From Dysbiosis to Immune Rewiring: Proteomics-Based Mechanistic Insights into PFAS Effects on Human Health

This thesis investigates the effects that Per- and Poly-fluoroalkyl substances (PFAS) have on human health using proteomics approaches. PFAS are a big and heterogeneous class of man-made chemicals that present an alkyl chain and at least a fluorinated carbon. They have been vastly used for their exceptional abilities to resist high temperatures and repel water. These properties made them employable in many everyday products, but their vast usage led to their release in the environment and bioaccumulation. Many are the concerns and the proven risks regarding these “forever chemicals”, mainly the ones regarding their immunotoxic and carcinogenic effects in humans. Because of the risks associated with these molecules, many of them have been phased out of the market but newer and modified versions have been created to replace them. Not enough is known about these newer PFAS and their risks and more insights are needed to understand the biological mechanisms involved in traditional PFAS’s harmfulness. The work here presented is divided in three sections that try to shed light on PFAS impact on the human gut microbiota and immune system. Part of these molecules, when ingested, travel through the intestine but there is not enough information about how they interact and impact microbial communities in our gut. Instead, the immunotoxic effects of traditional PFAS are well known, but the mechanisms and proteins affected, as well as the effects of newer PFAS are still unclear. For the gut microbiota study, anaerobic human fecal fermentations, preserving the taxonomic and functional profiles of the human gut microbiome, was employed to culture gut bacteria cells with 6 replacement PFAS and one traditional one (PFOA) to enable a direct comparison of their effects. Regarding the studies investigating the effect on the immune system, in the first one cytokine profiling and DIA proteomics were used to identify PFOS-induced perturbations in primary human peripheral blood mononuclear cells (PBMCs) under innate (LPS) and adaptive (PHA) stimulation. In the second immune-related study, the pre-exposure to a set of 27 different PFAS, comprises both legacy and new generation one, was tested, as done in the previous study, on PBMCs whose inflammation was triggered by LPS treatment. Changes in the proteomic and epiproteomic profiles were analyzed through mass spectrometry. PFAS exposures led to a multilayered disruption spanning across gut microbiota and immune system, in a molecule and dose-dependent manner. Gut-microbiota analysis showed that while PFOA-treated samples are similar to controls, shorter-chained replacement PFAS generated distinc and dysbiotic profiles, reducing beneficial taxa like Faecalibacterium Prausnitzii and Blautia Obeum and increasing potentially harmful species such as Desulfovibrio spp. and Ruminococcus Gauvreauiii. Funtional alterations toward flagellar assembly, membrane transport and oxidative stress pathways further highlight the disruptive potential of these newer PFAS and underline their health. In immune cells, PFOS led to a prismatic reprogramming of PBMC responses under both LPS and PHA stimulation, repressing antigen presentation, Th1/Th2 differentiation and interferon 1 signaling, while keeping and promoting regulatory pathways like PD-1/PD-L1 and IL-10/IL-4/IL-13 signaling. These combined cytokine and proteomic changes led to attenuated effector responses while leaving key regulatory pathways active, which is consistent with epidemiological evidence of weakened vaccine and infection responses. The broader PFAS panel, instead, exhibited an ambivalent pattern in which pro inflammatory pathways, like MHC presentation and IL-1/IL-8 signaling were increased, while essential immune-coordinating molecules are dampened. Epiproteomic results of both activating and inhibiting histone modifications confirms this duality, revealing a molecule-dependent reprogramming that increases sensitivity to inflammatory stimuli while limiting downstream responses. Together, these findings underscore that PFAS toxicity is heterogeneous and system-wide, underlining the importance for mechanistic, molecule specific evaluation to fully comprehend their biological public-health implications.