Bystander Cells Research Articles

Cellular and transcriptome signatures unveiled by single-cell RNA-Seq following ex vivo infection of murine splenocytes with Borrelia burgdorferi.

Lyme disease, the most common tick-borne infectious disease in the US, is caused by a spirochetal pathogen Borrelia burgdorferi (Bb). Distinct host responses are observed in susceptible and resistant strains of inbred of mice following infection with Bb reflecting a subset of inflammatory responses observed in human Lyme disease. The advent of post-genomic methodologies and genomic data sets enables dissecting the host responses to advance therapeutic options for limiting the pathogen transmission and/or treatment of Lyme disease. In this study, we used single-cell RNA-Seq analysis in conjunction with mouse genomics exploiting GFP-expressing Bb to sort GFP+ splenocytes and GFP- bystander cells to uncover novel molecular and cellular signatures that contribute to early stages of immune responses against Bb. These data decoded the heterogeneity of splenic neutrophils, macrophages, NK cells, B cells, and T cells in C3H/HeN mice in response to Bb infection. Increased mRNA abundance of apoptosis-related genes was observed in neutrophils and macrophages clustered from GFP+ splenocytes. Moreover, complement-mediated phagocytosis-related genes such as C1q and Ficolin were elevated in an inflammatory macrophage subset, suggesting upregulation of these genes during the interaction of macrophages with Bb-infected neutrophils. In addition, the role of DUSP1 in regulating the expression of Casp3 and pro-inflammatory cytokines Cxcl1, Cxcl2, Il1b, and Ccl5 in Bb-infected neutrophils were identified. These findings serve as a growing catalog of cell phenotypes/biomarkers among murine splenocytes that can be exploited for limiting spirochetal burden to limit the transmission of the agent of Lyme disease to humans via reservoir hosts.

IL-17RA promotes pathologic epithelial inflammation in a mouse model of upper respiratory influenza infection.

The upper respiratory tract (nasopharynx or NP) is the first site of influenza replication, allowing the virus to disseminate to the lower respiratory tract or promoting community transmission. The host response in the NP regulates an intricate balance between viral control and tissue pathology. The hyper-inflammatory responses promote epithelial injury, allowing for increased viral dissemination and susceptibility to secondary bacterial infections. However, the pathologic contributors to influenza upper respiratory tissue pathology are incompletely understood. In this study, we investigated the role of interleukin IL-17 recetor A (IL-17RA) as a modulator of influenza host response and inflammation in the upper respiratory tract. We used a combined experimental approach involving IL-17RA-/- mice and an air-liquid interface (ALI) epithelial culture model to investigate the role of IL-17 response in epithelial inflammation, barrier function, and tissue pathology. Our data show that IL-17RA-/- mice exhibited significantly reduced neutrophilia, epithelial injury, and viral load. The reduced NP inflammation and epithelial injury in IL-17RA-/- mice correlated with increased resistance against co-infection by Streptococcus pneumoniae (Spn). IL-17A treatment, while potentiating the apoptosis of IAV-infected epithelial cells, caused bystander cell death and disrupted the barrier function in ALI epithelial model, supporting the in vivo findings.

Primary and Secondary Bystander Effects of Proton Microbeam Irradiation on Human Lung Cancer Cells under Hypoxic Conditions.

Tumor hypoxia is the most common feature of radioresistance to the radiotherapy (RT) of lung cancer and results in poor clinical outcomes. High-linear energy transfer (LET) radiation is a novel RT technique to overcome this problem. However, a limited number of studies have been elucidated on the underlying mechanism(s) of RIBE and RISBE in cancer cells exposed to high-LET radiation under hypoxia. Here, we developed a new method to investigate the RIBE and RISBE under hypoxia using the SPICE-QST proton microbeams and a layered tissue co-culture system. Normal lung fibroblast (WI-38) and lung cancer (A549) cells were exposed in the range of 06 Gy of proton microbeams, wherein only ~0.04-0.15% of the cells were traversed by protons. Subsequently, primary bystander A549 cells were co-cultured with secondary bystander A549 cells in the presence or absence of a GJIC and NO inhibitor using co-culture systems. Studies show that there are differences in RIBE in A549 and WI-38 primary bystander cells under normoxia and hypoxia. Interestingly, treatment with a GJIC inhibitor showed an increase in the toxicity of primary bystander WI-38 cells but a decrease in A549 cells under hypoxia. Our results also show the induction of RISBE in secondary bystander A549 cells under hypoxia, where GJIC and NO inhibitors reduced the stressful effects on secondary bystander A549 cells. Together, these preliminary results, for the first time, represented the involvement of intercellular communications through GJIC in propagation of RIBE and RISBE in hypoxic cancer cells.

Abstract C115: Preclinical characterization of STRO-002, a clinical-stage anti-FolRα antibody-drug conjugate

Abstract STRO-002 is a novel folate receptor alpha (FolRα)-targeting antibody-drug conjugate (ADC) currently undergoing evaluation in clinical trials at various phases of development in ovarian and endometrial cancer and pediatric AML. Here, we describe the favorable properties of the STRO-002 ADC and summarize its activity in preclinical models. STRO-002 is an ADC composed of an anti-FolRα antibody conjugated to four tubulin-targeting hemiasterlin warheads. Incorporation of non-natural amino acids, using Sutro’s XpressCF® and XpressCF+® cell-free expression and conjugation systems, allows for site-specific conjugation of the hemiasterlin payload, yielding a homogenous and high-fidelity ADC. As an ADC, STRO-002 internalizes rapidly into FolRα+ tumor cells and exhibits potent target-dependent cell killing on xenograft tumor cell lines. Further characterization of STRO-002 and the hemiasterlin payload reveals significant ADC bystander activity and a lower sensitivity to drug efflux via the P-gp drug pump compared to other microtubule-targeting warheads. In addition to robust direct and bystander cell killing, STRO-002 is also capable of triggering immunogenic cell death (ICD). In in vitro cell killing assays, STRO-002 induces all three hallmarks of ICD—HMGB1 and ATP release and surface exposure of calreticulin—and in in vivo vaccination studies, injection of STRO-002-killed tumor cells was effective at establishing a protective immune response against subsequent tumor challenge, identifying STRO-002 as a bonafide ICD inducer.In vivo, STRO-002 treatment demonstrates robust efficacy in ovarian xenograft models, even when treatment is initiated in large, established tumors. In addition, combination therapy with STRO-002 and carboplatin, anti-VEGF, or anti-PD-L1 results in greater efficacy than treatment with single agents alone, demonstrating combination benefit with current standard-of-care therapies. To better understand the activity of STRO-002 in more-translatable preclinical models, we turned to patient-derived xenografts (PDX) and found that STRO-002 exhibits good anti-tumor activity in endometrial cancer and non-small cell lung cancer PDX studies, suggesting potential clinical benefit in these indications. In conclusion, STRO-002 exhibits multiple advantageous properties as an ADC and shows good anti-tumor activity, either in combination or as a single agent, in both cell-derived xenograft and PDX models. Citation Format: Robert Yuan, Andrew McGeehan, Sihong Zhou, Jennifer Smith, Dayson Moreira, Krishna Bajjuri, Cuong Tran, Gang Yin, Alice Yam, Helena Kiefel, Xiaofan Li. Preclinical characterization of STRO-002, a clinical-stage anti-FolRα antibody-drug conjugate [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C115.

Brain extracellular vesicles from Alzheimer’s Disease and Down Syndrome patients mediate spread of senescence‐like phenotype in vitro

AbstractBackgroundAlzheimer’s disease (AD) is the prevalent cause of dementia in older adults. Down Syndrome (DS) is the most common chromosomal disorder with strong predisposition to the development of AD. Cellular senescence contributes in the pathophysiology of both AD and DS but the mechanisms are unclear. Senescence associated secretory phenotype (SASP), which refers to a complex of secreted mediators, including soluble factors and extracellular vesicles (EVs) may contribute to the spread of senescence to bystander cells but its role in neurodegenerative disorders has not been evaluated.MethodParietal cortex tissue from patients with AD, DS, and normal/low‐pathology (NL) cases was obtained from the UC Irvine ADRC. The cryopreserved human tissue was used for small EV isolation by ultracentrifugation and size exclusive chromatography (SEC). Purity of the EV fractions was confirmed by immunoblotting against positive (CD63, CD9, Syntenin‐1) and negative (calnexin and GM130) EV/exosomal markers, transmission electron microscopy, and nanoparticle tracking analysis. Cell‐type specific EV fractions were isolated by immunoprecipitation (IP). Senescence transfer potential of brain‐derived EVs were assessed in neuroblastoma and glioma cells using senescence associate beta‐galactosidase activity assay, and confirmed by other senescence markers.ResultOur preliminary data demonstrated the ability of AD and DS, but not NL samples, to transfer senescence to neuroblastoma SH‐SY5Y and glioma U87‐MG cells. Senescence associate beta‐galactosidase activity was on average around 15% higher in cells treated with AD brain derived EVs (AD‐EVs) compared to NL brain EVs (NL‐EVs). This data correlates with around 20% elevation in p21 expression in AD‐EV treated cells. When senescence transfer potentials were compared between AD‐EVs and DS brain derived EVs (DS‐EVs), DS‐EVs exceeded AD‐EVs in their ability to transfer senescence‐like phenotype with around 40% increase in senescence associate beta‐galactosidase activity in cells treated with DS‐EVs. We are currently evaluating levels of senescence‐associated and SASP markers in AD and DS cells and cell‐type specific brain derived small EVs.ConclusionWe demonstrated an ability of AD and DS brain derived EVs to transfer senescence‐like phenotype in vitro. This phenomenon deserves further investigation to estimate its impact on the progression of AD in Down and non‐Down population along with evaluation its therapeutic potential.

Modular chimeric cytokine receptors with leucine zippers enhance the antitumour activity of CAR T cells via JAK/STAT signalling.

The limited availability of cytokines in solid tumours hinders maintenance of the antitumour activity of chimeric antigen receptor (CAR) T cells. Cytokine receptor signalling pathways in CAR T cells can be activated by transgenic expression or injection of cytokines in the tumour, or by engineering the activation of cognate cytokine receptors. However, these strategies are constrained by toxicity arising from the activation of bystander cells, by the suboptimal biodistribution of the cytokines and by downregulation of the cognate receptor. Here we show that replacement of the extracellular domains of heterodimeric cytokine receptors in T cells with two leucine zipper motifs provides optimal Janus kinase/signal transducer and activator of transcription signalling. Such chimeric cytokine receptors, which can be generated for common γ-chain receptors, interleukin-10 and -12 receptors, enabled T cells to survive cytokine starvation without induction of autonomous cell growth, and augmented the effector function of CAR T cells in vitro in the setting of chronic antigen exposure and in human tumour xenografts in mice. As a modular design, leucine zippers can be used to generate constitutively active cytokine receptors in effector immune cells.

Cellular Dynamics Following CAR T Cell Therapy Are Associated with Response, Resistance and Cytokine Release Syndrome in Relapsed/Refractory Multiple Myeloma

Introduction The introduction of B cell maturation antigen (BCMA)-targeting chimeric antigen receptor (CAR) T cells revolutionized the treatment of relapsed/refractory multiple myeloma (RRMM). While long-term follow-up of CD19 CAR T cells demonstrated a connection between CAR T cell expansion and persistence with outcome, very little is known about persistence and effects on bystander cells of BCMA-targeting CAR T cells. Methods We analyzed 27 RRMM patients (pts) treated with Idecabtagene vicleucel (Ide-cel) at our center. Pts were grouped based on their remission evaluated 100 days after infusion into responders (partial response or better, n=16) or non-responders (progressive disease, n=11). Peripheral blood (pb) was drawn at day of leukapheresis, at day of infusion/after lymphodepletion (LD, d0) and at several time points following Ide-cel infusion (d7, d14, d30 and d100). In order to reflect a comprehensive picture of the longitudinal dynamics of CAR and non-CAR T cells, we subjected pb mononuclear cells to advanced flow cytometry. Additionally, we assessed the impact of cellular dynamics on the occurrence of cytokine release syndrome (CRS), tocilizumab application and the incidence of cytopenia. Results The median number of infused CAR T cells was 430x10 6 (range: 248.8 - 489.1x10 6) with one exception (25.7x10 6). There was no correlation between the median number of infused cells and in vivo expansion of CAR T cells after infusion as well as response or the development of CRS or cytopenia. Significant differences between responders and non-responders were already detected on day of leukapheresis: While non-responders showed similar numbers of CD4+ and CD8+ T cells, responders had significantly higher CD8+ T cell counts as compared to CD4+ T cells (p<0.05, Figure 1). Additionally, significantly reduced percentages of effector memory CD8+ T cells were detected in responders as compared to non-responders. The latter group showed significantly increased numbers of CD3+CD8+ T cells at the day of infusion indicating a less effective LD in non-responders. Following CAR T cell infusion, peak expansion occurred two weeks after infusion and was significantly higher in responders as compared to non-responders (p<0.001, Figure 2). CD3+ CAR T cell compartment predominantly comprised of CD8+ T cells regardless of response (median: 85%). Analysis of the distribution of CAR and non-CAR T cells after infusion revealed that the majority of CD4+ T cells in responders and non-responders were untransfected. Similar results were detected for CD8+ T cells in non-responders. However, in responders CD8+ CAR T cells approximated CD8+ non-CAR T cells with no significant differences on d7 and d14. With regard to T cell differentiation, almost the entire CD4+ T cells exhibited either a central memory or effector memory phenotype after CAR T cell infusion. Shortly after CAR T cell infusion, effector memory CD8+ T cells outbalanced all other CD8+ T cell subtypes. Most pts had no detectable CAR T cells 100 days after infusion, regardless of remission. Overall, 22 pts (81%) developed CRS shortly after CAR T cell infusion and 7 of these (32%) required tocilizumab treatment. Whereas percentages of CD3+ CAR T cells were significantly elevated in both CRS positive groups (p<0.05) shortly after infusion, absolute numbers were significantly increased only in CRS positive pts without tocilizumab. Analysis of blood counts following CAR T cell infusion revealed that cytopenias persisted longer in CRS pts treated with tocilizumab, which was also reflected by significantly increased numbers of days below thresholds for all cell lines (anemia, thrombocytopenia and leukopenia, p<0.01). Even though the occurrence of CRS and especially the treatment with tocilizumab affected blood cell composition, it did not correlate to response. Conclusion We demonstrate that BCMA-targeting CAR T cells only transiently persist in pb after infusion. Still, initial CAR T cell expansion, predominantly caused by effector memory CD8+ CAR T cells, was linked to response and CRS in RRMM pts. A correlation between CRS and tocilizumab administration with prolonged cytopenias following CAR T cell infusion was also observed. Our data provide first evidence that responders and non-responders can early be distinguished by differential cellular composition in CAR and non-CAR T cell compartments with distinct features already present at day of apheresis.

Primary cilium participates in radiation-induced bystander effects through TGF-β1 signaling.

Many studies have indicated that tumor growth factor-beta (TGF-β) signaling mediates radiation-induced bystander effects (RIBEs). The primary cilium (PC) coordinates several signaling pathways including TGF-β signaling to regulate diverse cellular processes. But whether the PC participates in TGF-β induced RIBEs remains unclear.The cellular levels of TGF-β1 were detected by western blot analysis and the secretion of TGF-β1 was measured by ELISA kit. The ciliogenesis was altered by CytoD treatment, STIL siRNA transfection, IFT88 siRNA transfection, or KIF3a siRNA transfection, separately, and was detected by western blot analysis and immunofluorescence staining. G0 /G1 phase cells were arrested by serum starvation and S phase cells were induced by double thymidine block. The TGF-β1 signaling was interfered by LY2109761, a TGF-β receptor 1 (TβR1) inhibitor, or TGF-β1 neutral antibody. The DNA damages were induced by TGF-β1 or radiated conditional medium (RCM) from irradiated cells and were reflected by p21 expression, 53BP1 foci, and γH2AX foci.Compared with unirradiated control, both A549 and Beas-2B cells expressed and secreted more TGF-β1 after carbon ion beam or X-ray irradiation. RCM collected from irradiated cells or TGF-β1 treatment caused an increase of DNA damage in cocultured unirradiated Beas-2B cells while blockage of TGF-β signaling by TβR1 inhibitor or TGF-β1 neutral antibody alleviates this phenomenon. IFT88 siRNA or KIF3a siRNA impaired PC formation resulted in an aggravated DNA damage in bystander cells, while elevated PC formation by CytoD or STIL siRNA resulted in a decrease of DNA damage. Furthermore, TGF-β1 induced more DNA damages in S phases cells which showed lower PC formation rate and less DNA damages in G0 /G1 phase cells which showed higher PC formation rate.This study demonstrates the particular role of primary cilia during RCM induced DNA damages through TGF-β1 signaling restriction and thereby provides a functional link between primary cilia and RIBEs.

Toll-like receptor 9 signaling in chronic lymphocytic leukemia cell lines.

Chronic lymphocytic leukemia (CLL) is a prototypic neoplasia in which malignant cells strongly depend on microenvironmental stimulations in the lymphoid tissues where they accumulate; leukemic cells are exposed to interaction with bystander and accessory cells, as well as inflammatory soluble mediators. Cell lines are frequently used to model the pathobiology of this disease; however, they do not always recapitulate leukemic cell growth and response to stimulation, and no data are available on Toll-like receptors (TLR) signaling in CLL cell lines. To address this gap, we analyzed HG3, MEC2, and PCL12 cell lines, before and after CpG stimulation, by RNA-sequencing followed by bioinformatic analyses and validation experiments. We identified NFKBIZ mRNA and the corresponding IkBz protein as robust markers of TLR9 activation in both MEC2 and PCL12, but not in HG3 cells. Next, we compared our current results with previous results obtained with primary CLL patient samples and were able to conclude that MEC2 is most similar to the patients' cells in terms of global responsiveness to TLR stimulation; in particular, MEC2 better resembles the samples of patients, as it is characterized by high expression levels of IkBz, but with a lower number of genes regulated.

The COX-2/PGE2 Response Pathway Upregulates Radioresistance in A549 Human Lung Cancer Cells through Radiation-Induced Bystander Signaling.

This study aimed to determine the mechanism underlying the modulation of radiosensitivity in cancer cells by the radiation-induced bystander effect (RIBE). We hypothesized that the RIBE mediates cyclooxygenase-2 (COX-2) and its metabolite prostaglandin E2 (PGE2) in elevating radioresistance in unirradiated cells. In this study, we used the SPICE-QST microbeam irradiation system to target 0.07-0.7% cells by 3.4-MeV proton microbeam in the cell culture sample, such that most cells in the dish became bystander cells. Twenty-four hours after irradiation, we observed COX-2 protein upregulation in microbeam-irradiated cells compared to that of controls. Additionally, 0.29% of the microbeam-irradiated cells exhibited increased cell survival and a reduced micronucleus rate against X-ray irradiation compared to that of non-microbeam irradiated cells. The radioresistance response was diminished in both cell groups with the hemichannel inhibitor and in COX-2-knockout cells under cell-to-cell contact and sparsely distributed conditions. The results indicate that the RIBE upregulates the cell radioresistance through COX-2/PGE2 intercellular responses, thereby contributing to issues, such as the risk of cancer recurrence.

Systematic functional interrogation of SARS-CoV-2 host factors using Perturb-seq

Genomic and proteomic screens have identified numerous host factors of SARS-CoV-2, but efficient delineation of their molecular roles during infection remains a challenge. Here we use Perturb-seq, combining genetic perturbations with a single-cell readout, to investigate how inactivation of host factors changes the course of SARS-CoV-2 infection and the host response in human lung epithelial cells. Our high-dimensional data resolve complex phenotypes such as shifts in the stages of infection and modulations of the interferon response. However, only a small percentage of host factors showed such phenotypes upon perturbation. We further identified the NF-κB inhibitor IκBα (NFKBIA), as well as the translation factors EIF4E2 and EIF4H as strong host dependency factors acting early in infection. Overall, our study provides massively parallel functional characterization of host factors of SARS-CoV-2 and quantitatively defines their roles both in virus-infected and bystander cells.

PIM1 controls GBP1 activity to limit self-damage and to guard against pathogen infection.

Disruption of cellular activities by pathogen virulence factors can trigger innate immune responses. Interferon-γ (IFN-γ)-inducible antimicrobial factors, such as the guanylate binding proteins (GBPs), promote cell-intrinsic defense by attacking intracellular pathogens and by inducing programmed cell death. Working in human macrophages, we discovered that GBP1 expression in the absence of IFN-γ killed the cells and induced Golgi fragmentation. IFN-γ exposure improved macrophage survival through the activity of the kinase PIM1. PIM1 phosphorylated GBP1, leading to its sequestration by 14-3-3σ, which thereby prevented GBP1 membrane association. During Toxoplasma gondii infection, the virulence protein TgIST interfered with IFN-γ signaling and depleted PIM1, thereby increasing GBP1 activity. Although infected cells can restrain pathogens in a GBP1-dependent manner, this mechanism can protect uninfected bystander cells. Thus, PIM1 can provide a bait for pathogen virulence factors, guarding the integrity of IFN-γ signaling.

Typhoid toxin hijacks Wnt5a to establish host senescence and Salmonella infection

Damage to our genome causes acute senescence in mammalian cells, which undergo growth arrest and release a senescence-associated secretory phenotype (SASP) that propagates the stress response to bystander cells. Thus, acute senescence is a powerful tumor suppressor. Salmonella enterica hijacks senescence through its typhoid toxin, which usurps unidentified factors in the stress secretome of senescent cells to mediate intracellular infections. Here, transcriptomics of toxin-induced senescent cells (TxSCs) and proteomics of their secretome identify the factors as Wnt5a, INHBA, and GDF15. Wnt5a establishes a positive feedback loop, driving INHBA and GDF15 expression. In fibroblasts, Wnt5a and INHBA mediate autocrine senescence in TxSCs and paracrine senescence in naive cells. Wnt5a synergizes with GDF15 to increase Salmonella invasion. Intestinal TxSCs undergo apoptosis without Wnt5a, which is required for establishing intestinal TxSCs. The study reveals how an innate defense against cancer is co-opted by a bacterial pathogen to cause widespread damage and mediate infections.

IFN-λ derived from nonsusceptible enterocytes acts on tuft cells to limit persistent norovirus.

Norovirus is a leading cause of epidemic viral gastroenteritis, with no currently approved vaccines or antivirals. Murine norovirus (MNoV) is a well-characterized model of norovirus pathogenesis in vivo, and persistent strains exhibit lifelong intestinal infection. Interferon-λ (IFN-λ) is a potent antiviral that rapidly cures MNoV. We previously demonstrated that IFN-λ signaling in intestinal epithelial cells (IECs) controls persistent MNoV, and here demonstrate that IFN-λ acts on tuft cells, the exclusive site of MNoV persistence, to limit infection. While interrogating the source of IFN-λ to regulate MNoV, we confirmed that MDA5-MAVS signaling, required for IFN-λ induction to MNoV in vitro, controls persistent MNoV in vivo. We demonstrate that MAVS in IECs and not immune cells controls MNoV. MAVS in nonsusceptible enterocytes, but not in tuft cells, restricts MNoV, implicating noninfected cells as the IFN-λ source. Our findings indicate that host sensing of MNoV is distinct from cellular tropism, suggesting intercellular communication between IECs for antiviral signaling induction in uninfected bystander cells.

Secreted NS1 proteins of tick-borne encephalitis virus and West Nile virus block dendritic cell activation and effector functions.

The flavivirus non-structural protein 1 (NS1) is secreted from infected cells into the circulation and the serum levels correlate with disease severity. The effect of secreted NS1 (sNS1) on non-infected mammalian immune cells is largely unknown. Here, we expressed recombinant sNS1 proteins of tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) and investigated their effects on dendritic cell (DC) effector functions. Murine bone marrow-derived DCs (BMDCs) showed reduced surface expression of co-stimulatory molecules and decreased release of pro-inflammatory cytokines when treated with sNS1 of TBEV or WNV prior to poly(I:C) stimulation. Transcriptional profiles of BMDCs that were sNS1-exposed prior to poly(I:C) stimulation showed two gene clusters that were downregulated by TBEV or WNV sNS1 and that were associated with innate and adaptive immune responses. Functionally, both sNS1 proteins modulated the capacity for BMDCs to induce specific T-cell responses as indicated by reduced IFN-γ levels in both CD4+ and CD8+ T cells after BMDC co-cultivation. In human monocyte-derived DCs, poly(I:C)-induced upregulation of co-stimulatory molecules and cytokine responses were even more strongly impaired by TBEV sNS1 or WNV sNS1 pretreatment than in the murine system. Our findings indicate that exogenous flaviviral sNS1 proteins interfere with DC-mediated stimulation of T cells, which is crucial for the initiation of cell-mediated adaptive immune responses in human flavivirus infections. Collectively, our data determine soluble flaviviral NS1 as a virulence factor responsible for a dampened immune response to flavivirus infections. IMPORTANCE The effective initiation of protective host immune responses controls the outcome of infection, and dysfunctional T-cell responses have previously been associated with symptomatic human flavivirus infections. We demonstrate that secreted flavivirus NS1 proteins modulate innate immune responses of uninfected bystander cells. In particular, sNS1 markedly reduced the capacity of dendritic cells to stimulate T-cell responses upon activation. Hence, by modulating cellular host responses that are required for effective antigen presentation and initiation of adaptive immunity, sNS1 proteins may contribute to severe outcomes of flavivirus disease.

Unambiguous detection of SARS-CoV-2 subgenomic mRNAs with single-cell RNA sequencing.

Single-cell RNA sequencing (scRNA-Seq) studies have provided critical insight into the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). scRNA-Seq library preparation methods and data processing workflows are generally designed for the detection and quantification of eukaryotic host mRNAs and not viral RNAs. Here, we compare different scRNA-Seq library preparation methods for their ability to quantify and detect SARS-CoV-2 RNAs with a focus on subgenomic mRNAs (sgmRNAs). We show that compared to 10X Genomics Chromium Next GEM Single Cell 3' (10X 3') libraries or 10X Genomics Chromium Next GEM Single Cell V(D)J (10X 5') libraries sequenced with standard read configurations, 10X 5' libraries sequenced with an extended length read 1 (R1) that covers both cell barcode and transcript sequence (termed "10X 5' with extended R1") increase the number of unambiguous reads spanning leader-sgmRNA junction sites. We further present a data processing workflow, single-cell coronavirus sequencing (scCoVseq), which quantifies reads unambiguously assigned to viral sgmRNAs or viral genomic RNA (gRNA). We find that combining 10X 5' with extended R1 library preparation/sequencing and scCoVseq data processing maximizes the number of viral UMIs per cell quantified by scRNA-Seq. Corresponding sgmRNA expression levels are highly correlated with expression in matched bulk RNA-Seq data sets quantified with established tools for SARS-CoV-2 analysis. Using this scRNA-Seq approach, we find that SARS-CoV-2 gene expression is highly correlated across individual infected cells, which suggests that the proportion of viral sgmRNAs remains generally consistent throughout infection. Taken together, these results and corresponding data processing workflow enable robust quantification of coronavirus sgmRNA expression at single-cell resolution, thereby supporting high-resolution studies of viral RNA processes in individual cells. IMPORTANCE Single-cell RNA sequencing (scRNA-Seq) has emerged as a valuable tool to study host-virus interactions, especially for coronavirus disease 2019 (COVID-19). Here we compare the performance of different scRNA-Seq library preparation methods and sequencing strategies to detect SARS-CoV-2 RNAs and develop a data processing workflow to quantify unambiguous sequence reads derived from SARS-CoV-2 genomic RNA and subgenomic mRNAs. After establishing a workflow that maximizes the detection of SARS-CoV-2 subgenomic mRNAs, we explore patterns of SARS-CoV-2 gene expression across cells with variable levels of total viral RNA, assess host gene expression differences between infected and bystander cells, and identify non-canonical and lowly abundant SARS-CoV-2 RNAs. The sequencing and data processing strategies developed here can enhance studies of coronavirus RNA biology at single-cell resolution and thereby contribute to our understanding of viral pathogenesis.

Interplay of Ebola Virus With Immune Cells Leading to Their Death by Diverse Mechanisms.

Inflammation and cytopenia are commonly observed during Ebola virus (EBOV) infection; however, mechanisms responsible for EBOV-induced cell death remain obscure. While apoptosis and necrosis are already identified as mechanisms of cell death induced by the virus, our study demonstrates that THP-1 monocytes and SupT1 T cells exposed to EBOV undergo pyroptosis and necroptosis, respectively, through a direct contact with EBOV, and also mediate pyroptosis or necroptosis of uninfected bystander cells via indirect effects associated with secreted soluble factors. These results emphasize novel aspects of interactions between EBOV and immune cell populations and provide a better understanding of the immunopathogenesis of EBOV disease.

Ebola Virus Infection Induces HCAR2 Expression Leading to Cell Death.

Ebola virus (EBOV) induces cell death not only in infected permissive cells but also in nonpermissive, bystander cells by employing different mechanisms. Hydroxycarboxylic acid receptor 2 (HCAR2) has been reported to be involved in apoptotic cell death. We previously reported an increase in the expression of HCAR2-specific mRNA in EBOV-infected individuals with fatal outcomes. Here, we report that infection with an EBOV lacking the VP30 gene (EBOVΔVP30) results in the upregulation of HCAR2 mRNA expression in human hepatocyte Huh7.0 cells stably expressing VP30. Transient overexpression of HCAR2 reduced the viability of Huh7.0 cells and human embryonic kidney cells. Phosphatidylserine externalization and cell membrane permeabilization by HCAR2 overexpression was also observed. Interestingly, coexpression of HCAR2 with EBOV VP40 further reduced cell viability in transfected cells compared to HCAR2 coexpression with other viral proteins. Our data suggest that HCAR2 may contribute to EBOV-induced cell death.

CGAS-STING drives ageing-related inflammation and neurodegeneration.

Low-grade inflammation is a hallmark of old age and a central driver of ageing-associated impairment and disease1. Multiple factors can contribute to ageing-associated inflammation2; however, the molecular pathways that transduce aberrant inflammatory signalling and their impact in natural ageing remain unclear. Here we show that the cGAS-STING signalling pathway, which mediates immune sensing of DNA3, is a critical driver of chronic inflammation and functional decline during ageing. Blockade of STING suppresses the inflammatory phenotypes of senescent human cells and tissues, attenuates ageing-related inflammation in multiple peripheral organs and the brain in mice, and leads to an improvement in tissue function. Focusing on the ageing brain, we reveal that activation of STING triggers reactive microglial transcriptional states, neurodegeneration and cognitive decline. Cytosolic DNA released from perturbed mitochondria elicits cGAS activity in old microglia, defining a mechanism by which cGAS-STING signalling is engaged in the ageing brain. Single-nucleus RNA-sequencing analysis of microglia and hippocampi of a cGAS gain-of-function mouse model demonstrates that engagement of cGAS in microglia is sufficient to direct ageing-associated transcriptional microglial states leading to bystander cell inflammation, neurotoxicity and impaired memory capacity. Our findings establish the cGAS-STING pathway as a driver of ageing-related inflammation in peripheral organs and the brain, and reveal blockade of cGAS-STING signalling as a potential strategy to halt neurodegenerative processes during old age.

Pro-Tumor Activity of Endogenous Nitric Oxide in Anti-Tumor Photodynamic Therapy: Recently Recognized Bystander Effects.

Various studies have revealed that several cancer cell types can upregulate inducible nitric oxide synthase (iNOS) and iNOS-derived nitric oxide (NO) after moderate photodynamic treatment (PDT) sensitized by 5-aminolevulinic acid (ALA)-induced protoporphyrin-IX. As will be discussed, the NO signaled cell resistance to photokilling as well as greater growth and migratory aggressiveness of surviving cells. On this basis, it was predicted that diffusible NO from PDT-targeted cells in a tumor might enhance the growth, migration, and invasiveness of non- or poorly PDT-targeted bystander cells. This was tested using a novel approach in which ALA-PDT-targeted cancer cells on a culture dish were initially segregated from non-targeted bystander cells of the same type via impermeable silicone-rimmed rings. Several hours after LED irradiation, the rings were removed, and both cell populations were analyzed in the dark for various responses. After a moderate extent of targeted cell killing (~25%), bystander proliferation and migration were evaluated, and both were found to be significantly enhanced. Enhancement correlated with iNOS/NO upregulation in surviving PDT-targeted cancer cells in the following cell type order: PC3 > MDA-MB-231 > U87 > BLM. If occurring in an actual PDT-challenged tumor, such bystander effects might compromise treatment efficacy by stimulating tumor growth and/or metastatic dissemination. Mitigation of these and other negative NO effects using pharmacologic adjuvants that either inhibit iNOS transcription or enzymatic activity will be discussed.