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Unveiling Interindividual Variability of Human Fibroblast Innate Immune Response Using Robust Cell-Based Protocols.

ABSTRACT: The LabEx Milieu Interieur (MI) project is a clinical study centered on the detailed characterization of the baseline and induced immune responses in blood samples from 1,000 healthy donors. Analyses of these samples has lay ground for seminal studies on the genetic and environmental determinants of immunologic variance in a healthy cohort population. In the current study we developed in vitro methods enabling standardized quantification of MI-cohort-derived primary fibroblasts responses. Our results show that in vitro human donor cohort fibroblast responses to stimulation by different MAMPs analogs allows to characterize individual donor immune-phenotype variability. The results provide proof-of-concept foundation to a new experimental framework for such studies. A bio-bank of primary fibroblast lines was generated from 323 out of 1,000 healthy individuals selected from the MI-study cohort. To study inter-donor variability of innate immune response in primary human dermal fibroblasts we chose to measure the TLR3 and TLR4 response pathways, both receptors being expressed and previously studied in fibroblasts. We established high-throughput automation compatible methods for standardized primary fibroblast cell activation, using purified MAMPS analogs, poly I:C and LPS that stimulate TLR3 and TLR4 pathways respectively. These results were in turn compared with a stimulation method using infection by HSV-1 virus. Our "Add-only" protocol minimizes high-throughput automation system variability facilitating whole process automation from cell plating through stimulation to recovery of cell supernatants, and fluorescent labeling. Images were acquired automatically by high-throughput acquisition on an automated high-content imaging microscope. Under these methodological conditions standardized image acquisition provided for quantification of cellular responses allowing biological variability to be measured with low system noise and high biological signal fidelity. Optimal for automated analysis of immuno-phenotype of primary human cell responses our method and experimental framework as reported here is highly compatible to high-throughput screening protocols like those necessary for chemo-genomic screening. In context of primary fibroblasts derived from donors enrolled to the MI-clinical-study our results open the way to assert the utility of studying immune-phenotype characteristics relevant to a human clinical cohort.

SUBMITTER: Chansard A

PROVIDER: S-EPMC7829859 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Fibroblast growth factor-23 (FGF-23) is a bone-derived hormone that activates FGFR/?-Klotho binary complexes in the kidney renal tubules to regulate phosphate reabsorption and vitamin D metabolism. The objective of this review is to discuss the emerging data that show that FGF-23 has functions beyond regulation of mineral metabolism, including roles in innate immune and hemodynamic responses. Excess FGF-23 is associated with inflammation and adverse infectious outcomes, as well as increased morbidity and mortality, particularly in patients with chronic kidney disease. Enhancer elements in the FGF-23 promoter have been identified that mediate the effects of inflammatory cytokines to stimulate FGF-23 gene transcription in bone. In addition, inflammation induces ectopic expression of FGF-23 and ?-Klotho in macrophages that do not normally express FGF-23 or its binary receptor complexes. These observations suggest that FGF-23 may play an important role in regulating innate immunity through multiple potential mechanisms. Circulating FGF-23 acts as a counter-regulatory hormone to suppress 1,25D production in the proximal tubule of the kidney. Since vitamin D deficiency may predispose infectious and cardiovascular diseases, FGF-23 effects on innate immune responses may be due to suppression of 1,25D production. Alternatively, systemic and locally produced FGF-23 may modulate immune functions through direct interactions with myeloid cells, including macrophages and polymorphonuclear leukocytes to impair immune cell functions. Short-acting small molecules that reversibly inhibit FGF-23 offer the potential to block pro-inflammatory and cardiotoxic effects of FGF-23 with less side effects compared with FGF-23 blocking antibodies that have the potential to cause hyperphosphatemia and soft tissue calcifications in animal models. In conclusion, there are several mechanisms by which FGF-23 impacts the innate immune system and further investigation is critical for the development of therapies to treat diseases associated with elevated FGF-23.

Project description:The human innate immune response, particularly the type-I interferon (IFN) response, is highly robust and effective first line of defense against virus invasion. IFN molecules are produced and secreted from infected cells upon virus infection and recognition. They then act as signaling/communication molecules to activate an antiviral response in neighboring cells so that those cells become refractory to infection. Previous experimental studies have identified the detailed molecular mechanisms for the IFN signaling and response. However, the principles underlying how host cells use IFN to communicate with each other to collectively and robustly halt an infection is not understood. Here we take a multiplex network modeling approach to provide a theoretical framework to identify key factors that determine the effectiveness of the IFN response against virus infection of a host. In this approach, we consider the virus spread among host cells and the interferon signaling to protect host cells as a competition process on a two-layer multiplex network. We focused on two types of network topology, i.e., the Erdős-Rényi (ER) network and the Geometric Random (GR) network, which represent the scenarios when infection of cells is mostly well mixed (e.g., in the blood) and when infection is spatially segregated (e.g., in tissues), respectively. We show that in general, the IFN response works effectively to stop viral infection when virus infection spreads spatially (a most likely scenario for initial virus infection of a host at the peripheral tissue). Importantly, we show that the effectiveness of the IFN response is robust against large variations in the distance of IFN diffusion as long as IFNs diffuse faster than viruses and they can effectively induce antiviral responses in susceptible host cells. This suggests that the effectiveness of the IFN response is insensitive to the specific arrangement of host cells in peripheral tissues. Thus, our work provides a quantitative explanation of why the IFN response can serve an effective and robust response in different tissue types to a wide range of viral infections of a host.

Project description:BackgroundAlzheimer's disease (AD) is a progressive neurodegenerative disease that impacts nearly 400 million people worldwide. The accumulation of amyloid beta (Aβ) in the brain has historically been associated with AD, and recent evidence suggests that neuroinflammation plays a central role in its origin and progression. These observations have given rise to the theory that Aβ is the primary trigger of AD, and induces proinflammatory activation of immune brain cells (i.e., microglia), which culminates in neuronal damage and cognitive decline. To test this hypothesis, many in vitro systems have been established to study Aβ-mediated activation of innate immune cells. Nevertheless, the transcriptional resemblance of these models to the microglia in the AD brain has never been comprehensively studied on a genome-wide scale.MethodsWe used bulk RNA-seq to assess the transcriptional differences between in vitro cell types used to model neuroinflammation in AD, including several established, primary and iPSC-derived immune cell lines (macrophages, microglia and astrocytes) and their similarities to primary cells in the AD brain. We then analyzed the transcriptional response of these innate immune cells to synthetic Aβ or LPS and INFγ.ResultsWe found that human induced pluripotent stem cell (hIPSC)-derived microglia (IMGL) are the in vitro cell model that best resembles primary microglia. Surprisingly, synthetic Aβ does not trigger a robust transcriptional response in any of the cellular models analyzed, despite testing a wide variety of Aβ formulations, concentrations, and treatment conditions. Finally, we found that bacterial LPS and INFγ activate microglia and induce transcriptional changes that resemble many, but not all, aspects of the transcriptomic profiles of disease associated microglia (DAM) present in the AD brain.ConclusionsThese results suggest that synthetic Aβ treatment of innate immune cell cultures does not recapitulate transcriptional profiles observed in microglia from AD brains. In contrast, treating IMGL with LPS and INFγ induces transcriptional changes similar to those observed in microglia detected in AD brains.

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Unveiling Interindividual Variability of Human Fibroblast Innate Immune Response Using Robust Cell-Based Protocols.

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