Probes for INS

BACKGROUND: Severe early-onset erythroderma and gut inflammation, with massive tissue infiltration of oligoclonal activated T cells are the hallmark of Omenn Syndrome (OS). OBJECTIVE: The impact of altered gut homeostasis in the cutaneous manifestations of OS remains to be clarified. METHODS: We analyzed a cohort of 15 patients with OS and the Rag2R229Q mouse model. Homing phenotype of circulating lymphocytes were analyzed by flow cytometry. Inflammatory cytokines and chemokines were examined in the sera by ELISA and in skin biopsies by immunohistochemistry and in situ RNA hybridization. Experimental colitis was induced in mice by dextran sulfate sodium salt (DSS). RESULTS: We show that memory/activated T cells from OS patients and from the Rag2R229Q mouse model of OS abundantly express the skin homing receptors Cutaneous Lymphocyte Associated Antigen (CLA) and CCR4, associated with high levels of CCL17 and CCL22 chemokines. Serum levels of LPS are also elevated. A broad Th1/Th2/Th17 inflammatory signature is detected in the periphery and in the skin. Increased Tlr4 expression in the skin of Rag2R229Q mice is associated with enhanced cutaneous inflammation upon local and systemic administration of LPS. Likewise, boosting colitis in Rag2R229Q mice results in increased frequency of CCR4+ splenic T cells and worsening of skin inflammation, as indicated by epidermal thickening, enhanced epithelial cell activation and dermal infiltration by Th1 effector T cells. CONCLUSIONS: These results support the existence of an interplay between gut and skin that can sustain skin inflammation in O

Hepatic innate immune control of viral infections has largely been attributed to Kupffer cells, the liver macrophages. However, also hepatocytes, the parenchymal cells of the liver, possess potent immunological functions in addition to their known metabolic functions. Owing to their abundance in the liver and known immunological functions, we aimed to investigate the direct anti-viral mechanisms employed by hepatocytes. METHODS: Using lymphocytic choriomeningitis virus (LCMV) as a model of liver infection, we first assessed the role of myeloid cells by depletion prior to infection. We investigated the role of hepatocyte-intrinsic innate immune signaling by infecting mice lacking canonical NF-?B signaling (IKK??Hep) specifically in hepatocytes. In addition, mice lacking hepatocyte-specific interferon-?/? signaling-(IFNAR?Hep), or interferon-?/? signaling in myeloid cells-(IFNAR?Myel) were infected. RESULTS: Here, we demonstrate that LCMV activates NF-?B signaling in hepatocytes. LCMV-triggered NF-?B activation in hepatocytes did not depend on Kupffer cells or TNFR1- but rather on TLR-signaling. LCMV-infected IKK??Hep livers displayed strongly elevated viral titers due to LCMV accumulation within hepatocytes, reduced interferon-stimulated gene (ISG) expression, delayed intrahepatic immune cell influx and delayed intrahepatic LCMV-specific CD8+ T-cell responses. Notably, viral clearance and ISG expression were also reduced in LCMV-infected primary hepatocytes lacking IKK?, demonstrating a hepatocyte-intrinsic effect. Similar to livers of IKK??Hep mice, enhanced hepatocytic LCMV accumulation was observed in livers of IFNAR?Hep, whereas IFNAR?Myel mice were able to control LCMV-infection. Hepatocytic NF-?B signaling was also required for efficient ISG induction in HDV-infected dHepaRG cells and interferon-?/?-mediated inhibition of HBV replication in vitro. CONCLUSIONS: Together, these data show that hepatocyte-intrinsic NF-?B is a vital amplifier of interferon-?/? signaling pivotal for early, strong ISG responses, influx of immune cells and hepatic viral clearance.

The development of an effective vaccine against SARS-CoV-2, the etiologic agent of COVID-19, is a global priority. Here, we compared the protective capacity of intranasal and intramuscular delivery of a chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike protein (ChAd-SARS-CoV-2-S) in Golden Syrian hamsters. While immunization with ChAd-SARS-CoV-2-S induced robust spike protein specific antibodies capable of neutralizing the virus, antibody levels in serum were higher in hamsters vaccinated by an intranasal compared to intramuscular route. Accordingly, against challenge with SARS-CoV-2, ChAd-SARS-CoV-2-S immunized hamsters were protected against less weight loss and had reduced viral infection in nasal swabs and lungs, and reduced pathology and inflammatory gene expression in the lungs, compared to ChAd-Control immunized hamsters. Intranasal immunization with ChAd-SARS-CoV-2-S provided superior protection against SARS-CoV-2 infection and inflammation in the upper respiratory tract. These findings support intranasal administration of the ChAd-SARS-CoV-2-S candidate vaccine to prevent SARS-CoV-2 infection, disease, and possibly transmission.

Post-mortem examination plays a pivotal role in understanding the pathobiology of the SARS-CoV-2; thus, the optimization of virus detection on the post-mortem formalin-fixed paraffin-embedded (FFPE) tissue is needed. Different techniques are available for the identification of the SARS-CoV-2, including reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy. The main goal of this study is to compare ISH versus RT-PCR to detect SARS-CoV-2 on post-mortem lung samples of positive deceased subjects. A total of 27 samples were analyzed by RT-PCR targeting different viral RNA sequences of SARS-CoV-2, including envelope (E), nucleocapsid (N), spike (S), and open reading frame (ORF1ab) genes and ISH targeting S and Orf1ab. All 27 cases showed the N gene amplification, 22 out of 27 the E gene amplification, 26 out of 27 the S gene amplification, and only 6 the ORF1ab gene amplification. The S ISH was positive only in 12 out of 26 cases positive by RT-PCR. The S ISH positive cases with strong and diffuse staining showed a correlation with low values of the number of the amplification cycles by S RT-PCR suggesting that ISH is a sensitive assay mainly in cases carrying high levels of S RNA. In conclusion, our findings demonstrated that ISH assay has lower sensitivity to detect SARS-CoV-2 in FFPE compared to RT-PCR; however, it is able to localize the virus in the cellular context since it preserves the morphology.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a historic pandemic of respiratory disease (coronavirus disease 2019 [COVID-19]), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2 signaling restricts the viral burden in the lung. We find that a recently developed mouse-adapted SARS-CoV-2 (MA-SARS-CoV-2) strain as well as the emerging B.1.351 variant trigger an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using intravital antibody labeling, we demonstrate that MA-SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. Single-cell RNA sequencing (scRNA-Seq) analysis of lung homogenates identified a hyperinflammatory monocyte profile. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified a potential CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2-dependent infiltration of monocytes restricts the viral burden in the lung. We find that SARS-CoV-2 triggers an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using RNA sequencing and flow cytometry approaches, we demonstrate that SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. Mechanistically, CCR2 signaling promoted the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified that the CCR2 pathway is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.

Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human induced pluripotent stem cell-derived kidney organoids with SARS-CoV-2. Single cell RNA-sequencing indicated injury and dedifferentiation of infected cells with activation of pro-fibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in Long-COVID.

(A) Myc-CaP wild-type (WT) and _Atg5_ knockout (_Atg5_ KO) cells were treated with increasing concentrations of ESK981 for 24 hours. Atg5 and LC3 levels were assessed by western blot from three independent experiments. GAPDH served as a loading control. (B) Representative morphology of vacuolization in Myc-CaP wild-type (WT) and _Atg5_ knockout (_Atg5_ KO) cells after treatment with control or 100 nM ESK981 for 24 hours from three independent experiments. (C) Autophagosome content of Myc-CaP WT and _Atg5_ KO cells were measured by CYTO-ID assay after being treated with increasing concentrations of ESK981 for 24 hours. Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated. (D) Mouse cytokine array using Myc-CaP WT and _Atg5_ KO cell supernatant after treatment with 10 ng/ml mouse interferon gamma (mIFNγ) or mIFNγ + 100 nM ESK981 for 24 hours. Differential expression candidate dots are highlighted by boxes. (E) Mouse CXCL10 protein levels were measured by ELISA in Myc-CaP WT and _Atg5_ KO conditioned medium with the indicated treatment for 24 hours. Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated. (F) mRNA levels of _Cxcl10_ and _Cxcl9_ were measured by qPCR in Myc-CaP WT and _Atg5_ KO cells with 50 nM or 100 nM ESK981 and 10 ng/ml mIFNγ treatment for 24 hours. Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated.

Single-cell RNA sequencing (scRNA-seq) is a powerful technique for describing cell states. Identifying the spatial arrangement of these states in tissues remains challenging, with the existing methods requiring niche methodologies and expertise. Here, we describe segmentation by exogenous perfusion (SEEP), a rapid and integrated method to link surface proximity and environment accessibility to transcriptional identity within three-dimensional (3D) disease models. The method utilizes the steady-state diffusion kinetics of a fluorescent dye to establish a gradient along the radial axis of disease models. Classification of sample layers based on dye accessibility enables dissociated and sorted cells to be characterized by transcriptomic and regional identities. Using SEEP, we analyze spheroid, organoid, and in vivo tumor models of high-grade serous ovarian cancer (HGSOC). The results validate long-standing beliefs about the relationship between cell state and position while revealing new concepts regarding how spatially unique microenvironments influence the identity of individual cells within tumors.