Endothelial cell expression of a STING gain-of-function mutation initiates pulmonary lymphocytic infiltration.

Patients afflicted with Stimulator of interferon gene (STING) gain-of-function mutations frequently present with debilitating interstitial lung disease (ILD) that is recapitulated in mice expressing the STINGV154M mutation (VM). Prior radiation chimera studies revealed an unexpected and critical role for non-hematopoietic cells in initiating ILD. To identify STING-expressing non-hematopoietic cell types required for the development of ILD, we use a conditional knockin (CKI) model and direct expression of the VM allele to hematopoietic cells, fibroblasts, epithelial cells, or endothelial cells. Only endothelial cell-targeted VM expression results in enhanced recruitment of immune cells to the lung associated with elevated chemokine expression and the formation of bronchus-associated lymphoid tissue, as seen in the parental VM strain. These findings reveal the importance of endothelial cells as instigators of STING-driven lung disease and suggest that therapeutic targeting of STING inhibitors to endothelial cells could potentially mitigate inflammation in the lungs of STING-associated vasculopathy with onset in infancy (SAVI) patients or patients afflicted with other ILD-related disorders.

Activation of Autoreactive Lymphocytes in the Lung by STING Gain-of-function Mutation Radioresistant Cells.

Gain-of-function mutations in the dsDNA sensing adaptor STING lead to a severe autoinflammatory syndrome known as STING-associated vasculopathy with onset in Infancy (SAVI). SAVI patients develop interstitial lung disease (ILD) and commonly produce anti-nuclear antibodies (ANAs), indicative of concomitant autoimmunity. Mice heterozygous for the most common SAVI mutation, V154M (VM), also develop ILD, triggered by nonhematopoietic VM cells, but exhibit severe peripheral lymphopenia, low serum Ig titers and fail to produce autoantibodies. In contrast, we now show that lethally irradiated VM mice reconstituted with WT stem cells (WT→VM chimeras) develop ANAs and lung-reactive autoantibodies associated with accumulation of activated lymphocytes and formation of germinal centers in lung tissues. Moreover, when splenocytes from WT→VM chimeras were adoptively transferred into unmanipulated Rag1 -/- mice, donor T cells accumulated in the lung. Overall, these findings demonstrate that expression of the VM mutation in non-hematopoietic cells can promote the activation of immunocompetent autoreactive lymphocytes. Summary: Chimeric mice expressing STING only in non-hematopoietic cells develop systemic and lung directed autoimmunity which recapitulates what is seen in pediatric patients with SAVI disease.

Expression of a STING Gain-of-function Mutation in Endothelial Cells Initiates Lymphocytic Infiltration of the Lungs.

Patients afflicted with STING gain-of-function mutations frequently present with debilitating interstitial lung disease ( ILD ) that is recapitulated in mice expressing the STING V154M mutation ( VM ). Prior radiation chimera studies revealed an unexpected and critical role for non-hematopoietic cells in the initiation of ILD. To identify STING-expressing non-hematopoietic cell types relevant to ILD, we generated a conditional knock-in ( CKI ) model in which expression of the VM allele was directed to hematopoietic cells, fibroblasts, epithelial cells, or endothelial cells. Only endothelial cell-targeted expression of the mutant allele resulted in the recruitment of immune cells to the lung and the formation of bronchus-associated lymphoid tissue, as seen in the parental VM strain. These findings reveal the importance of endothelial cells as instigators of STING-driven lung disease and suggest that therapeutic targeting of STING inhibitors to endothelial cells could potentially mitigate inflammation in the lungs of SAVI patients or patients afflicted with other ILD-related disorders. Summary: Patients with STING gain-of-function (GOF) mutations develop life-threatening lung autoinflammation. In this study, Gao et al. utilize a mouse model of conditional STING GOF to demonstrate a role for endothelial STING GOF in initiating immune cell recruitment into lung tissues of SAVI mice.

Type-1 interferon-dependent and -independent mechanisms in cyclic GMP-AMP synthase-stimulator of interferon genes-driven auto-inflammation.

The cyclic cyclic gaunosine monophosphate adenosine monophosphate (GMP-AMP) synthase-stimulator of interferon genes (cGAS-STING) pathway senses cytosolic dsDNA and initiates immune responses against pathogens. It is also implicated in several auto-inflammatory diseases known as monogenic interferonopathies, specifically Three prime repair exonuclease 1 (Trex1) loss-of-function (LOF), Dnase2 LOF, and stimulator of interferon genes-associated-vasculopathy-with-onset-in-infancy (SAVI). Although monogenic interferonopathies have diverse clinical presentations, they are distinguished by the elevation of type-1 interferons (T1IFNs). However, animal models have demonstrated that T1IFNs contribute to only some disease outcomes and that cGAS-STING activation also promotes T1IFN-independent pathology. For example, while T1IFNs drive the immunopathology associated with Trex1 LOF, disease in Dnase2 LOF is partially independent of T1IFNs, while disease in SAVI appears to occur entirely independent of T1IFNs. Additionally, while the cGAS-STING pathway is well characterized in hematopoietic cells, these animal models point to important roles for STING activity in nonhematopoietic cells in disease. Together, these models illustrate the complex role that cGAS-STING-driven responses play in the pathogenesis of inflammatory diseases across tissues.

Human nasal wash RNA-Seq reveals distinct cell-specific innate immune responses in influenza versus SARS-CoV-2.

BACKGROUNDInfluenza A virus (IAV) and SARS-CoV-2 are pandemic viruses causing millions of deaths, yet their clinical manifestations are distinctly different.METHODSWith the hypothesis that upper airway immune and epithelial cell responses are also distinct, we performed single-cell RNA sequencing (scRNA-Seq) on nasal wash cells freshly collected from adults with either acute COVID-19 or influenza or from healthy controls. We focused on major cell types and subtypes in a subset of donor samples.ResultsNasal wash cells were enriched for macrophages and neutrophils for both individuals with influenza and those with COVID-19 compared with healthy controls. Hillock-like epithelial cells, M2-like macrophages, and age-dependent B cells were enriched in COVID-19 samples. A global decrease in IFN-associated transcripts in neutrophils, macrophages, and epithelial cells was apparent in COVID-19 samples compared with influenza samples. The innate immune response to SARS-CoV-2 appears to be maintained in macrophages, despite evidence for limited epithelial cell immune sensing. Cell-to-cell interaction analyses revealed a decrease in epithelial cell interactions in COVID-19 and highlighted differences in macrophage-macrophage interactions for COVID-19 and influenza.ConclusionsOur study demonstrates that scRNA-Seq can define host and viral transcriptional activity at the site of infection and reveal distinct local epithelial and immune cell responses for COVID-19 and influenza that may contribute to their divergent disease courses.FundingMassachusetts Consortium on Pathogen Readiness, the Mathers Foundation, and the Department of Defense (W81XWH2110029) "COVID-19 Expansion for AIRe Program."

cGAS-STING Pathway Does Not Promote Autoimmunity in Murine Models of SLE.

Detection of DNA is an important determinant of host-defense but also a driver of autoinflammatory and autoimmune diseases. Failure to degrade self-DNA in DNAseII or III(TREX1)-deficient mice results in activation of the cGAS-STING pathway. Deficiency of cGAS or STING in these models ameliorates disease manifestations. However, the contribution of the cGAS-STING pathway, relative to endosomal TLRs, in systemic lupus erythematosus (SLE) is controversial. In fact, STING deficiency failed to rescue, and actually exacerbated, disease manifestations in Fas-deficient SLE-prone mice. We have now extended these observations to a chronic model of SLE induced by the i.p. injection of TMPD (pristane). We found that both cGAS- and STING-deficiency not only failed to rescue mice from TMPD-induced SLE, but resulted in increased autoantibody production and higher proteinuria levels compared to cGAS STING sufficient mice. Further, we generated cGASKOFaslpr mice on a pure MRL/Faslpr background using Crispr/Cas9 and found slightly exacerbated, and not attenuated, disease. We hypothesized that the cGAS-STING pathway constrains TLR activation, and thereby limits autoimmune manifestations in these two models. Consistent with this premise, mice lacking cGAS and Unc93B1 or STING and Unc93B1 developed minimal systemic autoimmunity as compared to cGAS or STING single knock out animals. Nevertheless, TMPD-driven lupus in B6 mice was abrogated upon AAV-delivery of DNAse I, implicating a DNA trigger. Overall, this study demonstrated that the cGAS-STING pathway does not promote systemic autoimmunity in murine models of SLE. These data have important implications for cGAS-STING-directed therapies being developed for the treatment of systemic autoimmunity.

The FasLane to ocular pathology-metalloproteinase cleavage of membrane-bound FasL determines FasL function.

Fas ligand (FasL) is best known for its ability to induce cell death in a wide range of Fas-expressing targets and to limit inflammation in immunoprivileged sites such as the eye. In addition, the ability of FasL to induce a much more extensive list of outcomes is being increasingly explored and accepted. These outcomes include the induction of proinflammatory cytokine production, T cell activation, and cell motility. However, the distinct and opposing functions of membrane-associated FasL (mFasL) and the C-terminal soluble FasL fragment (sFasL) released by metalloproteinase cleavage is less well documented and understood. Both mFasL and sFasL can form trimers that engage the trimeric Fas receptor, but only mFasL can form a multimeric complex in lipid rafts to trigger apoptosis and inflammation. By contrast, a number of reports have now documented the anti-apoptotic and anti-inflammatory activity of sFasL, pointing to a critical regulatory function of the soluble molecule. The immunomodulatory activity of FasL is particularly evident in ocular pathology where elimination of the metalloproteinase cleavage site and the ensuing increased expression of mFasL can severely exacerbate the extent of inflammation and cell death. By contrast, both homeostatic and increased expression of sFasL can limit inflammation and cell death. The mechanism(s) responsible for the protective activity of sFasL are discussed but remain controversial. Nevertheless, it will be important to consider therapeutic applications of sFasL for the treatment of ocular diseases such as glaucoma.

A small peptide antagonist of the Fas receptor inhibits neuroinflammation and prevents axon degeneration and retinal ganglion cell death in an inducible mouse model of glaucoma.

BACKGROUND: Glaucoma is a complex, multifactorial disease where apoptosis, microglia activation, and inflammation have been linked to the death of retinal ganglion cells (RGCs) and axon degeneration. We demonstrated previously that FasL-Fas signaling was required for axon degeneration and death of RGCs in chronic and inducible mouse models of glaucoma and that Fas activation triggered RGC apoptosis, glial activation, and inflammation. Here, we investigated whether targeting the Fas receptor with a small peptide antagonist, ONL1204, has anti-inflammatory and neuroprotective effects in a microbead-induced mouse model of glaucoma. METHODS: Intracameral injection of microbeads was used to elevate intraocular pressure (IOP) in Fas-deficient (Faslpr) mice and WT C57BL/6J mice that received an intravitreal injection of the Fas inhibitor, ONL1204 (2 µg/1 µl) (or vehicle only), on day 0 or day 7 after microbead injection. The IOP was monitored by rebound tonometry, and at 28 days post-microbead injection, Brn3a-stained RGCs and paraphenylenediamine (PPD)-stained axons were analyzed. The effects of ONL1204 on retinal microglia activation and the expression of inflammatory genes were analyzed by immunostaining of retinal flatmounts and quantitative PCR (qPCR). RESULTS: Rebound tonometry showed equivalent elevation of IOP in all groups of microbead-injected mice. At 28 days post-microbead injection, the RGC and axon counts from microbead-injected Faslpr mice were equivalent to saline-injected (no IOP elevation) controls. Treatment with ONL1204 also significantly reduced RGC death and loss of axons in microbead-injected WT mice when compared to vehicle-treated controls, even when administered after IOP elevation. Confocal analysis of Iba1-stained retinal flatmounts and qPCR demonstrated that ONL1204 also abrogated microglia activation and inhibited the induction of multiple genes implicated in glaucoma, including cytokines and chemokines (GFAP, Caspase-8, TNFα, IL-1ß, IL-6, IL-18, MIP-1α, MIP-1ß, MIP-2, MCPI, and IP10), components of the complement cascade (C3, C1Q), Toll-like receptor pathway (TLR4), and inflammasome pathway (NLRP3). CONCLUSIONS: These results serve as proof-of-principal that the small peptide inhibitor of the Fas receptor, ONL1204, can provide robust neuroprotection in an inducible mouse model of glaucoma, even when administered after IOP elevation. Moreover, Fas signaling contributes to the pathogenesis of glaucoma through activation of both apoptotic and inflammatory pathways.

Soluble Fas ligand blocks destructive corneal inflammation in mouse models of corneal epithelial debridement and LPS induced keratitis.

Neutrophil-mediated inflammation plays a critical role in corneal damage following injury or infection. Previous studies demonstrated that membrane-bound FasL (mFasL) induces neutrophil chemokine production. However, the extracellular domain of mFasL is normally cleaved by matrix metalloproteinases to release a soluble form of FasL (sFasL) and sFasL antagonizes mFasL-mediated chemokine production. Therefore, we hypothesized that sFasL could be used to prevent neutrophil-mediated corneal inflammation associated with injury and bacterial keratitis. To test this hypothesis, GFP-only, sFasL-GFP, or mFasL-GFP were expressed in the corneal stroma of C57BL/6 mice, using intra-stromal injections of plasmid DNA or adenoviral vectors (AV) and the role of mFasL and sFasL in corneal inflammation was examined in models of corneal injury and LPS-induced keratitis. Our work addresses an important area of disagreement in the field of FasL, with regard to the mechanism by which sFasL regulates ocular inflammation. Herein, we demonstrate that an intrastromal injection of GFP-only, sFasL-GFP, or mFasL-GFP plasmid DNA resulted in GFP expression throughout the corneal stroma for up to two weeks with little to no evidence of inflammation in the GFP-only and sFasL-GFP groups and mild corneal inflammation in the mFasL-GFP group. Similarly, following epithelial debridement, corneas expressing GFP-only or sFasL-GFP showed no significant signs of corneal inflammation, with clear corneas at 15 days post debridement. By contrast, epithelial debridement of corneas expressing mFasL-GFP triggered persistent corneal inflammation and the development of central corneal opacities that was blocked by sFasL. Similar to the mFasL-GFP plasmid DNA, intrastromal injection of mFasL-GFP AV triggered mild corneal inflammation, but it was transient and resolved by day 10 with corneas remaining clear out to 30 days post injection. Nevertheless, intrastromal expression of mFasL-GFP AV exacerbated LPS-induced keratitis, corneal opacity, and neovascularization, while sFasL-GFP AV expression prevented LPS-induced keratitis, resulting in a clear cornea. Histological analysis of corneas with LPS-induced keratitis revealed a robust infiltration of macrophages and neutrophils and sFasL expression specifically blocked the neutrophil influx. Overall, our data demonstrate that stromal expression of mFasL is inflammatory, while sFasL is non-inflammatory, and opposes the effects of mFasL in mouse models of epithelial debridement and LPS-induced keratitis. These data demonstrate that a delicate balance between sFasL and mFasL regulates ocular inflammation. This study further identifies sFasL as a potent inhibitor of neutrophil-mediated corneal damage, and supports the potential use of sFasL in the treatment of neutrophil-mediated keratitis. These results strongly support the hypothesis that, in the immune privileged environment of the eye, the isoform of FasL regulates immune privilege and determines the extent of inflammation: mFasL promotes inflammation and sFasL blocks inflammation.

Fas ligand promotes an inducible TLR-dependent model of cutaneous lupus-like inflammation.

Toll-like receptors TLR7 and TLR9 are both implicated in the activation of autoreactive B cells and other cell types associated with systemic lupus erythematosus (SLE) pathogenesis. However, Tlr9-/- autoimmune-prone strains paradoxically develop more severe disease. We have now leveraged the negative regulatory role of TLR9 to develop an inducible rapid-onset murine model of systemic autoimmunity that depends on T cell detection of a membrane-bound OVA fusion protein expressed by MHC class II+ cells, expression of TLR7, expression of the type I IFN receptor, and loss of expression of TLR9. These mice are distinguished by a high frequency of OVA-specific Tbet+, IFN-γ+, and FasL-expressing Th1 cells as well as autoantibody-producing B cells. Unexpectedly, contrary to what occurs in most models of SLE, they also developed skin lesions that are very similar to those of human cutaneous lupus erythematosus (CLE) as far as clinical appearance, histological changes, and gene expression. FasL was a key effector mechanism in the skin, as the transfer of FasL-deficient DO11gld T cells completely failed to elicit overt skin lesions. FasL was also upregulated in human CLE biopsies. Overall, our model provides a relevant system for exploring the pathophysiology of CLE as well as the negative regulatory role of TLR9.

The Chaperone UNC93B1 Regulates Toll-like Receptor Stability Independently of Endosomal TLR Transport.

Unc-93 homolog B1 (UNC93B1) is a key regulator of nucleic acid (NA)-sensing Toll-like receptors (TLRs). Loss of NA-sensing TLR responses in UNC93B1-deficient patients facilitates Herpes simplex virus type 1 (HSV-1) encephalitis. UNC93B1 is thought to guide NA-sensing TLRs from the endoplasmic reticulum (ER) to their respective endosomal signaling compartments and to guide the flagellin receptor TLR5 to the cell surface, raising the question of how UNC93B1 mediates differential TLR trafficking. Here, we report that UNC93B1 regulates a step upstream of the differential TLR trafficking process. We discovered that UNC93B1 deficiency resulted in near-complete loss of TLR3 and TLR7 proteins in primary splenic mouse dendritic cells and macrophages, showing that UNC93B1 is critical for maintaining TLR expression. Notably, expression of an ER-retained UNC93B1 version was sufficient to stabilize TLRs and largely restore endosomal TLR trafficking and activity. These data are critical for an understanding of how UNC93B1 can regulate the function of a broad subset of TLRs.

Dendritic Cell RIPK1 Maintains Immune Homeostasis by Preventing Inflammation and Autoimmunity.

Necroptosis is a form of cell death associated with inflammation; however, the biological consequences of chronic necroptosis are unknown. Necroptosis is mediated by RIPK1, RIPK3, and MLKL kinases but in hematopoietic cells RIPK1 has anti-inflammatory roles and functions to prevent necroptosis. Here we interrogate the consequences of chronic necroptosis on immune homeostasis by deleting Ripk1 in mouse dendritic cells. We demonstrate that deregulated necroptosis results in systemic inflammation, tissue fibrosis, and autoimmunity. We show that inflammation and autoimmunity are prevented upon expression of kinase inactive RIPK1 or deletion of RIPK3 or MLKL. We provide evidence that the inflammation is not driven by microbial ligands, but depends on the release of danger-associated molecular patterns and MyD88-dependent signaling. Importantly, although the inflammation is independent of type I IFN and the nucleic acid sensing TLRs, blocking these pathways rescues the autoimmunity. These mouse genetic studies reveal that chronic necroptosis may underlie human fibrotic and autoimmune disorders.

A TLR9-dependent checkpoint governs B cell responses to DNA-containing antigens.

Mature B cell pools retain a substantial proportion of polyreactive and self-reactive clonotypes, suggesting that activation checkpoints exist to reduce the initiation of autoreactive B cell responses. Here, we have described a relationship among the B cell receptor (BCR), TLR9, and cytokine signals that regulate B cell responses to DNA-containing antigens. In both mouse and human B cells, BCR ligands that deliver a TLR9 agonist induce an initial proliferative burst that is followed by apoptotic death. The latter mechanism involves p38-dependent G1 cell-cycle arrest and subsequent intrinsic mitochondrial apoptosis and is shared by all preimmune murine B cell subsets and CD27- human B cells. Survival or costimulatory signals rescue B cells from this fate, but the outcome varies depending on the signals involved. B lymphocyte stimulator (BLyS) engenders survival and antibody secretion, whereas CD40 costimulation with IL-21 or IFN-γ promotes a T-bet+ B cell phenotype. Finally, in vivo immunization studies revealed that when protein antigens are conjugated with DNA, the humoral immune response is blunted and acquires features associated with T-bet+ B cell differentiation. We propose that this mechanism integrating BCR, TLR9, and cytokine signals provides a peripheral checkpoint for DNA-containing antigens that, if circumvented by survival and differentiative cues, yields B cells with the autoimmune-associated T-bet+ phenotype.

Taking the STING out of TLR-driven autoimmune diseases: good, bad, or indifferent?

Both endosomal and cytosolic-nucleic acid-sensing receptors can detect endogenous ligands and promote autoimmunity and autoinflammation. These responses involve a complex interplay among and between the cytosolic and endosomal sensors involving both hematopoietic and radioresistant cells. Cytosolic sensors directly promote inflammatory responses through the production of type I IFNs and proinflammatory cytokines. Inflammation-associated tissue damage can further promote autoimmune responses indirectly, as receptor-mediated internalization of the resulting cell debris can activate endosomal Toll-like receptors (TLR). Both endosomal and cytosolic receptors can also negatively regulate inflammatory responses. A better understanding of the factors and pathways that promote and constrain autoimmune diseases will have important implications for the development of agonists and antagonists that modulate these pathways.

Overexpression of Soluble Fas Ligand following Adeno-Associated Virus Gene Therapy Prevents Retinal Ganglion Cell Death in Chronic and Acute Murine Models of Glaucoma.

Glaucoma is a multifactorial disease resulting in the death of retinal ganglion cells (RGCs) and irreversible blindness. Glaucoma-associated RGC death depends on the proapoptotic and proinflammatory activity of membrane-bound Fas ligand (mFasL). In contrast to mFasL, the natural cleavage product, soluble Fas ligand (sFasL) inhibits mFasL-mediated apoptosis and inflammation and, therefore, is an mFasL antagonist. DBA/2J mice spontaneously develop glaucoma and, predictably, RGC destruction is exacerbated by expression of a mutated membrane-only FasL gene that lacks the extracellular cleavage site. Remarkably, one-time intraocular adeno-associated virus-mediated gene delivery of sFasL provides complete and sustained neuroprotection in the chronic DBA/2J and acute microbead-induced models of glaucoma, even in the presence of elevated intraocular pressure. This protection correlated with inhibition of glial activation, reduced production of TNF-α, and decreased apoptosis of RGCs and loss of axons. These data indicate that cleavage of FasL under homeostatic conditions, and the ensuing release of sFasL, normally limits the neurodestructive activity of FasL. The data further support the notion that sFasL, and not mFasL, contributes to the immune-privileged status of the eye.

Synergy between Hematopoietic and Radioresistant Stromal Cells Is Required for Autoimmune Manifestations of DNase II-/-IFNaR-/- Mice.

Detection of endogenous nucleic acids by cytosolic receptors, dependent on STING, and endosomal sensors, dependent on Unc93b1, can provoke inflammatory responses that contribute to a variety of autoimmune and autoinflammatory diseases. In DNase II-deficient mice, the excessive accrual of undegraded DNA leads to both a STING-dependent inflammatory arthritis and additional Unc93b1-dependent autoimmune manifestations, including splenomegaly, extramedullary hematopoiesis, and autoantibody production. In this study, we use bone marrow chimeras to show that clinical and histological inflammation in the joint depends upon DNase II deficiency in both donor hematopoietic cells and host radioresistant cells. Additional features of autoimmunity in these mice, known to depend on Unc93b1 and therefore endosomal TLRs, also require DNase II deficiency in both donor and host compartments, but only require functional TLRs in the hematopoietic cells. Collectively, our data demonstrate a major role of both stromal and hematopoietic cells in all aspects of DNA-driven autoimmunity. These findings further point to the importance of cytosolic nucleic acid sensors in creating an inflammatory environment that facilitates the development of Unc93b1-dependent autoimmunity.

Cutting edge: AIM2 and endosomal TLRs differentially regulate arthritis and autoantibody production in DNase II-deficient mice.

Innate immune pattern recognition receptors sense nucleic acids from microbes and orchestrate cytokine production to resolve infection. Inappropriate recognition of host nucleic acids also results in autoimmune disease. In this study, we use a model of inflammation resulting from accrual of self DNA (DNase II(-/-) type I IFN receptor [Ifnar](-/-)) to understand the role of pattern recognition receptor-sensing pathways in arthritis and autoantibody production. Using triple knockout (TKO) mice deficient in DNase II/IFNaR together with deficiency in either stimulator of IFN genes (STING) or absent in melanoma 2 (AIM2), we reveal central roles for the STING and AIM2 pathways in arthritis. AIM2 TKO mice show limited inflammasome activation and, similar to STING TKO mice, have reduced inflammation in joints. Surprisingly, autoantibody production is maintained in AIM2 and STING TKO mice, whereas DNase II(-/-) Ifnar(-/-) mice also deficient in Unc93b, a chaperone required for TLR7/9 endosomal localization, fail to produce autoantibodies to nucleic acids. Collectively, these data support distinct roles for cytosolic and endosomal nucleic acid-sensing pathways in disease manifestations.

Gadolinium-based compounds induce NLRP3-dependent IL-1ß production and peritoneal inflammation.

OBJECTIVE: Nephrogenic systemic fibrosis (NSF) is a progressive fibrosing disorder that may develop in patients with chronic kidney disease after administration of gadolinium (Gd)-based contrast agents (GBCAs). In the setting of impaired renal clearance of GBCAs, Gd deposits in various tissues and fibrosis subsequently develops. However, the precise mechanism by which fibrosis occurs in NSF is incompletely understood. Because other profibrotic agents, such as silica or asbestos, activate the nucleotide-binding oligomerisation domain (NOD)-like receptor protein 3 (NLRP3) inflammasome and initiate interleukin (IL)-1ß release with the subsequent development of fibrosis, we evaluated the effects of GBCAs on inflammasome activation. METHODS: Bone marrow derived macrophages from C57BL/6, Nlrp3(-/-) and Asc(-/-) mice were incubated with three Gd-containing compounds and IL-1ß activation and secretion was detected by ELISA and western blot analysis. Inflammasome activation and regulation was investigated in IL-4- and interferon (IFN)γ-polarised macrophages by ELISA, quantitative real time (qRT)-PCR and NanoString nCounter analysis. Furthermore, C57BL/6 and Nlrp3(-/-)mice were intraperitoneally injected with GBCA and recruitment of inflammatory cells to the peritoneum was analysed by fluorescence-activated cell sorting (FACS). RESULTS: Free Gd and GBCAs activate the NLRP3 inflammasome and induce IL-1ß secretion in vitro. Gd-diethylenetriaminepentaacetic acid also induces the recruitment of neutrophils and inflammatory monocytes to the peritoneum in vivo. Gd activated IL-4-polarised macrophages more effectively than IFNγ-polarised macrophages, which preferentially expressed genes known to downregulate inflammasome activity. CONCLUSIONS: These data suggest that Gd released from GBCAs triggers a NLRP3 inflammasome-dependent inflammatory response that leads to fibrosis in an appropriate clinical setting. The preferential activation of IL-4-differentiated macrophages is consistent with the predominantly fibrotic presentation of NSF.

Cutting edge: the UNC93B1 tyrosine-based motif regulates trafficking and TLR responses via separate mechanisms.

Sensing of nucleic acids by TLRs is crucial in the host defense against viruses and bacteria. Unc-93 homolog B1 (UNC93B1) regulates the trafficking of nucleic acid-sensing TLRs from the endoplasmic reticulum to endolysosomes, where the TLRs encounter their respective ligands and become activated. In this article, we show that a carboxyl-terminal tyrosine-based sorting motif (YxxΦ) in UNC93B1 differentially regulates human nucleic acid-sensing TLRs in a receptor- and ligand-specific manner. Destruction of YxxΦ abolished TLR7, TLR8, and TLR9 activity toward nucleic acids in human B cells and monocytes, whereas TLR8 responses toward small molecules remained intact. YxxΦ in UNC93B1 influenced the subcellular localization of human UNC93B1 via both adapter protein complex (AP)1- and AP2-dependent trafficking pathways. However, loss of AP function was not causal for altered TLR responses, suggesting AP-independent functions of YxxΦ in UNC93B1.

The adaptor ASC has extracellular and 'prionoid' activities that propagate inflammation.

Microbes or danger signals trigger inflammasome sensors, which induce polymerization of the adaptor ASC and the assembly of ASC specks. ASC specks recruit and activate caspase-1, which induces maturation of the cytokine interleukin 1ß (IL-1ß) and pyroptotic cell death. Here we found that after pyroptosis, ASC specks accumulated in the extracellular space, where they promoted further maturation of IL-1ß. In addition, phagocytosis of ASC specks by macrophages induced lysosomal damage and nucleation of soluble ASC, as well as activation of IL-1ß in recipient cells. ASC specks appeared in bodily fluids from inflamed tissues, and autoantibodies to ASC specks developed in patients and mice with autoimmune pathologies. Together these findings reveal extracellular functions of ASC specks and a previously unknown form of cell-to-cell communication.