Immune response to COVID-19 vaccination in a population with a history of elevated exposure to per- and polyfluoroalkyl substances (PFAS) through drinking water.

BACKGROUND: Exposure to per- and polyfluoroalkyl substances (PFAS) has been linked to lower vaccine-induced antibody concentrations in children, while data from adults remains limited and equivocal. Characteristics of PFAS exposure and age at vaccination may modify such effects. OBJECTIVE: We used the mass administration of novel COVID-19 vaccines to test the hypothesis that prior exposure to environmentally-relevant concentrations of PFAS affect antibody response to vaccines in adolescents and adults. METHODS: Between April and June 2021, 226 participants aged 12-90 years with a history of exposure to PFAS in drinking water and who received an mRNA COVID-19 vaccine participated in our prospective cohort study. SARS-CoV-2 anti-spike and anti-nucleocapsid antibodies (IgG) were quantified before the first and second vaccine doses and again at two follow-ups in the following months (up to 103 days post dose 1). Serum PFAS concentrations (n = 39 individual PFAS) were measured once for each participant during baseline, before their first vaccination. The association between PFAS exposure and immune response to vaccination was investigated using linear regression and generalized estimating equation (GEE) models with adjustment for covariates that affect antibody response. PFAS mixture effects were assessed using weighted quantile sum and Bayesian kernel machine regression methods. RESULTS: The geometric mean (standard deviation) of perfluorooctane sulfonate and perfluorooctanoic acid serum concentrations in this population was 10.49 (3.22) and 3.90 (4.90) µg/L, respectively. PFAS concentrations were not associated with peak anti-spike antibody response, the initial increase in anti-spike antibody response following vaccination, or the waning over time of the anti-spike antibody response. Neither individual PFAS concentrations nor their evaluation as a mixture was associated with antibody response to mRNA vaccination against COVID-19. IMPACT STATEMENT: Given the importance of understanding vaccine response among populations exposed to environmental contaminants and the current gaps in understanding this relationship outside of early life/childhood vaccinations, our manuscript contributes meaningful data from an adolescent and adult population receiving a novel vaccination.

Hematopoietic cell kinase associates with the 40S ribosomal subunit and mediates the ribotoxic stress response to deoxynivalenol in mononuclear phagocytes.

The trichothecene deoxynivalenol (DON) binds to eukaryotic ribosomes and triggers p38-driven proinflammatory gene expression in the macrophage-a response that is dependent on both double-stranded RNA-activated protein kinase (PKR) and hematopoietic cell kinase (Hck). Here we elucidated critical linkages that exist among the ribosome and these kinases during the course of DON-induced ribotoxic stress in mononuclear phagocytes. Similar to PKR inhibitors, Hck inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine (PP2) suppressed p38 activation and p38-driven interleukin 8 (IL-8) expression in the U937 human monocyte cell line. U937 cells stably transfected with a PKR antisense vector (U9K-A1) displayed marked reduction of DON-induced p38 activation and IL-8 expression as compared to cells transfected with empty vector (U9K-C2), with both responses being completely ablated by PP2. Western analysis of sucrose density gradient fractions revealed that PKR and Hck interacted with the 40S ribosomal subunit in U9K-C2 but not U9K-A1 cells. Subsequent transfection and immunoprecipitation studies with HeLa cells indicated that Hck interacted with ribosomal protein S3. Consistent with U937 cells, DON induced p38 association with the ribosome and phosphorylation in peritoneal macrophages from wild-type but not PKR-deficient mice. DON-induced phosphorylation of ribosome-associated Hck in RAW 264.7 murine macrophages was also suppressed by 2-aminopurine (2-AP). Both 2-AP and PP2 inhibited DON-induced phosphorylation of p38 as well as two kinases, apoptosis signal-regulating kinase 1 and mitogen-activated protein kinase 3/6, known to be upstream of p38. Taken together, PKR and Hck were critical for DON-induced ribosomal recruitment of p38, its subsequent phosphorylation, and, ultimately, p38-driven proinflammatory cytokine expression.

Double-stranded RNA-activated protein kinase mediates induction of interleukin-8 expression by deoxynivalenol, Shiga toxin 1, and ricin in monocytes.

Translational inhibitors such as the trichothecene mycotoxin deoxynivalenol (DON) and ribosomal inhibitory proteins (RIPs) induce mitogen-activated protein kinase (MAPK)-driven chemokine and cytokine production by a mechanism known as the ribotoxic stress response (RSR). Double-stranded RNA-activated protein kinase (PKR) associates with the ribosome making it uniquely positioned to sense 28S ribosomal RNA damage and initiate the RSR. We have previously shown that PKR mediates DON-induced MAPK phosphorylation in macrophages and monocytes. The purpose of this study was to test the hypothesis that PKR is essential for induction of interleukin (IL)-8 expression in monocytes by DON and two prototypical RIPs, ricin, and Shiga toxin 1 (Stx1). Preincubation of human monocytic U937 cells with the PKR inhibitors C16 and 2-aminopurine (2-AP) blocked DON-induced expression of IL-8 protein and mRNA. Induction of IL-8 expression was similarly impaired in U937 cells stably transfected with a dominant negative PKR plasmid (UK9M) as compared with cells transfected with control plasmid (UK9C). Nuclear factor-kappa B binding, which has been previously shown to be a requisite for DON-induced IL-8 transcription, was markedly reduced in UK9M cells as compared with UK9C cells. As observed for DON, ricin-, and Stx1-induced IL-8 expression was suppressed by the PKR inhibitors C16 and 2-AP as well as impaired in UK9M cells. Taken together, these data indicate that PKR plays a common role in IL-8 induction by DON and the two RIPs, suggesting that this kinase might be a critical factor in RSR.

Transcriptional regulation of deoxynivalenol-induced IL-8 expression in human monocytes.

The trichothecene mycotoxin deoxynivalenol (DON), commonly present in contaminated grains worldwide, induces expression of the chemokine interleukin (IL)-8 in human monocytes. The purpose of this study was to test the hypothesis that DON modulates transcriptional and posttranscriptional regulation of IL-8 expression in the U937 human monocyte model. When U937 cells were transfected with a wild-type IL-8 promoter luciferase construct (-162/+44 IL-8 LUC) and incubated with DON (1 mug/ml) or the positive control, lipopolysaccharide (LPS) (1 mug/ml), there was a significant increase in luciferase expression. Mutation of the nuclear factor-kappaB (NF-kappaB) binding site significantly impaired both DON- and LPS-induced luciferase expression. In contrast, mutating the activator protein-1 binding site resulted in significantly increased DON- and LPS-induced luciferase expression. CCAAT/enhancer-binding protein beta, octamer-1, or NF-kappaB repressing factor binding site mutations did not affect DON-induced luciferase activity. Consistent with reporter studies, the NF-kappaB inhibitor caffeic acid phenethyl ester completely ablated both DON-induced IL-8 mRNA and protein expression. When NF-kappaB subunit binding to a specific IL-8 promoter probe was evaluated by enzyme-linked immunosorbent assay (ELISA), DON was observed to increase p65 binding by 21-fold, have no effect on p50 binding and decrease p52 binding. DON was not found to stabilize IL-8 mRNA in U937 cells. Taken together, these data suggest that DON-induced IL-8 expression is likely to be mediated at the transcriptional level by NF-kappaB, specifically p65, but does not appear to involve mRNA stabilization.

p38 Mitogen-activated protein kinase mediates IL-8 induction by the ribotoxin deoxynivalenol in human monocytes.

The effects of the ribotoxic trichothecene deoxynivalenol (DON) on mitogen-activated protein kinase (MAPK)-mediated IL-8 expression were investigated in cloned human monocytes and peripheral blood mononuclear cells (PBMC). DON (250 to 1000 ng/ml) induced both IL-8 mRNA and IL-8 heteronuclear RNA (hnRNA), an indicator of IL-8 transcription, in the human U937 monocytic cell line in a concentration-dependent manner. Expression of IL-8 hnRNA, mRNA and protein correlated with p38 phosphorylation and was completely abrogated by the p38 MAPK inhibitor SB203580. DON at 500 ng/ml similarly induced p38-dependent IL-8 protein and mRNA expression in PBMC cultures from healthy volunteers. Significantly increased IL-6 and IL-1beta intracellular protein and mRNA expression was also observed in PBMC treated with DON (500 ng/ml) which were also partially p38-dependent. Flow cytometry of PBMC revealed that DON-induced p38 phosphorylation varied among individuals relative to both threshold toxin concentrations (25-100 ng/ml) and relative increases in percentages of phospho-p38(+) cells. DON-induced p38 activation occurred exclusively in the CD14(+) monocyte population. DON was devoid of agonist activity for human Toll-like receptors 2, 3, 4, 5, 7, 8 and 9. However, two other ribotoxins, emetine and anisomycin, induced p38 phosphorylation in PBMC similarly to DON. Taken together, these data suggest that (1) p38 activation was required for induction of IL-8 and proinflammatory gene expression in the monocyte and (2) DON induced p38 activation in human monocytes via the ribotoxic stress response.