The cellular localisation of many innate signalling events following viral infection has yet to be elucidated, however there has been a few cases in which membranes of certain cellular organelles have acted as platforms to these events. Of these, lipid droplets (LDs) have recently been identified as signalling platforms for innate TLR7 and 9 signalling. Despite their wide range of similar roles in various metabolic pathways, LDs have been overlooked as potential platforms for antiviral innate signalling events. This study established an in vitro model to evaluate the efficiency of the early innate immune response in cells with reduced LD content to the viral mimics, dsDNA and dsRNA, and Sendai viral infection. Using RT-qPCR, the expression of IFN-β and IFN-λ was quantified following stimulation along with the expression of specific ISGs. Luciferase based assays evaluated the combined expression of ISRE-promoter driven ISGs under IFN-β stimulation. Cellular LD content did not alter the entry of fluorescently labelled viral mimics into cells, but significantly decreased the ability of both Huh-7 and HeLa cells to produce type I and III IFN, as well as downstream ISG expression, indicative of an impeded innate immune response. This observation was also seen during Sendai virus infection of HeLa cells, where both control and LD reduced cells replicated the virus to the same level, but a significantly impaired type I and III IFN response was observed in the LD reduced cells. In addition to altered IFN production, cells with reduced LD content exhibited decreased expression of specific antiviral ISGs: Viperin, IFIT-1 and OAS-1 under IFN-β stimulation; However the overall induction of the ISRE-promoter was not effected. This study implicates a role for LDs in an efficient early innate host response to viral infection and future work will endeavour to determine the precise role these important organelles play in induction of an antiviral response.

Significance Statement: Missense variants of NPHS2 that cause mistrafficking of the encoded protein, PODOCIN, have been associated with steroid-resistant nephrotic syndrome. However, most studies have overexpressed such variants in 2D nonpodocyte cells. This study describes the generation and characterization of human kidney organoids representing an allelic series of homozygous NPHS2 missense variants. The strategy revealed a previously unappreciated reduction in variant PODOCIN protein, variant-specific subcellular localization, and specific effects on NEPHRIN association. All variants showed apoptosis in the absence of endoplasmic reticulum stress. Engineering endogenous NPHS2 variants to model in 3D human organoids provides a more accurate view of the pathobiology and a toolkit to screen compounds for reduction of variant protein degradation and mistrafficking. Background: NPHS2 variants are the most common cause of steroid-resistant nephrotic syndrome in children >1 month old. Missense NPHS2 variants were reported to cause mistrafficking of the encoded protein, PODOCIN, but this conclusion was on the basis of overexpression in some nonpodocyte cell lines. Methods: We generated a series of human induced pluripotent stem cell (iPSC) lines bearing pathogenic missense variants of NPHS2 , encoding the protein changes p.G92C, p.P118L, p.R138Q, p.R168H, and p.R291W, and control lines. iPSC lines were also generated from a patient with steroid-resistant nephrotic syndrome (p.R168H homozygote) and a healthy heterozygous parent. All lines were differentiated into kidney organoids. Immunofluorescence assessed PODOCIN expression and subcellular localization. Podocytes were transcriptionally profiled and PODOCIN-NEPHRIN interaction interrogated. Results: All variant lines revealed reduced levels of PODOCIN protein in the absence of reduced transcription. Although wild-type PODOCIN localized to the membrane, distinct variant proteins displayed unique patterns of subcellular protein trafficking, some unreported. P118L and R138Q were preferentially retained in the endoplasmic reticulum (ER); R168H and R291W accumulated in the Golgi. Podocyte profiling demonstrated minimal disease-associated transcriptional change. All variants displayed podocyte-specific apoptosis, which was not linked to ER stress. NEPHRIN-PODOCIN colocalization elucidated the variant-specific effect on NEPHRIN association and hence NEPHRIN trafficking. Conclusions: Specific variants of endogenous NPHS2 result in distinct subcellular PODOCIN localization within organoid podocytes. Understanding the effect of each variant on protein levels and localization and the effect on NEPHRIN provides additional insight into the pathobiology of NPHS2 variants. Podcast: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2023_01_05_JASN2022060707.mp3

Lung disease in cystic fibrosis (CF) is often exacerbated following acute upper respiratory tract infections caused by the human rhinovirus (HRV). Pathophysiology of these exacerbations is presently unclear and may involve deficient innate antiviral or exaggerated inflammatory responses in CF airway epithelial cells. Furthermore, responses of CF cells to HRV may be adversely affected by pre-exposure to virulence factors of Pseudomonas (P.) aeruginosa, the microorganism that frequently colonizes CF airways. Here we examined production of antiviral cytokine interferon-β and inflammatory chemokine interleukin-8, expression of the interferon-responsive antiviral gene 2’-5’-oligoadenylate synthetase 1 (OAS1), and intracellular virus RNA load in primary CF (delF508 CFTR) and healthy airway epithelial cells following inoculation with HRV16. Parallel cells were exposed to virulence factors of P. aeruginosa prior to and during HRV16 inoculation. CF cells exhibited production of interferon-β and interleukin-8, and expression of OAS1 at levels comparable to those in healthy cells, yet significantly higher HRV16 RNA load during early hours post-inoculation with HRV16. In line with this, HRV16 RNA load was higher in the CFBE41o- dF cell line overexpessing delF508 CFTR, compared with the isogenic control CFBE41o- WT (wild-type CFTR). Pre-exposure to virulence factors of P. aeruginosa did not affect OAS1 expression or HRV16 RNA load, but potentiated interleukin-8 production. In conclusion, CF cells demonstrate elevated HRV RNA load despite preserved interferon-β and OAS1 responses. High HRV load in CF airway epithelial cells appears to be due to deficiencies manifesting early during HRV infection, and may not be related to interferon-β.

Zika virus (ZIKV) is a pathogenic neurotropic virus that infects the central nervous system (CNS) and results in various neurological complications. Astrocytes are the dominant CNS cell producer of the antiviral cytokine IFN-β, however little is known about the factors involved in their ability to mediate viral infection control. Recent studies have displayed differential responses in astrocytes to ZIKV infection, and this study sought to elucidate astrocyte cell-specific responses to ZIKV using a variety of cell models infected with either the African (MR766) or Asian (PRVABC59) ZIKV strains. Expression levels of pro-inflammatory (TNF-α and IL-1β) and inflammatory (IL-8) cytokines following viral infection were low and mostly comparable within the ZIKV-resistant and ZIKV-susceptible astrocyte models, with better control of proinflammatory cytokines displayed in resistant astrocyte cells, synchronising with the viral infection level at specific timepoints. Astrocyte cell lines displaying ZIKV-resistance also demonstrated early upregulation of multiple antiviral genes compared with susceptible astrocytes. Interestingly, pre-stimulation of ZIKV-susceptible astrocytes with either poly(I:C) or poly(dA:dT) showed efficient protection against ZIKV compared with pre-stimulation with either recombinant IFN-β or IFN-λ, perhaps indicating that a more diverse antiviral gene expression is necessary for astrocyte control of ZIKV, and this is driven in part through interferon-independent mechanisms.