Increased Inflammasome Activation Is Associated with Aging and Chronic Myelomonocytic Leukemia Disease Severity.

Aging causes chronic low-grade inflammation known as inflamm-aging. It is a risk factor for several chronic disorders, including chronic myelomonocytic leukemia (CMML), a hematological malignancy that is most prevalent in older people. Recent studies suggest a critical role for the NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome in inflamm-aging. However, the mechanisms involved in NLRP3 activation in aging and its involvement in CMML progression are not fully understood. In this study, we report that aging increases IL-1ß production upon NLRP3 activation in human CD14+ monocytes. Interestingly, we found that the TLR1/2 agonist Pam3CSK4 directly activates the NLRP3 inflammasome in monocytes from older but not from younger healthy donors. Furthermore, we observed a dichotomous response to NLRP3 inflammasome activation in monocytes from a small cohort of CMML patients, and some patients produced high levels of IL-1ß and some patients produced low levels of IL-1ß compared with older healthy donors. Intriguingly, CMML patients with heightened NLRP3 activation showed increased treatment dependency and disease severity. Collectively, our results suggest that aging causes increased sensitivity to NLRP3 inflammasome activation at a cellular level, which may explain increased inflammation and immune dysregulation in older individuals. Furthermore, NLRP3 inflammasome activation was dysregulated in a small cohort of CMML patients and was positively correlated with disease severity.

Diagnostic and Prognostic Implications of Caspase-1 and PD-L1 Co-Expression Patterns in Myelodysplastic Syndromes.

BACKGROUND: The inflammasome plays an essential role in lower risk MDS and immune subversion, with the up-regulation of immune checkpoint molecules in the progression to higher-risk disease. In this study, we explored the utility of immune-related biomarkers for the diagnosis and prognosis of MDS. METHODS: We performed an exploratory, case-control study with 20 randomly selected MDS patients and nine controls with non-inflammatory (n = 3) and inflammatory conditions (n = 6). Patients were stratified in groups of lower (n = 10) and higher risk (n = 10) using IPSS-R. For the exploration of inflammasome and immune checkpoint activities, the expression of caspase-1 (Casp1), programmed cell death protein 1 (PD-1) and its ligand (PD-L1) were assessed in bone marrow samples using immunohistochemistry. RESULTS: In multivariate analysis, we observed significant differences for Casp1 but not PD1/PD-L1 expression in our four conditions (p = 0.003). We found a discordant co-expression of Casp1/PD-L1 in MDS (rho = -0.41, p = 0.07) compared with a concordant co-expression in controls (rho = 0.64, p = 0.06). Neutrophil counts correlated directly with Casp1 (rho = 0.57, p = 0.009) but inversely with PD-L1 expression (rho = -0.58, p = 0.007). CONCLUSION: We identified characteristic discordant co-expression patterns in lower- (Casp1high/PD-L1low) and higher-risk MDS (Casp1low/PD-L1high), contrasting with concordant patterns in the non-inflammatory (Casp1low/PD-L1low) and inflammatory conditions (Casp1high/PD-L1high). Further validation is warranted in larger, prospective studies.

Inference of kinase-signaling networks in human myeloid cell line models by Phosphoproteomics using kinase activity enrichment analysis (KAEA).

BACKGROUND: Despite the introduction of targeted therapies, most patients with myeloid malignancies will not be cured and progress. Genomics is useful to elucidate the mutational landscape but remains limited in the prediction of therapeutic outcome and identification of targets for resistance. Dysregulation of phosphorylation-based signaling pathways is a hallmark of cancer, and therefore, kinase-inhibitors are playing an increasingly important role as targeted treatments. Untargeted phosphoproteomics analysis pipelines have been published but show limitations in inferring kinase-activities and identifying potential biomarkers of response and resistance. METHODS: We developed a phosphoproteomics workflow based on titanium dioxide phosphopeptide enrichment with subsequent analysis by liquid chromatography tandem mass spectrometry (LC-MS). We applied a novel Kinase-Activity Enrichment Analysis (KAEA) pipeline on differential phosphoproteomics profiles, which is based on the recently published SetRank enrichment algorithm  with reduced false positive rates. Kinase activities were inferred by this algorithm using an extensive reference database comprising five experimentally validated kinase-substrate meta-databases complemented with the NetworKIN in-silico prediction tool. For the proof of concept, we used human myeloid cell lines (K562, NB4, THP1, OCI-AML3, MOLM13 and MV4-11) with known oncogenic drivers and exposed them to clinically established kinase-inhibitors. RESULTS: Biologically meaningful over- and under-active kinases were identified by KAEA in the unperturbed human myeloid cell lines (K562, NB4, THP1, OCI-AML3 and MOLM13). To increase the inhibition signal of the driving oncogenic kinases, we exposed the K562 (BCR-ABL1) and MOLM13/MV4-11 (FLT3-ITD) cell lines to either Nilotinib or Midostaurin kinase inhibitors, respectively. We observed correct detection of expected direct (ABL, KIT, SRC) and indirect (MAPK) targets of Nilotinib in K562 as well as indirect (PRKC, MAPK, AKT, RPS6K) targets of Midostaurin in MOLM13/MV4-11, respectively. Moreover, our pipeline was able to characterize unexplored kinase-activities within the corresponding signaling networks. CONCLUSIONS: We developed and validated a novel KAEA pipeline for the analysis of differential phosphoproteomics MS profiling data. We provide translational researchers with an improved instrument to characterize the biological behavior of kinases in response or resistance to targeted treatment. Further investigations are warranted to determine the utility of KAEA to characterize mechanisms of disease progression and treatment failure using primary patient samples.

Myelodysplastic Syndromes in the Postgenomic Era and Future Perspectives for Precision Medicine.

Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal disorders caused by sequential accumulation of somatic driver mutations in hematopoietic stem and progenitor cells (HSPCs). MDS is characterized by ineffective hematopoiesis with cytopenia, dysplasia, inflammation, and a variable risk of transformation into secondary acute myeloid leukemia. The advent of next-generation sequencing has revolutionized our understanding of the genetic basis of the disease. Nevertheless, the biology of clonal evolution remains poorly understood, and the stochastic genetic drift with sequential accumulation of genetic hits in HSPCs is individual, highly dynamic and hardly predictable. These continuously moving genetic targets pose substantial challenges for the implementation of precision medicine, which aims to maximize efficacy with minimal toxicity of treatments. In the current postgenomic era, allogeneic hematopoietic stem cell transplantation remains the only curative option for younger and fit MDS patients. For all unfit patients, regeneration of HSPCs stays out of reach and all available therapies remain palliative, which will eventually lead to refractoriness and progression. In this review, we summarize the recent advances in our understanding of MDS pathophysiology and its impact on diagnosis, risk-assessment and disease monitoring. Moreover, we present ongoing clinical trials with targeting compounds and highlight future perspectives for precision medicine.

Angiogenin (ANG)-Ribonuclease Inhibitor (RNH1) System in Protein Synthesis and Disease.

Protein synthesis is a highly complex process executed by well-organized translation machinery. Ribosomes, tRNAs and mRNAs are the principal components of this machinery whereas RNA binding proteins and ribosome interacting partners act as accessory factors. Angiogenin (ANG)-Ribonuclease inhibitor (RNH1) system is one such accessory part of the translation machinery that came into focus afresh due to its unconventional role in the translation. ANG is conventionally known for its ability to induce blood vessel formation and RNH1 as a "sentry" to protect RNAs from extracellular RNases. However, recent studies suggest them to be important in translation regulation. During cell homeostasis, ANG in the nucleus promotes rRNA transcription. While under stress, ANG translocates to the cytosol and cleaves tRNA into fragments which inhibit ribosome biogenesis and protein synthesis. RNH1, which intimately interacts with ANG to inhibit its ribonucleolytic activity, can also bind to the 40S ribosomes and control translation by yet to be known mechanisms. Here, we review recent advancement in the knowledge of translation regulation by the ANG-RNH1 system. We also gather information about this system in cell homeostasis as well as in pathological conditions such as cancer and ribosomopathies. Additionally, we discuss the future research directions and therapeutic potential of this system.

Inflammasome Activation in Myeloid Malignancies-Friend or Foe?

Myeloid malignancies including myelodysplastic syndromes, myeloproliferative neoplasms and acute myeloid leukemia are heterogeneous disorders originating from mutated hematopoietic stem and progenitor cells (HSPCs). Genetically, they are very heterogeneous and characterized by uncontrolled proliferation and/or blockage of differentiation of abnormal HSPCs. Recent studies suggest the involvement of inflammasome activation in disease initiation and clonal progression. Inflammasomes are cytosolic innate immune sensors that, upon activation, induce caspase-1 mediated processing of interleukin (IL) -1-cytokine members IL-1ß and IL-18, as well as initiation of gasdermin D-dependent pyroptosis. Inflammasome activation leads to a pro-inflammatory microenvironment in the bone marrow, which drives proliferation and may induce clonal selection of mutated HSPCs. However, there are also contradictory data showing that inflammasome activation actually counteracts leukemogenesis. Overall, the beneficial or detrimental effect of inflammasome activation seems to be highly dependent on mutational, environmental, and immunological contexts and an improved understanding is fundamental to advance specific therapeutic targeting strategies. This review summarizes current knowledge about this dichotomous effect of inflammasome activation in myeloid malignancies and provides further perspectives on therapeutic targeting.

Ribonuclease inhibitor 1 regulates erythropoiesis by controlling GATA1 translation.

Ribosomal proteins (RP) regulate specific gene expression by selectively translating subsets of mRNAs. Indeed, in Diamond-Blackfan anemia and 5q- syndrome, mutations in RP genes lead to a specific defect in erythroid gene translation and cause anemia. Little is known about the molecular mechanisms of selective mRNA translation and involvement of ribosomal-associated factors in this process. Ribonuclease inhibitor 1 (RNH1) is a ubiquitously expressed protein that binds to and inhibits pancreatic-type ribonucleases. Here, we report that RNH1 binds to ribosomes and regulates erythropoiesis by controlling translation of the erythroid transcription factor GATA1. Rnh1-deficient mice die between embryonic days E8.5 and E10 due to impaired production of mature erythroid cells from progenitor cells. In Rnh1-deficient embryos, mRNA levels of Gata1 are normal, but GATA1 protein levels are decreased. At the molecular level, we found that RNH1 binds to the 40S subunit of ribosomes and facilitates polysome formation on Gata1 mRNA to confer transcript-specific translation. Further, RNH1 knockdown in human CD34+ progenitor cells decreased erythroid differentiation without affecting myelopoiesis. Our results reveal an unsuspected role for RNH1 in the control of GATA1 mRNA translation and erythropoiesis.

Hepatocyte growth factor secreted by bone marrow stem cell reduce ER stress and improves repair in alveolar epithelial II cells.

Idiopathic Pulmonary Fibrosis (IPF) is a progressive, irreversible lung disease with complex pathophysiology. Evidence of endoplasmic reticulum (ER) stress has been reported in alveolar epithelial cells (AEC) in IPF patients. Secreted mediators from bone marrow stem cells (BMSC-cm) have regenerative properties. In this study we investigate the beneficial effects of BMSC-cm on ER stress response in primary AEC and ER stressed A549 cells. We hypothesize that BMSC-cm reduces ER stress. Primary AEC isolated from IPF patients were treated with BMSC-cm. To induce ER stress A549 cells were incubated with Tunicamycin or Thapsigargin and treated with BMSC-cm, or control media. Primary IPF-AEC had high Grp78 and CHOP gene expression, which was lowered after BMSC-cm treatment. Similar results were observed in ER stressed A549 cells. Alveolar epithelial repair increased in presence of BMSC-cm in ER stressed A549 cells. Hepatocyte growth factor (HGF) was detected in biologically relevant levels in BMSC-cm. Neutralization of HGF in BMSC-cm attenuated the beneficial effects of BMSC-cm including synthesis of surfactant protein C (SP-C) in primary AEC, indicating a crucial role of HGF in ER homeostasis and alveolar epithelial repair. Our data suggest that BMSC-cm may be a potential therapeutic option for treating pulmonary fibrosis.

Epithelial NAIPs protect against colonic tumorigenesis.

NLR family apoptosis inhibitory proteins (NAIPs) belong to both the Nod-like receptor (NLR) and the inhibitor of apoptosis (IAP) families. NAIPs are known to form an inflammasome with NLRC4, but other in vivo functions remain unexplored. Using mice deficient for all NAIP paralogs (Naip1-6(Δ/Δ)), we show that NAIPs are key regulators of colorectal tumorigenesis. Naip1-6(Δ/Δ) mice developed increased colorectal tumors, in an epithelial-intrinsic manner, in a model of colitis-associated cancer. Increased tumorigenesis, however, was not driven by an exacerbated inflammatory response. Instead, Naip1-6(Δ/Δ) mice were protected from severe colitis and displayed increased antiapoptotic and proliferation-related gene expression. Naip1-6(Δ/Δ) mice also displayed increased tumorigenesis in an inflammation-independent model of colorectal cancer. Moreover, Naip1-6(Δ/Δ) mice, but not Nlrc4-null mice, displayed hyper-activation of STAT3 and failed to activate p53 18 h after carcinogen exposure. This suggests that NAIPs protect against tumor initiation in the colon by promoting the removal of carcinogen-elicited epithelium, likely in a NLRC4 inflammasome-independent manner. Collectively, we demonstrate a novel epithelial-intrinsic function of NAIPs in protecting the colonic epithelium against tumorigenesis.

Mitochondrial apoptosis is dispensable for NLRP3 inflammasome activation but non-apoptotic caspase-8 is required for inflammasome priming.

A current paradigm proposes that mitochondrial damage is a critical determinant of NLRP3 inflammasome activation. Here, we genetically assess whether mitochondrial signalling represents a unified mechanism to explain how NLRP3 is activated by divergent stimuli. Neither co-deletion of the essential executioners of mitochondrial apoptosis BAK and BAX, nor removal of the mitochondrial permeability transition pore component cyclophilin D, nor loss of the mitophagy regulator Parkin, nor deficiency in MAVS affects NLRP3 inflammasome function. In contrast, caspase-8, a caspase essential for death-receptor-mediated apoptosis, is required for efficient Toll-like-receptor-induced inflammasome priming and cytokine production. Collectively, these results demonstrate that mitochondrial apoptosis is not required for NLRP3 activation, and highlight an important non-apoptotic role for caspase-8 in regulating inflammasome activation and pro-inflammatory cytokine levels.

Extracellular histones in tissue injury and inflammation.

Neutrophil NETosis is an important element of host defense as it catapults chromatin out of the cell to trap bacteria, which then are killed, e.g., by the chromatin's histone component. Also, during sterile inflammation TNF-alpha and other mediators trigger NETosis, which elicits cytotoxic effects on host cells. The same mechanism should apply to other forms of regulated necrosis including pyroptosis, necroptosis, ferroptosis, and cyclophilin D-mediated regulated necrosis. Beyond these toxic effects, extracellular histones also trigger thrombus formation and innate immunity by activating Toll-like receptors and the NLRP3 inflammasome. Thereby, extracellular histones contribute to the microvascular complications of sepsis, major trauma, small vessel vasculitis as well as acute liver, kidney, brain, and lung injury. Finally, histones prevent the degradation of extracellular DNA, which promotes autoimmunization, anti-nuclear antibody formation, and autoimmunity in susceptible individuals. Here, we review the current evidence on the pathogenic role of extracellular histones in disease and discuss how to target extracellular histones to improve disease outcomes.

Analysis of TNF-mediated recruitment and activation of glomerular dendritic cells in mouse kidneys by compartment-specific flow cytometry.

Renal dendritic cells (DCs) form an interstitial network contributing to inflammatory and adaptive immune responses in the kidney. The presence and functional role of DC-like glomerular CD11c(+) mononuclear phagocytes is a matter of debate. Using compartment-specific flow cytometry we found that healthy mouse kidneys contained 1.3 CD11c(+) cells per 100 glomeruli and these increased by 4.6-fold and 13-fold after TNF stimulation and immune complex deposition, respectively. Compartment-specific mRNA expression revealed a predominantly glomerular expression of TNF receptors, chemokines, and adhesion molecules; all upregulated after TNF exposure. Intraperitoneal TNF injection induced influx of neutrophils and mononuclear phagocytes including DC-like CD11c(+) cells into both the glomerular and tubulointerstitial compartments, but reduced in TNF receptor (Tnfr) 1-deficient mice. Additionally, Tnfr2 deficiency impaired glomerular infiltration of CD11c(+) cells, but not neutrophils. Interstitial CD11c(+) cells infiltrated in the presence of Tnfr1 or Tnfr2. TNF exposure also induced similar maturation of glomerular and interstitial CD11c(+) cells as demonstrated by increased surface expression of MHC II, CD54, and costimulatory molecules CD40, CD80, and CD86. Thus, by compartment-specific flow cytometry we could demonstrate the constitutive presence of DC-like CD11c(+) mononuclear phagocytes in normal mouse glomeruli and their TNF-induced accumulation and activation.

Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4.

In AKI, dying renal cells release intracellular molecules that stimulate immune cells to secrete proinflammatory cytokines, which trigger leukocyte recruitment and renal inflammation. Whether the release of histones, specifically, from dying cells contributes to the inflammation of AKI is unknown. In this study, we found that dying tubular epithelial cells released histones into the extracellular space, which directly interacted with Toll-like receptor (TLR)-2 (TLR2) and TLR4 to induce MyD88, NF-κB, and mitogen activated protein kinase signaling. Extracellular histones also had directly toxic effects on renal endothelial cells and tubular epithelial cells in vitro. In addition, direct injection of histones into the renal arteries of mice demonstrated that histones induce leukocyte recruitment, microvascular vascular leakage, renal inflammation, and structural features of AKI in a TLR2/TLR4-dependent manner. Antihistone IgG, which neutralizes the immunostimulatory effects of histones, suppressed intrarenal inflammation, neutrophil infiltration, and tubular cell necrosis and improved excretory renal function. In summary, the release of histones from dying cells aggravates AKI via both its direct toxicity to renal cells and its proinflammatory effects. Because the induction of proinflammatory cytokines in dendritic cells requires TLR2 and TLR4, these results support the concept that renal damage triggers an innate immune response, which contributes to the pathogenesis of AKI.

Inhibitor of apoptosis proteins limit RIP3 kinase-dependent interleukin-1 activation.

Interleukin-1ß (IL-1ß) is a potent inflammatory cytokine that is usually cleaved and activated by inflammasome-associated caspase-1. To determine whether IL-1ß activation is regulated by inhibitor of apoptosis (IAP) proteins, we treated macrophages with an IAP-antagonist "Smac mimetic" compound or genetically deleted the genes that encode the three IAP family members cIAP1, cIAP2, and XIAP. After Toll-like receptor priming, IAP inhibition triggered cleavage of IL-1ß that was mediated not only by the NLRP3-caspase-1 inflammasome, but also by caspase-8 in a caspase-1-independent manner. In the absence of IAPs, rapid and full generation of active IL-1ß by the NLRP3-caspase-1 inflammasome, or by caspase-8, required the kinase RIP3 and reactive oxygen species production. These results demonstrate that activation of the cell death-inducing ripoptosome platform and RIP3 can generate bioactive IL-1ß and implicate them as additional targets for the treatment of pathological IL-1-driven inflammatory responses.

Mdm2 promotes systemic lupus erythematosus and lupus nephritis.

Systemic lupus erythematosus (SLE) is a polyclonal autoimmune syndrome directed against multiple nuclear autoantigens. Although RNA and DNA seem to have identical immunostimulatory effects on systemic and intrarenal inflammation, each seems to differ with regard to the propensity to induce mitogenic effects such as lymphoproliferation. To identify potential mechanisms by which DNA specifically contributes to the pathogenesis of lupus nephritis, we stimulated cells with immunostimulatory DNA or RNA in vitro and used microarray to compare the transcriptomes of RNA- and DNA-induced genes. Immunostimulatory DNA, but not RNA, induced Mdm2, which is a negative regulator of p53. In vivo, we observed greater expression and activation of Mdm2 in the spleen and kidneys in a mouse model of lupus (MRL-Fas(lpr) mice) than healthy controls. Treatment of MRL-Fas(lpr) mice with the Mdm2 inhibitor nutlin-3a prevented nephritis and lung disease and significantly prolonged survival. Inhibition of Mdm2 reduced systemic inflammation and abrogated immune complex disease by suppressing plasma cells and the production of lupus autoantibodies. In addition, nutlin-3a suppressed the abnormal expansion of all T cell subsets, including CD3(+)CD4(-)CD8(-) T cells, which associated with attenuated systemic inflammation. However, inhibiting Mdm2 did not cause myelosuppression or affect splenic regulatory T cells, neutrophils, dendritic cells, or monocytes. Taken together, these data suggest that the induction of Mdm2 promotes the expansion of plasma cells and CD3(+)CD4(-)CD8(-) T cells, which cause autoantibody production and immune complex disease in MRL-Fas(lpr) mice. Antagonizing Mdm2 may have therapeutic potential in lupus nephritis.

Polyene macrolide antifungal drugs trigger interleukin-1ß secretion by activating the NLRP3 inflammasome.

The use of antimycotic drugs in fungal infections is based on the concept that they suppress fungal growth by a direct killing effect. However, amphotericin and nystatin have been reported to also trigger interleukin-1ß (IL-1ß) secretion in monocytes but the molecular mechanism is unknown. Here we report that only the polyene macrolides amphotericin B, nystatin, and natamycin but none of the tested azole antimycotic drugs induce significant IL-1ß secretion in-vitro in dendritic cells isolated from C57BL/6 mouse bone marrow. IL-1ß release depended on Toll-like receptor-mediated induction of pro-IL-1ß as well as the NLRP3 inflammasome, its adaptor ASC, and caspase-1 for enzymatic cleavage of pro-IL-1ß into its mature form. All three drugs induced potassium efflux from the cells as a known mechanism for NLRP3 activation but the P2X7 receptor was not required for this process. Natamycin-induced IL-1ß secretion also involved phagocytosis, as cathepsin activation as described for crystal-induced IL-1ß release. Together, the polyene macrolides amphotericin B, nystatin, and natamycin trigger IL-1ß secretion by causing potassium efflux from which activates the NLRP3-ASC-caspase-1. We conclude that beyond their effects on fungal growth, these antifungal drugs directly activate the host's innate immunity.

Cutting edge: cyclic polypeptide and aminoglycoside antibiotics trigger IL-1ß secretion by activating the NLRP3 inflammasome.

Clinical use of antibiotics is based on their capacity to inhibit bacterial growth via bacteriostatic or bacteriocidal effects. In this article, we show that the aminoglycoside antibiotic neomycin, the cyclic lipopeptide antibiotic polymyxin B, and the cyclic peptide antibiotics gramicidin and tyrothricin can induce IL-1ß secretion in bone marrow dendritic cells and macrophages. LPS priming was required to trigger the transcription and translation of pro-IL-1ß but was independent of TNFR or IL-1R signaling. All four antibiotics required the NLRP3 inflammasome, the adaptor ASC, and caspase-1 activation to secrete IL-1ß, a process that depended on potassium efflux but was independent of P2X7 receptor. All four antibiotics induced neutrophil influx into the peritoneal cavity of mice, which required NLRP3 only in the case of polymyxin B. Together, certain antibiotics have the potential to directly activate innate immunity of the host.

Viral RNA induces type I interferon-dependent cytokine release and cell death in mesangial cells via melanoma-differentiation-associated gene-5: Implications for viral infection-associated glomerulonephritis.

Viral RNA can trigger interferon signaling in dendritic cells via the innate recognition receptors melanoma-differentiation-associated gene (MDA)-5 and retinod-inducible gene (RIG)-I in the cytosol or via Toll-like receptors (TLRs) in intracellular endosomes. We hypothesized that viral RNA would also activate glomerular mesangial cells to produce type I interferon (IFN) via TLR-dependent and TLR-independent pathways. To test this hypothesis, we examined Toll/Interleukin-1 receptor domain-containing adaptor-inducing interferon-beta (TRIF)-deficient mice, which lack a key adaptor for TLR3 signaling. In primary mesangial cells, poly I:C RNA-mediated IFN-beta induction was partially TRIF dependent; however, when poly I:C RNA was complexed with cationic lipids to enhance cytosolic uptake, mesangial cells produced large amounts of IFN-alpha and IFN-beta independent of TRIF. Mesangial cells expressed RIG-I and MDA-5 and their mitochondrial adaptor IFN-beta promoter stimulator-1 as well, and small interfering RNA studies revealed that MDA5 but not RIG-I was required for cytosolic poly I:C RNA signaling. In addition, mesangial cells produced Il-6 on stimulation with IFN-alpha and IFN-beta, suggesting an autocrine proinflammatory effect. Indeed, blockade of IFN-alphabeta or lack of the IFNA receptor reduced viral RNA-induced Il-6 production and apoptotic cell death in mesangial cells. Furthermore, viral RNA/cationic lipid complexes increased focal necrosis in murine nephrotoxic serum nephritis in association with increased renal mRNA expression of IFN-related genes. Thus, TLR-independent recognition of viral RNA is a potent inducer of type I interferon in mesangial cells, which can be an important mediator of virally induced glomerulonephritis.

Resident dendritic cells prevent postischemic acute renal failure by help of single Ig IL-1 receptor-related protein.

Ischemia-reperfusion (IR) triggers tissue injury by activating innate immunity, for example, via TLR2 and TLR4. Surprisingly, TLR signaling in intrinsic renal cells predominates in comparison to intrarenal myeloid cells in the postischemic kidney. We hypothesized that immune cell activation is specifically suppressed in the postischemic kidney, for example, by single Ig IL-1-related receptor (SIGIRR). SIGIRR deficiency aggravated postischemic acute renal failure in association with increased renal CXCL2/MIP2, CCL2/MCP-1, and IL-6 mRNA expression 24 h after IR. Consistent with this finding interstitial neutrophil and macrophage counts were increased and tubular cell necrosis was aggravated in Sigirr-deficient vs wild-type IR kidneys. In vivo microscopy revealed increased leukocyte transmigration in the postischemic microvasculature of Sigirr-deficient mice. IL-6 and CXCL2/MIP2 release was much higher in Sigirr-deficient renal myeloid cells but not in Sigirr-deficient tubular epithelial cells after transient hypoxic culture conditions. Renal IR studies with chimeric mice confirmed this finding, as lack of SIGIRR in myeloid cells largely reproduced the phenotype of renal IR injury seen in Sigirr(-/-) mice. Additionally, clodronate depletion of dendritic cells prevented the aggravated renal failure in Sigirr(-/-) mice. Thus, loss of function mutations in the SIGIRR gene predispose to acute renal failure because SIGIRR prevents overshooting tissue injury by suppressing the postischemic activation of intrarenal myeloid cells.

Viral RNA and DNA trigger common antiviral responses in mesangial cells.

Extrarenal viral infections commonly trigger glomerulonephritis, usually in association with immune complex disease. The Ig component of immune complexes can activate glomerular cell Fc receptors, but whether complexed viral nucleic acids contribute to glomerular inflammation remains unknown. Because of the types of Toll-like receptors (Tlrs) expressed by glomerular mesangial cells, we hypothesized that viral single-stranded RNA and DNA would activate mesangial cells via Tlr-independent pathways and trigger overlapping antiviral immune responses. Consistent with this hypothesis, 5'-triphosphate RNA (3P-RNA) and non-CpG DNA activated murine primary glomerular mesangial cells to secrete Cxcl10 and Il-6 even in cells derived from mice deficient in the Tlr adaptor proteins Myd88 and Trif. Transcriptome analysis revealed that 3P-RNA and non-CpG-DNA triggered almost identical gene expression programs, especially the proinflammatory cytokine Il-6, several chemokines, and genes related to type I IFN. We observed similar findings in glomerular preparations after injecting 3P-RNA and non-CpG-DNA in vivo. These effects depended on the formation of complexes with cationic lipids, which enhanced nucleic acid uptake into the cytosol of mesangial cells. Small interfering RNA studies revealed that 3P-RNA recognition involves Rig-1, whereas non-CpG-DNA did not require Rig-1 or Dai to activate glomerular mesangial cells. We conclude that 3P-RNA and double-stranded DNA trigger a common, TLR-independent, antiviral response in glomerular mesangial cells, which may promote glomerulonephritis in the setting of viral infection.