Dysfunction in Ataxia-telangiectasia mutated (ATM), a central component of the DNA repair machinery, results in Ataxia Telangiectasia (AT), a cancer-prone disease with a variety of inflammatory ...manifestations. By analyzing AT patient samples and Atm(-/-) mice, we found that unrepaired DNA lesions induce type I interferons (IFNs), resulting in enhanced anti-viral and anti-bacterial responses in Atm(-/-) mice. Priming of the type I interferon system by DNA damage involved release of DNA into the cytoplasm where it activated the cytosolic DNA sensing STING-mediated pathway, which in turn enhanced responses to innate stimuli by activating the expression of Toll-like receptors, RIG-I-like receptors, cytoplasmic DNA sensors, and their downstream signaling partners. This study provides a potential explanation for the inflammatory phenotype of AT patients and establishes damaged DNA as a cell intrinsic danger signal that primes the innate immune system for a rapid and amplified response to microbial and environmental threats.
Localized therapeutic modalities that subvert the tumor microenvironment from immune‐suppressive to pro‐immunogenic can elicit systemic antitumor immune responses that induce regression of directly ...treated as well as nontreated distal tumors. A key toward generating robust antitumor T cell responses is the activation of dendritic cells (DCs) in the tumor microenvironment. Treatment with agonists triggering various pattern recognition receptors is very efficient to activate DCs, yet suffers from the induction of serious immune‐related adverse effects, which is closely linked to their unfavorable PK/PD profile causing systemic immune activation and cytokine release. Here, it is reported that nanoparticle conjugation of a highly potent TLR7/8 agonist restricts immune activation to the tumor bed and its sentinel lymph nodes without hampering therapeutic antitumor efficacy. On a mechanistic level, it is confirmed that localized treatment with a nanoparticle‐conjugated TLR7/8 agonist leads to potent activation of DCs in the sentinel lymph nodes and promotes proliferation of tumor antigen‐specific CD8 T cells. Furthermore, therapeutic improvement upon combination with anti‐PDL1 checkpoint inhibition and Flt3L, a growth factor that expands and mobilizes DCs from the bone marrow, is demonstrated. The findings provide a rational base for localized tumor engineering by nanomedicine strategies that provide spatial control over immune‐activation.
Conjugation of TLR7/8 agonist provides spatial control of immune activation to avoid systemic inflammation. Local immune stimulation allows the generation of tumor‐specific immune responses that show therapeutic improvement in combination with checkpoint inhibitor treatment to combat immune suppression.
Developing safe and efficient non-viral delivery systems remains a major challenge for in vivo applications of gene therapy, especially in cystic fibrosis. Unlike conventional cationic polymers or ...lipids, the emerging poloxamine-based copolymers display promising in vivo gene delivery capabilities. However, poloxamines are invalid for in vitro applications and their in vivo transfection efficiency is still low compared with viral vectors. Here, we show that peptides developed by modular design approaches can spontaneously form compact and monodisperse nanoparticles with poloxamines and nucleic acids via self-assembly. Both messenger RNA and plasmid DNA expression mediated by peptide-poloxamine nanoparticles are greatly boosted in vitro and in the lungs of cystic fibrosis mice with negligible toxicity. Peptide-poloxamine nanoparticles containing integrating vectors enable successful in vitro and in vivo long-term restoration of cystic fibrosis transmembrane conductance regulator deficiency with a safe integration profile. Our dataset provides a new framework for designing non-viral gene delivery systems qualified for in vivo genetic modifications.
Among innovative adjuvants conferring a Th1-shift, RNAdjuvant is a promising candidate. This adjuvant consists of a 547-nt uncapped noncoding ssRNA containing polyU repeats that is stabilized by a ...cationic carrier peptide. Whereas vaccination of mice with an influenza subunit vaccine induced moderate virus-specific IgG1, vaccination together with RNAdjuvant significantly enhanced this IgG1 and additionally promoted the formation of IgG2b/c, which is indicative of Th1 responses. Furthermore, such sera neutralized influenza virus, whereas this effect was not detected upon vaccination with the subunit vaccine alone. Similarly, upon vaccination with virus-like particles displaying vesicular stomatitis virus G protein, RNAdjuvant promoted the formation of virus-specific IgG2b/c and enhanced neutralizing IgG responses to an extent that mice were protected against lethal virus infection. RNAdjuvant induced dendritic cells to upregulate activation markers and produce IFN-I. Although these effects were strictly TLR7 dependent, RNAdjuvant-mediated augmentation of vaccine responses needed concurrent TLR and RIG-I-like helicase signaling. This was indicated by the absence of the adjuvant effect in vaccinated MyD88
Cardif
mice, which are devoid of TLR (with the exception of TLR3) and RIG-I-like helicase signaling, whereas in vaccinated MyD88
mice the adjuvant effect was reduced. Notably, i.m. RNAdjuvant injection induced local IFN-I responses and did not induce systemic effects, implying good tolerability and a favorable safety profile for RNAdjuvant.
Overexpression of C-X-C chemokine receptor type 4 (CXCR4) has been shown in several cancers, including non-small cell lung cancer (NSCLC) and is linked to early metastasis and worse prognosis. The ...crosstalk between cancer cells and tumor stroma promotes the growth and metastasis and CXCR4 signaling is a key element of this crosstalk.
To test the effects of CXCR4 overexpression (CXCR4-OE), we transduced the human NSCLC cell line A549 by using a lentiviral vector. A 3D cell culture model showed generations of tumorspheres and the effects derived by the co-culturing of lung fibroblasts. Using a xenograft mouse model, we also studied the effects of CXCR4-OE in pulmonary cell engraftment and tumor burden in vivo.
Our data indicate that CXCR4-OE leads to increased tumorsphere formation and epithelial-mesenchymal transition (EMT). CXCR4-OE by A549 cells resulted in a significant increase in the production of the CXCR4-ligand macrophage migration inhibitory factor (MIF) compared to those transduced with an empty vector (EV) or in which the CXCR4 expression was deleted (KO). In our in vitro system, we did not detect any production of the canonical CXCR4 ligand CXCL12. Autocrine MIF production and CXCR4 signaling are part of a self-perpetuating loop that amplifies tumor growth and EMT. Co-culture with lung fibroblasts further increased tumorsphere formation, partially driven by an increase in IL-6 production. When A549 cells were injected into murine lungs, we observed more abundant and significantly larger tumor lesions in recipients of CXCR4-OE A549 cells compared to those receiving EV or KO cells, consistent with our in vitro findings. Treatment of mice with the MIF antagonist ISO-1 resulted in significantly less tumor burden.
In conclusion, our data highlight the role of the CXCR4-OE/MIF/IL-6 axis in epithelial mesenchymal crosstalk and NSCLC progression.
•CXCR4 overexpression of the NSCLC line A549 resulted in increased tumorsphere formation•CXCR4 overexpression of A549 induced autocrine MIF but not CXCL12 production•CXCR4 overexpression or stimulation with MIF lead to increased EMT of A549•The MIF antagonist ISO-1 reduced tumor growth in vitro and in vivo•Crosstalk with fibroblasts increased tumorsphere counts and IL-6 production suggesting a CXCR4/MIF/IL-6 amplification loop
Metastases are the major cause of death from cancer. Thus, understanding the regulation of metastatic processes is of utmost importance. Here we show that mice with impaired type I IFN signaling ...(Ifnar1‐/‐) develop more lung metastases in the 4T1 mammary and LLC lung carcinoma model, compared to control mice. In Ifnar1‐/‐ mice, higher metastasis load is accompanied by massive neutrophil accumulation in lungs. Elevated G‐CSF levels in serum and enhanced CXCR2 expression on neutrophils are most likely responsible for this phenomenon. Lung infiltrating neutrophils facilitate an improved pre‐metastatic niche formation, supporting more efficient tumor cell extravasation and proliferation in this organ. This is due to the enhanced expression of pro‐metastatic proteins, like Bv8, MMP9, S100A8 and S100A9. Development of pre‐metastatic niche together with reduced neutrophil cytotoxicity against tumor cells results in enhanced metastatic processes in Ifnar1‐/‐ mice. Overall, our findings describe a novel role for IFN during metastasis development and suggest that new treatment strategies should be considered for prevention of metastasis formation in patients.
What's new?
The mechanisms regulating metastasis formation, whereby cancer cells from primary sites disseminate into target organs to form secondary tumors, remain unclear. Here, the authors show that type I IFNs hamper metastasis formation by inhibiting pre‐metastatic niche development in the lung and augmenting neutrophil cytotoxicity against tumor cells. A general shift of the neutrophil phenotype toward a pro‐metastatic phenotype is observed in absence of type I IFNs. The results confirm and extend the role of the type I IFN system in cancer immune surveillance, and reveal the therapeutic potential of type I IFNs in preventing cancer metastasis after standard surgical therapy.
Toll-like receptors (TLRs) are among the first-line sentinels for immune detection and responsiveness to pathogens. The TLR2 subfamily of TLRs (TLR1, TLR2, TLR6) form heterodimers with each other and ...are thus able to recognize a broad range of components from several microbes such as yeast, Gram-positive bacteria and protozoa. Until now, TLR2 activation by bacterial ligands has long been associated with pro-inflammatory cytokines but not type I interferon responses.
Using a variety of transgenic mice, here we provide in vivo and in vitro data showing that TLR2 activation does in fact induce interferon-beta and that this occurs via MyD88-IRF1 and -IRF7 pathways. Interestingly, by microscopy we demonstrate that although a cell surface receptor, TLR2 dependent induction of type I interferons occurs in endolysosomal compartments where it is translocated to upon ligand engagement. Furthermore, we could show that blocking receptor internalization or endolysosomal acidification inhibits the ability of TLR2 to trigger the induction type I interferon but not pro-inflammatory responses.
The results indicate that TLR2 activation induces pro-inflammatory and type I interferon responses from distinct subcellular sites: the plasma membrane and endolysosomal compartments respectively. Apart from identifying and characterizing a novel pathway for induction of type I interferons, the present study offers new insights into how TLR signaling discriminates and regulates the nature of responses to be elicited against extracellular and endocytosed microbes. These findings may also have clinical implication. Excessive production of pro-inflammatory cytokines and type I IFNs following activation of TLRs is a central pathologic event in several hyper-inflammatory conditions. The discovery that the induction of pro-inflammatory and type I IFN responses can be uncoupled through pharmacological manipulation of endolysosomal acidification suggests new avenues for potential therapeutic intervention against inflammations and sepsis.
Sickness behavior and cognitive dysfunction occur frequently by unknown mechanisms in virus-infected individuals with malignancies treated with type I interferons (IFNs) and in patients with ...autoimmune disorders. We found that during sickness behavior, single-stranded RNA viruses, double-stranded RNA ligands, and IFNs shared pathways involving engagement of melanoma differentiation-associated protein 5 (MDA5), retinoic acid-inducible gene 1 (RIG-I), and mitochondrial antiviral signaling protein (MAVS), and subsequently induced IFN responses specifically in brain endothelia and epithelia of mice. Behavioral alterations were specifically dependent on brain endothelial and epithelial IFN receptor chain 1 (IFNAR). Using gene profiling, we identified that the endothelia-derived chemokine ligand CXCL10 mediated behavioral changes through impairment of synaptic plasticity. These results identified brain endothelial and epithelial cells as natural gatekeepers for virus-induced sickness behavior, demonstrated tissue specific IFNAR engagement, and established the CXCL10-CXCR3 axis as target for the treatment of behavioral changes during virus infection and type I IFN therapy.
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•Viruses induce depressive behavior and ISG15 expression at the blood-brain barrier•IFNAR1 expression on neural cells is not involved in IFN-β-induced sickness behavior•IFNAR1 expression on brain endothelial and epithelial cells drives behavioral changes•Brain endothelia- and epithelia-derived CXCL10 inhibits hippocampal synaptic plasticity
Sickness behavior and cognitive dysfunction occur frequently during RNA virus infection by unknown mechanisms. Prinz and colleagues show that virus-induced sickness behavior is induced by interferon receptor chain 1 (IFNAR1) engagement on brain endothelial and epithelial cells that in turn influence neuronal signaling to drive cognitive impairment and depression-like behavior.
Small-molecular Toll-like receptor 7/8 (TLR7/8) agonists hold promise as immune modulators for a variety of immune therapeutic purposes including cancer therapy or vaccination. However, due to their ...rapid systemic distribution causing difficult-to-control inflammatory off-target effects, their application is still problematic, in particular systemically. To address this problem, we designed and robustly fabricated pH-responsive nanogels serving as versatile immunodrug nanocarriers for safe delivery of TLR7/8-stimulating imidazoquinolines after intravenous administration. To this aim, a primary amine-reactive methacrylamide monomer bearing a pendant squaric ester amide is introduced, which is polymerized under controlled RAFT polymerization conditions. Corresponding PEG-derived squaric ester amide block copolymers self-assemble into precursor micelles in polar protic solvents. Their cores are amine-reactive and can sequentially be transformed by acid-sensitive cross-linkers, dyes, and imidazoquinolines. Remaining squaric ester amides are hydrophilized affording fully hydrophilic nanogels with profound stability in human plasma but stimuli-responsive degradation upon exposure to endolysosomal pH conditions. The immunomodulatory behavior of the imidazoquinolines alone or conjugated to the nanogels was demonstrated by macrophages in vitro. In vivo, however, we observed a remarkable impact of the nanogel: After intravenous injection, a spatially controlled immunostimulatory activity was evident in the spleen, whereas systemic off-target inflammatory responses triggered by the small-molecular imidazoquinoline analogue were absent. These findings underline the potential of squaric ester-based, pH-degradable nanogels as a promising platform to permit intravenous administration routes of small-molecular TLR7/8 agonists and, thus, the opportunity to explore their adjuvant potency for systemic vaccination or cancer immunotherapy purposes.
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