The DNA Sensor, Cyclic GMP–AMP Synthase, Is Essential for Induction of IFN-β during Chlamydia trachomatis Infection

IFNβ has been implicated as an effector of oviduct pathology resulting from genital chlamydial infection in the mouse model. In this study, we investigated the role of cytosolic DNA and engagement of DNA sensors in IFNβ expression during chlamydial infection. We determined that TREX-1, a host 3’to 5’ exonuclease, reduced IFNβ expression significantly during chlamydial infection using siRNA and gene knock out fibroblasts, implicating cytosolic DNA as a ligand for this response. The DNA sensor cGAS has been shown to bind cytosolic DNA to generate cGAMP, which binds to the signaling adaptor STING to induce IFNβ expression. We determined that cGAS is required for IFNβ expression during chlamydial infection in multiple cell types. Interestingly, although infected cells deficient for STING or cGAS alone failed to induce IFNβ, co-culture of cells depleted for either STING or cGAS rescued IFNβ expression. These data demonstrate that cGAMP produced in infected cGAS+STING− cells can migrate into adjacent cells via gap junctions to function in trans in cGAS−STING+ cells. Further, we observed cGAS localized in punctate regions on the cytosolic side of the chlamydial inclusion membrane in association with STING, indicating that chlamydial DNA is likely recognized outside the inclusion as infection progresses. These novel findings provide evidence that cGAS-mediated-DNA sensing directs IFNβ expression during C.trachomatis infection and suggests that effectors from infected cells can directly upregulate IFNβ expression in adjacent uninfected cells during in vivo infection, contributing to pathogenesis

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Cytoplasmic recognition of Chlamydia muridarum: Beyond toll -like receptors

Fallopian tube pathology resulting from infection with the obligate intracellular human pathogen Chlamydia trachomatis has been proposed to be caused by excessive host cytokine production. Using the mouse chlamydiae Chlamydia muridarum, this study was undertaken to better understand the mechanistic basis for production of the pathology associated cytokines IFN-P and IL-1 p. Although cytosolic pathogen recognition receptors are crucial for expression of both cytokines, the chlamydial contributions and host proteins involved are distinct. Using a combination of knockout macrophages and RNAi mediated gene silencing, it was determined that IFN-β expression was TLR independent, requiring chlamydial growth, signaling through the ER protein STING, and the transcription factor IRF3. Cytosolic NOD1 also contributed to maximal IFN-G3, likely by activation of the transcription factor NF-κB and p38 MAPK signaling. The role of IRF3, the key transcription factor in IFN-β induction, was further examined during an in vivo genital infection. In contrast to IFN-β, activation of IRF3 is ultimately beneficial to the host as its absence leads to exacerbated uterine horn pathology during infection of the mouse female genital tract, suggesting an IFN-β independent role for IRF3. In contrast to IFN-β, IL-1β secretion in primed macrophages was independent of chlamydial entry and growth. Using gene knockout mouse macrophages, it was determined that C. muridarum was capable of activating the inflammasome for subsequent IL-1β secretion via multiple pathways utilizing the adaptor protein ASC, including cryopyrin and a putative pyrin domain containing protein(s). To further understand the bacterial contribution in host cytokine induction, the role of the chlamydial Type III secretion (T3S) apparatus in the expression of IL-1β and IFN-β was addressed. Administration of a T3S antagonist during infection decreased expression of IFN-β, cxc110, and IL-6. In contrast, IL-1β secretion occurred independently of the T3S rod sensing inflammasome protein IPAF. However the fact that T3S is present on the chlamydial elementary body in the absence of de novo protein synthesis, suggests it could still play a role, possibly by an alternate T3S dependent inflammasome. These latter studies illustrate that T3S could be a unifying concept in activation of the cytosolic pathways required for IFN-β and IL-1β expression

Anti-Mesothelin Nanobodies for Both Conventional and Nanoparticle-Based Biomedical Applications

International audienc

Anti-Mesothelin Nanobodies for Both Conventional and Nanoparticle-Based Biomedical Applications

International audienc

The \u3b8-defensin retrocyclin 101 inhibits TLR4- and TLR2-dependent signaling and protects mice against influenza infection

Despite widespread use of annual influenza vaccines, seasonal influenza-associated deaths number in the thousands each year, in part because of exacerbating bacterial superinfections. Therefore, discovering additional therapeutic options would be a valuable aid to public health. Recently, TLR4 inhibition has emerged as a possible mechanism for protection against influenza-associated lethality and acute lung injury. Based on recent data showing that rhesus macaque \u3b8-defensins could inhibit TLR4-dependent gene expression, we tested the hypothesis that a novel \u3b8-defensin, retrocyclin (RC)-101, could disrupt TLR4-dependent signaling and protect against viral infection. In this study, RC-101, a variant of the humanized \u3b8-defensin RC-1, blocked TLR4-mediated gene expression in mouse and human macrophages in response to LPS, targeting both MyD88- and TRIF-dependent pathways. In a cell-free assay, RC-101 neutralized the biologic activity of LPS at doses ranging from 0.5 to 50 EU/ml, consistent with data showing that RC-101 binds biotinylated LPS. The action of RC-101 was not limited to the TLR4 pathway because RC-101 treatment of macrophages also inhibited gene expression in response to a TLR2 agonist, Pam3CSK4, but failed to bind that biotinylated agonist. Mouse macrophages infected in vitro with mouse-adapted A/PR/8/34 influenza A virus (PR8) also produced lower levels of proinflammatory cytokine gene products in a TLR4-independent fashion when treated with RC-101. Finally, RC-101 decreased both the lethality and clinical severity associated with PR8 infection in mice. Cumulatively, our data demonstrate that RC-101 exhibits therapeutic potential for the mitigation of influenza-related morbidity and mortality, potentially acting through TLR-dependent and TLR-independent mechanisms

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) activates stimulator of interferon gene (STING)-dependent innate immune pathways and is regulated by mitochondrial membrane potential

The chemotherapeutic agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a potent inducer of type I IFNs and other cytokines. This ability is essential for its chemotherapeutic benefit in a mouse cancer model and suggests that it might also be useful as an antiviral agent. However, the mechanism underlying DMXAA-induced type I IFNs, including the host proteins involved, remains unclear. Recently, it was reported that the antioxidant N-acetylcysteine (NAC) decreased DMXAA-induced TNF-alpha and IL-6, suggesting that oxidative stress may play a role. The goal of this study was to identify host proteins involved in DMXAA-dependent signaling and determine how antioxidants modulate this response. We found that expression of IFN-beta in response to DMXAA in mouse macrophages requires the mitochondrial and endoplasmic reticulum resident protein STING. Addition of the antioxidant diphenylene iodonium (DPI) diminished DMXAA-induced IFN-beta, but this decrease was independent of both the NADPH oxidase, Nox2, and de novo generation of reactive oxygen species. Additionally, IFN-beta up-regulation by DMXAA was inhibited by agents that target the mitochondrial electron transport chain and, conversely, loss of mitochondrial membrane potential correlated with diminished innate immune signaling in response to DMXAA. Up-regulation of Ifnb1 gene expression mediated by cyclic dinucleotides was also impaired by DPI, whereas up-regulation of Ifnb1 mRNA due to cytosolic double-stranded DNA was not. Although both stimuli signal through STING, cyclic dinucleotides interact directly with STING, suggesting that recognition of DMXAA by STING may also be mediated by direct interaction