Paternal cytokine administration alters sperm small ncRNAs and offspring brain and behavior.

Paternal pre-conceptual exposure to pathogenic infection can alter offspring phenotypes via changes to sperm epigenetics, including small non-coding RNAs (ncRNAs). This phenomenon occurs even in the absence of infection when pathogen mimetics trigger paternal immune activation (PIA). While this implicates a shared component of the immune response, the specific mechanisms responsible remain unknown. Cytokines are a key component of innate immunity and are highly upregulated following exposure to both pathogens and their mimetics. Here we investigate whether paternal administration of the pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) recapitulates alterations to sperm small ncRNAs and offspring phenotypic changes from other models of PIA. C57BL/6J male mice were administered a single dose of IL-1β, TNF-α, or vehicle, prior to mating with naïve female mice, to produce offspring. Offspring from IL-1β-treated fathers exhibited altered fasting responses and female-specific alterations to stress-coping behavior. In response to paternal TNF-α, male offspring showed increased anxiety-like behavior. Analysis of paternal sperm small ncRNA revealed that IL-1β significantly downregulated several transfer RNA-derived small RNAs (tsRNAs) and P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) clusters. Surprisingly, paternal TNF-α only slightly altered small ncRNAs, downregulating a single piRNA cluster. These results partially recapitulate offspring phenotypic changes following paternal exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, in a COVID-19 mouse model) and polyinosinic:polycytidylic acid (poly I:C, a viral mimetic). Ultimately, we provide the first evidence that cytokines have intergenerational effects on brain and behavior, and that they contribute to altered offspring phenotypes in viral-like PIA. This study demonstrates that paternal cytokines are causally involved in a mechanism of epigenetic inheritance following PIA, and highlights the importance of PIA as a pre-conceptual factor that may modulate offspring health, including risk of neuropsychiatric disorders.