Fsl 1

phKCs (Lonza) were cultured in KBM‐Gold medium supplemented with KBM‐Gold SingleQuot KC (Lonza) for 3 days before use.¹⁰ Murine neurons were isolated from postnatal d5 C57BL/6 mice after deep anesthetization. The ganglia were digested and cultured in DMEM supplemented with 5% (v/v) FBS (Sigma), 100 U/ml penicillin, 100 μg/ml streptomycin, 1 × B27, and 50 ng/ml nerve growth factor (Sigma). Seven days of culture in the presence of 10 µM cytosine β‐d‐arabinofuranoside (Sigma) enriched >95% neurons characterized by NeuN staining. For RNA‐seq, neurons were incubated with a medium with either Pam3CSK4 (1 µg/ml, Novus), FSL‐1 (1 µg/ml, Sigma), Serpin E1 (20 µg/ml, Raybiotech), or the appropriate vehicle for 6 h.

Mice were provided with 4 mg/mL OVA in drinking water or drinking water alone ad libitum for 7 days. Prior to immunization (day −2), all mice were returned to normal water (Fig. 1a). At this dose, BALB/c mice consumed on average 13.75 mg ± 0.79 SEM of OVA/mouse/day (n = 8).
All groups were immunized i.p. (day 0) with 50 μg (C57BL/6 mice) or 10 μg (BALB/c mice) of OVA precipitated to alum (Fig. 1a) and boosted by i.p. injection of 10 μg (C57BL/6 mice) or 1 μg (BALB/c mice) soluble OVA in PBS on day 14. Blood and faecal samples were harvested on day 21. In studies examining the role of TLR activators on tolerance, mice were additionally treated with OVA gavage (1 mg in 100 μL PBS) 3 times during the week of ad libitum OVA treatment (days −9, −6, and −3) to ensure precise, concurrent delivery of OVA and TLR activators (Fig. 3a). In some groups, OVA gavage treatments were supplemented with one of the following: 10 μg TLR2/1 activator Pam₃CSK₄, 5 μg TLR2/6 activator FSL‐1, or 10 μg TLR4 activator LPS derived from E. coli (Sigma‐Aldrich, catalogue number L4524). These groups were compared to tolerized mice receiving 3 gavage treatments of OVA in PBS alone, and control mice receiving 3 gavage treatments of PBS. Oral treatment with FSL‐1 was performed at a 5‐μg dose due to toxic effects observed at higher doses.