Figure 5. L. reuteri-derived histamine inhibited TNF production via the H₂ receptor.

A. Histamine inhibited TNF production, and suppression of TNF was blocked by specific H₂ receptor antagonists in a concentration-dependent manner. L. reuteri 6475 conditioned media (CM) containing histamine inhibited TNF production, an effect partially blocked by specific H₂R antagonists. The presence or absence of histamine (10 nM) or L. reuteri 6475 CM is indicated by a “+” or “−” respectively. Colored bars indicate treatment with select antagonists - ranitidine (H₂R; blue), cimetidine (H₂R; green), chlorpheniramine (H₁R; red), and pyrilamine (H₁R; purple). H₁R antagonists were tested at 100 nM. Results represent the mean ± SD (n = 3), ^*p value<0.05 compared to medium control, ^#p value<0.05 compared to histamine, ^##p value<0.05 compared to L. reuteri 6475 CM. B. TFA-treated cell pellets containing histamine suppressed TNF production, an effect partially blocked by specific H₂R antagonists. HILIC-HPLC fraction B3, which was enriched for histamine, inhibited TNF production, an effect completely blocked by specific H₂R antagonists. All antagonists were tested at 100 nM. The presence or absence of L. reuteri 6475 CP or Fraction B3 is indicated by a “+” or “−” respectively. Results represent the mean ± SD (n = 3), ^*p value<0.05 compared to medium control, ^#p value<0.05 compared to L. reuteri 6475 CP, ^##p value<0.05 compared to fraction B3. C. Histamine receptor antagonists alone did not affect TNF production. All antagonists were tested at 100 nM. The H₁R antagonist, chlorpheniramine (CPM), did not prevent the H₂R antagonist, ranitidine (RAN), from blocking TNF suppression by histamine (10 nM) and L. reuteri 6475 CM. Blue bars with red stripes indicate combined treatment with CPM plus RAN. Results represent the mean ± SD (n = 3), ^*p value<0.05 compared to medium control, ^#p value<0.05 compared to histamine, ^##p value<0.05 compared to L. reuteri 6475 CM.