Gout is characterized by an acute inflammatory reaction and the accumulation of neutrophils in response to monosodium urate (MSU) crystals. Inflammation resolves spontaneously within a few days, although MSU crystals can still be detected in the synovial fluid and affected tissues. Here we report that neutrophils recruited to sites of inflammation undergo oxidative burst and form neutrophil extracellular traps (NETs). Under high neutrophil densities, these NETs aggregate and degrade cytokines and chemokines via serine proteases. Tophi, the pathognomonic structures of chronic gout, share characteristics with aggregated NETs, and MSU crystals can induce NETosis and aggregation of NETs. In individuals with impaired NETosis, MSU crystals induce uncontrolled production of inflammatory mediators from neutrophils and persistent inflammation. Furthermore, in models of neutrophilic inflammation, NETosis-deficient mice develop exacerbated and chronic disease that can be reduced by adoptive transfer of aggregated NETs. These findings suggest that aggregated NETs promote the resolution of neutrophilic inflammation by degrading cytokines and chemokines and disrupting neutrophil recruitment and activation.

Background Coronavirus induced disease 2019 (COVID-19) can be complicated by severe organ damage leading to dysfunction of the lungs and other organs. The processes that trigger organ damage in COVID-19 are incompletely understood. Methods Samples were donated from hospitalized patients. Sera, plasma, and autopsy-derived tissue sections were examined employing flow cytometry, enzyme-linked immunosorbent assays, and immunohistochemistry. Patient findings Here, we show that severe COVID-19 is characterized by a highly pronounced formation of neutrophil extracellular traps (NETs) inside the micro-vessels. Intravascular aggregation of NETs leads to rapid occlusion of the affected vessels, disturbed microcirculation, and organ damage. In severe COVID-19, neutrophil granulocytes are strongly activated and adopt a so-called low-density phenotype, prone to spontaneously form NETs. In accordance, markers indicating NET turnover are consistently increased in COVID-19 and linked to disease severity. Histopathology of the lungs and other organs from COVID-19 patients showed congestions of numerous micro-vessels by aggregated NETs associated with endothelial damage. Interpretation These data suggest that organ dysfunction in severe COVID-19 is associated with excessive NET formation and vascular damage. Funding Deutsche Forschungsgemeinschaft (DFG), EU, Volkswagen-Stiftung

Kaposi’s sarcoma associated herpesvirus (KSHV) is the human oncovirus which causes Kaposi’s sarcoma (KS), a highly vascularised tumour originating from lymphatic endothelial cells. Amongst others, the dimeric complex formed by the KSHV virion envelope glycoproteins H and L (gH/gL) is required for entry of herpesviruses into the host cell. We show that the Ephrin receptor tyrosine kinase A2 (EphA2) is a cellular receptor for KSHV gH/gL. EphA2 co-precipitated with both gH/gL and KSHV virions. KSHV infection rates were increased upon over-expression of EphA2. In contrast, antibodies against EphA2 and siRNAs directed against EphA2 inhibited KSHV infection of lymphatic endothelial cells. Pretreatment of KSHV virions with soluble EphA2 resulted in a dose-dependent inhibition of KSHV infection by up to 90%. Similarly, pretreating cells with the soluble EphA2 ligand EphrinA4 but not with EphA2 itself impaired KSHV infection. Notably, deletion of the EphA2 gene essentially abolished KSHV infection of murine vascular endothelial cells. Binding of gH/gL to EphA2 triggered EphA2 phosphorylation and endocytosis, a major pathway of KSHV entry. Quantitative RT-PCR and situ histochemistry revealed a close correlation between KSHV infection and EphA2 expression both in cultured cells derived from KS or lymphatic endothelium and in KS specimens, respectively. Taken together, these results identify EphA2, a tyrosine kinase with known functions in neo-vascularisation and oncogenesis, as receptor for KSHV gH/gL and implicate an important role for EphA2 in KSHV infection especially of endothelial cells and in KS.

SARS-CoV-2 infection poses a major threat to the lungs and multiple other organs, occasionally causing death. Until effective vaccines are developed to curb the pandemic, it is paramount to define the mechanisms and develop protective therapies to prevent organ dysfunction in patients with COVID-19. Individuals that develop severe manifestations have signs of dysregulated innate and adaptive immune responses. Emerging evidence implicates neutrophils and the disbalance between neutrophil extracellular trap (NET) formation and degradation plays a central role in the pathophysiology of inflammation, coagulopathy, organ damage, and immunothrombosis that characterize severe cases of COVID-19. Here, we discuss the evidence supporting a role for NETs in COVID-19 manifestations and present putative mechanisms, by which NETs promote tissue injury and immunothrombosis. We present therapeutic strategies, which have been successful in the treatment of immunο-inflammatory disorders and which target dysregulated NET formation or degradation, as potential approaches that may benefit patients with severe COVID-19.

Guanylate-binding proteins (GBPs) belong to the dynamin family of large GTPases and represent the major IFN-γ-induced proteins. Here we systematically investigated the mechanisms regulating the subcellular localization of GBPs. Three GBPs (GBP-1, GBP-2 and GBP-5) carry a C-terminal CaaX-prenylation signal, which is typical for small GTPases of the Ras family, and increases the membrane affinity of proteins. In this study, we demonstrated that GBP-1, GBP-2 and GBP-5 are prenylated in vivo and that prenylation is required for the membrane association of GBP-1, GBP-2 and GBP-5. Using co-immunoprecipitation, yeast-two-hybrid analysis and fluorescence complementation assays, we showed for the first time that GBPs are able to homodimerize in vivo and that the membrane association of GBPs is regulated by dimerization similarly to dynamin. Interestingly, GBPs could also heterodimerize. This resulted in hierarchical positioning effects on the intracellular localization of the proteins. Specifically, GBP-1 recruited GBP-5 and GBP-2 into its own cellular compartment and GBP-5 repositioned GBP-2. In addition, GBP-1, GBP-2 and GBP-5 were able to redirect non-prenylated GBPs to their compartment in a prenylation-dependent manner. Overall, these findings prove in vivo the ability of GBPs to dimerize, indicate that heterodimerization regulates sub-cellular localization of GBPs and underscore putative membrane-associated functions of this family of proteins.

in (partial) fulfillment of the requirements for obtaining the degree Dr. rer. nat. by Victoria Langer. Supporting grants were from the German Research Foundation (DFG) (KFO 257 [subproject 4 to M Stürzl and MJW], FOR 2438 [subproject 2 to EN and M Stürzl], SFB/TRR241 [subproject A06 to M Stürzl and NBL], and BR 5196/2-1 [to NBL]); the Interdisciplinary Center for Clinical Research (IZKF) of the Clinical Center Erlangen (D28 to EN and M Stürzl); the W. Lutz Stiftung (to M Stürzl); and the Forschungsstiftung Medizin am Universitätsklinikum Erlangen (to M Stürzl).

During angiogenesis and inflammatory processes, endothelial cells acquire different activation phenotypes, whose identification may help in understanding the complex network of angiogenic and inflammatory interactions in vivo. To this goal we investigated the expression of the human guanylatebinding protein (GBP)-1 that is highly induced by inflammatory cytokines (ICs) and, therefore, may characterize IC-activated cells. Using a new rat monoclonal antibody raised against GBP-1, we show that GBP-1 is a cytoplasmic protein and that its expression in endothelial cells is selectively induced by interferon-␥, interleukin-1␣, interleukin-1␤, or tumor necrosis factor-␣, but not by other cytokines, chemokines, or growth factors. Moreover, we found that GBP-1 expression is highly associated with vascular endothelial cells as confirmed by the simultaneous detection of GBP-1 and the endothelial cell-associated marker CD31 in a broad range of human tissues. Notably, GBP-1 expression was undetectable in the skin, but it was highly induced in vessels of skin diseases with a high-inflammatory component including pso-

Fibroblasts are polymorphic cells with pleiotropic roles in organ morphogenesis, tissue homeostasis and immune responses. In fibrotic diseases, fibroblasts synthesize abundant amounts of extracellular matrix which lead to scaring and organ failure. In contrast, the hallmark feature of fibroblasts in arthritis is matrix degradation by the release of metalloproteinases and degrading enzymes, and subsequent tissue destruction. The mechanisms driving these functionally opposing pro-fibrotic and pro-inflammatory phenotypes of fibroblasts are enigmatic. We identified the transcription factor PU.1 as an essential orchestrator of the pro-fibrotic gene expression program. The interplay between transcriptional and post-transcriptional mechanisms which normally control PU.1 expression is perturbed in various fibrotic diseases, resulting in upregulation of PU.1, induction of fibrosis-associated gene sets, and a phenotypic switch in matrix-producing pro-fibrotic fibroblasts. In contrast, pharmacological and genetic inactivation of PU.1 disrupts the fibrotic network and enables re-programming of fibrotic fibroblasts into resting fibroblasts with regression of fibrosis in different organs.