binding site for IRF-3 named interferon-stimulated response element-1 (ISRE-1) was identified in the human IL-12p35 promoter region between nucleotides -251 and -240. The ISRE-1 site was required for IL-12p35 gene activation in RAW 264.7 cells stimulated by lipopolysaccharide (LPS) or PolyI:C. Ectopic expression of IRF-3 was found to up-regulate IL-12p35 gene activation in the same system. Furthermore, chromatin immunoprecipitation (ChIP) studies demonstrated that IRF-3 is recruited to ISRE-1 site in TLR4- or TLR3-stimulated human DCs. Finally, experiments on DCs from IRF-3-deficient mice established that TLR4-induced IL-12p35 mRNA and IL-12p70 synthesis are impaired in absence of IRF-3. We conclude that IRF-3 binds to a critical cis-acting element in the IL-12p35 gene promoter and thereby represents a key factor for the induction of IL-12p70 synthesis in DCs.