Paternal immune activation by Poly I:C modulates sperm noncoding RNA profiles and causes transgenerational changes in offspring behavior

•The viral mimetic Poly I:C induces immune activation in male mice and changes their sperm small noncoding RNA profiles.•This paternal immune activation (PIA) alters the behavior of offspring and grand-offspring.•Paternal epigenetic inheritance via PIA affects phenotypes relevant to human brain function and physiology.•Changes in small noncoding RNA profiles are also found in the sperm of F1 mice.•The hippocampus of the F1 mice show transcriptomic changes relevant to their altered behavior. Paternal pre-conceptual environmental experiences, such as stress and diet, can affect offspring brain and behavioral phenotypes via epigenetic modifications in sperm. Furthermore, maternal immune activation due to infection during gestation can reprogram offspring behavior and brain functioning in adulthood. However, the effects of paternal pre-conceptual exposure to immune activation on the behavior and physiology of offspring (F1) and grand-offspring (F2) are not currently known. We explored effects of paternal pre-conceptual exposure to viral-like immune activation on F1 and F2 behavioral and physiological phenotypes using a C57BL/6 mouse model. Males were treated with a single injection (intraperitoneal) of the viral mimetic Polyinosinic:polycytidylic acid (Poly I:C: 12mg/kg) then bred with naïve female mice four weeks after the Poly I:C (or 0.9% saline control) injection. The F1 offspring of Poly I:C treated fathers displayed increased depression-like behavior in the Porsolt swim test, an altered stress response in the novelty-suppressed feeding test, and significant transcriptomic changes in their hippocampus. Additionally, the F1 male offspring only showed significantly increased immune responsivity after a Poly I:C immune challenge (12mg/kg). Furthermore, the F2 male grand-offspring took longer to enter and travelled significantly shorter distances in the light zone of the light/dark box. An analysis of the small noncoding RNA profiles in sperm from Poly I:C treated males and their male offspring revealed significant effects of Poly I:C on the sperm microRNA content at the time of conception and on the sperm PIWI-interacting RNA content of the male offspring. Notably, eight miRNAs with an FDR < 0.05 (miR-141-3p, miR-126b-5p, miR-669o-5p, miR-10b-3p, miR-471-5p, miR-463-5p, miR-148b-3p, and miR-181c-5p) were found to be significantly downregulated in the sperm of Poly I:C treated males. Collectively, we demonstrate that paternal pre-conceptual exposure to a vira