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Abstract Human cytomegalovirus (HCMV) is an opportunistic pathogen causing severe diseases in immunosuppressed individuals. To replicate its double-stranded DNA genome, HCMV induces profound changes in cellular homeostasis that may resemble senescence. However, it remains to be determined whether HCMV-induced senescence contributes to organ-specific pathogenesis. Here, we show a direct cytopathic effect of HCMV on primary renal proximal tubular epithelial cells (RPTECs), a natural setting of HCMV disease. We find that RPTECs are fully permissive for HCMV replication, which endows them with an inflammatory gene signature resembling the senescence-associated secretory phenotype (SASP), as confirmed by the presence of the recently established SenMayo gene set, which is not observed in retina-derived epithelial (ARPE-19) cells. Although HCMV-induced senescence is not cell-type specific, as it can be observed in both RPTECs and human fibroblasts (HFFs), only infected RPTECs show downregulation of LAMINB1 and KI67 mRNAs, and enhanced secretion of IL-6 and IL-8, which are well-established hallmarks of senescence. Finally, HCMV-infected RPTECs have the ability to trigger a senescence/inflammatory loop in an IL-6-dependent manner, leading to the development of a similar senescence/inflammatory phenotype in neighboring uninfected cells. Overall, our findings raise the intriguing possibility that this unique inflammatory loop contributes to HCMV-related pathogenesis in the kidney.

ABSTRACT In this prospective study, we used droplet digital (dd)PCR to determine the SARS-CoV-2 viral load determination in saliva samples collected from two cohorts of Italian health care workers (HCWs) who had tested positive for SARS-CoV-2 breakthrough infections in nasopharyngeal swabs (NPS). The study encompasses two distinct periods: one from February to May 2021 (n = 16), characterized by Alpha variant predominance, and another from January to March 2022 (n = 23), marked by the prevalence of the Omicron variant. To assess the temporal pattern of virus shedding, we performed dd-PCR analysis of self-collected longitudinal saliva harvested the day after NPS testing and every 3 days until SARS-CoV-2 negativization, determined by real-time PCR (RT-PCR). Using saliva harvested within 24 h from the initial SARS-CoV-2-positive NPS tested by real-time quantitative PCR (RT-qPCR), we found that 11/16 [68.7%, confidence interval (CI) 95% 44.0%–93.4%] HCWs in the “Alpha wave” and 6/23 (26.1%, CI 95% 7.1%–45.1%) in the “Omicron wave” were negative for the presence of SARS-CoV-2 by ddPCR. The viral loads obtained with these samples were negatively correlated with the cycle threshold (Ct) values measured in the NPS specimens (P < 0.0001). The median Ct values of the double-positive (RT-qPCR and ddPCR) subjects were significantly higher when compared to the single-positive (RT-qPCR only) ones. In addition, we found that using ddPCR in self-collected saliva, 80% of the HCWs in the “Alpha wave” and 53% in the “Omicron wave” would have been freed from isolation within four working days from the initial positive NPS, sparing 176 working days among the HCWs enrolled in the study. Overall, this study highlights the effectiveness of using ddPCR in self-collected saliva as part of a surveillance program. It ultimately aims to identify individuals who still test positive by RT-PCR in NPS but retains a residual, non-active infection. This diagnostic approach is likely to not only prevent unnecessary isolation but also spare working days. IMPORTANCE Real-time quantitative PCR (RT-qPCR) on nasopharyngeal swabs (NPS) has been used as the standard method for detecting and monitoring SARS-CoV-2 infection during the pandemic. However, NPS collection often causes discomfort and poses a higher risk of transmission to health care workers (HCW). Furthermore, RT-qPCR only provides relative quantification and does not allow distinguishing those samples with residual, no longer active infection, whereas droplet digital PCR (ddPCR) allows for precise quantification of viral load, offering greater sensitivity and reproducibility. This study highlights the effectiveness of using self-collected saliva as a convenient and reliable sampling method. By utilizing ddPCR to measure the SARS-CoV-2 viral load in saliva samples, individuals with low or undetectable viral loads can be quickly identified. This approach is particularly advantageous for surveillance programs targeting HCW, as it enables the early identification and release of uninfected personnel, minimizing lost workdays. Additionally, analyzing viral load in saliva samples by ddPCR is valuable in determining virus shedding duration across different SARS-CoV-2 variants, informing transmission and disease control. Finally, testing saliva could overcome the detection of historic cases due to prolonged RNA swabbing past-infection and the unnecessary exclusion of those individuals from the workplace.

Damage-associated molecular patterns (DAMPs) are endogenous molecules activating the immune system upon release from injured cells. Here we show that the IFI16 protein, once freely released in the extracellular milieu of chronically inflamed tissues, can function as a DAMP either alone or upon binding to lipopolysaccharide (LPS). Specifically, using pull-down and saturation binding experiments, we show that IFI16 binds with high affinity to the lipid A moiety of LPS. Remarkably, IFI16 DAMP activity is potentiated upon binding to subtoxic concentrations of strong TLR4-activating LPS variants, as judged by TLR4-MD2/TIRAP/MyD88-dependent IL-6, IL-8 and TNF-α transcriptional activation and release in stimulated monocytes and renal cells. Consistently, using co-immunoprecipitation (co-IP) and surface plasmon resonance (SPR) approaches, we show that IFI16 is a specific TLR4-ligand and that IFI16/LPS complexes display a faster stimulation turnover on TLR4 than LPS alone. Altogether, our findings point to a novel pathomechanism of inflammation involving the formation of multiple complexes between extracellular IFI16 and subtoxic doses of LPS variants, which then signal through TLR4.

Longitudinal mapping of antibody-based SARS-CoV-2 immunity is critical for public health control of the pandemic and vaccine development. We performed a longitudinal analysis of the antibody-based immune response in a cohort of 100 COVID-19 individuals who were infected during the first wave of infection in northern Italy. The SARS-CoV-2 humoral response was tested using the COVID-SeroIndex, Kantaro Quantitative SARS-CoV-2 IgG Antibody RUO Kit (R&D Systems, Bio-Techne, Minneapolis, USA) and pseudotype-based neutralizing antibody assay. Using sequential serum samples collected from 100 COVID-19 recovered individuals from northern Italy—mostly with mild disease—at 2 and 10 months after their first positive PCR test, we show that 93% of them seroconverted at 2 months, with a geometric mean (GeoMean) half-maximal neutralization titer (NT50) of 387.9. Among the 35 unvaccinated subjects retested at 10 months, 7 resulted seronegative, with an 80% drop in seropositivity, while 28 showed decreased anti-receptor binding domain (RBD) and anti-spike (S) IgG titers, with a GeoMean NT50 neutralization titer dropping to 163.5. As an NT50 > 100 is known to confer protection from SARS-CoV-2 re-infection, our data show that the neutralizing activity elicited by the natural infection has lasted for at least 10 months in a large fraction of subjects.

Absent in melanoma 2 (AIM2)-like receptors (ALRs) are a newly characterized class of pathogen recognition receptors (PRRs) involved in cytosolic and nuclear pathogen DNA recognition. In recent years, two ALR family members, the interferon (IFN)-inducible protein 16 (IFI16) and AIM2, have been linked to the pathogenesis of various autoimmune diseases, among which systemic lupus erythematosus (SLE) has recently gained increasing attention. SLE patients are indeed often characterized by constitutively high serum IFN levels and increased expression of IFN-stimulated genes due to an abnormal response to pathogens and/or incorrect self-DNA recognition process. Consistently, we and others have shown that IFI16 is overexpressed in a wide range of autoimmune diseases where it triggers production of specific autoantibodies. In addition, evidence from mouse models supports a model whereby ALRs are required for IFN-mediated host response to both exogenous and endogenous DNA. Following interaction with cytoplasmic or nuclear nucleic acids, ALRs can form a functional inflammasome through association with the adaptor ASC [apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD)] and with procaspase-1. Importantly, inflammasome-mediated upregulation of IL-1β and IL-18 production positively correlates with SLE disease severity. Therefore, targeting ALR sensors and their downstream pathways represents a promising alternative therapeutic approach for SLE and other systemic autoimmune diseases.

IFI16, a nuclear pathogenic DNA sensor induced by several pro-inflammatory cytokines, is a multifaceted protein with various functions. It is also a target for autoantibodies as specific antibodies have been demonstrated in the sera of patients affected by systemic autoimmune diseases. Following transfection of virus-derived DNA, or treatment with UVB, IFI16 delocalizes from the nucleus to the cytoplasm and is then eventually released into the extracellular milieu. In this study, using an in-house capture enzyme-linked immunsorbent assay we demonstrate that significant levels of IFI16 protein can also exist as circulating form in the sera of autoimmune patients. We also show that the rIFI16 protein, when added in-vitro to endothelial cells, does not affect cell viability, but severely limits their tubulogenesis and transwell migration activities. These inhibitory effects are fully reversed in the presence of anti-IFI16 N-terminal antibodies, indicating that its extracellular activity resides within the N-terminus. It was further demonstrated that endogenous IFI16 released by apoptotic cells bind neighboring cells in a co-culture. Immunofluorescence assays revealed existence of high-affinity binding sites on the plasma membrane of endothelial cells. Free recombinant IFI16 binds these sites on HUVEC with dissociation constant of 2.7 nM, radioiodinated and unlabeled IFI16 compete for binding sites, with inhibition constant (Ki) of 14.43 nM and half maximal inhibitory concentration (IC50) of 67.88 nM; these data allow us to estimate the presence of 250,000 to 450,000 specific binding sites per cell. Corroborating the results from functional assays, this binding could be completely inhibited using anti-IFI16 N-terminal antibody, but not with an antibody raised against the IFI16 C-terminal. Altogether, these data demonstrate that IFI16 may exist as circulating protein in the sera of autoimmune patients which binds endothelial cells causing damage, suggesting a new pathogenic and alarmin function through which this protein triggers the development of autoimmunity.