Histamine derived from probiotic Lactobacillus reuteri suppresses TNF via modulation of PKA and ERK signaling

Carissa M Thomas, Teresa Hong, Jan Peter van Pijkeren, Peera Hemarajata, Dan V Trinh, Weidong Hu, Robert A Britton, Markus Kalkum, James Versalovic, Carissa M Thomas, Teresa Hong, Jan Peter van Pijkeren, Peera Hemarajata, Dan V Trinh, Weidong Hu, Robert A Britton, Markus Kalkum, James Versalovic

Abstract

Beneficial microbes and probiotic species, such as Lactobacillus reuteri, produce biologically active compounds that can modulate host mucosal immunity. Previously, immunomodulatory factors secreted by L. reuteri ATCC PTA 6475 were unknown. A combined metabolomics and bacterial genetics strategy was utilized to identify small compound(s) produced by L. reuteri that were TNF-inhibitory. Hydrophilic interaction liquid chromatography-high performance liquid chromatography (HILIC-HPLC) separation isolated TNF-inhibitory compounds, and HILIC-HPLC fraction composition was determined by NMR and mass spectrometry analyses. Histamine was identified and quantified in TNF-inhibitory HILIC-HPLC fractions. Histamine is produced from L-histidine via histidine decarboxylase by some fermentative bacteria including lactobacilli. Targeted mutagenesis of each gene present in the histidine decarboxylase gene cluster in L. reuteri 6475 demonstrated the involvement of histidine decarboxylase pyruvoyl type A (hdcA), histidine/histamine antiporter (hdcP), and hdcB in production of the TNF-inhibitory factor. The mechanism of TNF inhibition by L. reuteri-derived histamine was investigated using Toll-like receptor 2 (TLR2)-activated human monocytoid cells. Bacterial histamine suppressed TNF production via activation of the H(2) receptor. Histamine from L. reuteri 6475 stimulated increased levels of cAMP, which inhibited downstream MEK/ERK MAPK signaling via protein kinase A (PKA) and resulted in suppression of TNF production by transcriptional regulation. In summary, a component of the gut microbiome, L. reuteri, is able to convert a dietary component, L-histidine, into an immunoregulatory signal, histamine, which suppresses pro-inflammatory TNF production. The identification of bacterial bioactive metabolites and their corresponding mechanisms of action with respect to immunomodulation may lead to improved anti-inflammatory strategies for chronic immune-mediated diseases.

Conflict of interest statement

Competing Interests: James Versalovic receives unrestricted research support from BioGaia AB. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1. Identification of histamine in TNF-inhibitory…
Figure 1. Identification of histamine in TNF-inhibitory fractions of L. reuteri 6475 by NMR.
1H NMR analysis demonstrated differences in the composition of HILIC-HPLC fractions from L. reuteri 6475. A. Top and bottom spectra show the 1D 1H NMR of TNF-inhibitory fraction B3 and fraction B4, respectively. The assigned peaks of phenylalanine (Phe), tryptophan (Trp), and histamine from fraction B3 are labeled on the top spectrum. Two complementary 2D NMR techniques were used to identify compounds unique to TNF-inhibitory fraction B3. B. Aromatic peaks of 1H-13C-HSQC from Phe, Trp, and histamine. C. Aliphatic peaks of 1H-13C-HSQC from Phe, Trp, and histamine. D. The TOCSY spectrum further identified the spin system of Phe, Trp, and histamine unique to TNF-inhibitory fraction B3.
Figure 2. The production of histamine by…
Figure 2. The production of histamine by L. reuteri 6475 was confirmed by mass spectrometry.
Examination of TNF-inhibitory HILIC-HPLC fractions from L. reuteri 6475 grown in LDMIIIG confirmed the presence of histamine by ESI qTOF MS. A. ESI qTOF MS of TNF-inhibitory HILIC-HPLC fraction B3 from strain 6475 grown in glucose. Histamine was detected at m/z 112.08. B. ESI qTOF MS of non-TNF inhibitory HILIC-HPLC fraction B3 from strain 6475 grown in sucrose. Histamine was not detected at m/z 112.08. C. MS/MS analysis of m/z 112.08 confirmed that the compound is histamine. D.L. reuteri culture supernatants from sucrose or glucose-containing media were analyzed for histamine concentration by triple quadrupole mass spectrometry. Histamine (pg/mL) was detected only in culture supernatants from glucose-containing medium cultures.
Figure 3. Quantification of histamine by triple…
Figure 3. Quantification of histamine by triple quadrupole mass spectrometry.
Histamine in HILIC-HPLC fractions B2–B7 from L. reuteri 6475 grown in a glucose-containing medium was quantified by triple quadrupole mass spectrometry. The y-axis indicates TNF production from activated human monocytoid cells. The concentration of histamine (pg/µL) is indicated above each bar graph. Elevated levels of histamine correlated with TNF inhibition.
Figure 4. The histidine decarboxylase gene cluster…
Figure 4. The histidine decarboxylase gene cluster contributed to the TNF-inhibitory phenotype of L. reuteri 6475.
The histidine decarboxylase gene cluster consists of three genes, hdcP (RPRB3004), hdcA (RPRB3005), and hdcB (RPRB3006). The organization of the cluster is depicted above the bar graph. Inactivation by targeted mutagenesis in any one of these genes resulted in a partial loss (approximately 40%) of TNF suppression by L. reuteri 6475. Results represent the mean ± SD (n = 9), *p value<0.05 compared to medium control, #p value<0.05 compared to L. reuteri 6475.
Figure 5. L. reuteri -derived histamine inhibited…
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.
Figure 6. Stimulation of the H 2…
Figure 6. Stimulation of the H2 receptor increased cAMP and PKA activity was required for TNF suppression.
Signaling pathway studies were performed to determine the effect of histamine on cAMP production and the role of PKA activity in TNF suppression. A. Treatment of activated THP-1 cells with L. reuteri 6475 CM or histamine increased intracellular cAMP. The increase in cAMP was blocked by a specific H2 receptor antagonist, ranitidine. Results represent the mean ± SD (n = 2), *p value<0.05 compared to medium control, **p value<0.05 compared to L. reuteri 6475, #p value<0.05 compared to histamine. B. Inhibition of PKA activity partially blocked TNF suppression by TNF-inhibitory compounds L. reuteri 6475 CM, HILIC-HPLC fraction B3, histamine, and dcAMP. Results represent the mean ± SD (n = 9), *p value<0.05 compared to H89 treated samples.
Figure 7. Stimulation of the H 2…
Figure 7. Stimulation of the H2 receptor inhibited the ERK signaling pathway.
Immunoblot studies were performed to determine the effect of histamine on activation of the ERK signaling pathway. ERK was activated by phosphorylation on Thr202/Tyr204 (P-ERK). Treatment of TLR-stimulated THP-1 cells with L. reuteri 6475, histamine, or U0126 suppressed activation of ERK1/2, an effect that was significantly blocked in the presence of ranitidine. The β-actin antibody served as the protein loading control.
Figure 8. Pathway diagram of the mechanism…
Figure 8. Pathway diagram of the mechanism of histamine inhibition of ERK activation.
THP-1 cells activated with a TLR2 agonist activated the MEK/ERK MAPK signaling cascade to induce production of TNF. Histamine produced by L. reuteri 6475 engaged the H2 receptor on activated THP-1 cells. This G protein-coupled receptor activated adenylate cyclase (AC) to increase intracellular cAMP, which subsequently activated protein kinase A (PKA). PKA inhibited MEK/ERK activation and suppressed production of TNF. It was predicted that PKA inhibited MEK/ERK activation by inhibiting the activity of c-Raf. Specific H2 receptor antagonists, such as ranitidine, prevented histamine from activating the H2 receptor and blocked TNF suppression by histamine. H89, a specific inhibitor of PKA, prevented TNF suppression by histamine, demonstrating that PKA activity was necessary for histamine's effect on TNF. Blocking MEK activity with U0126 suppressed activation of ERK and downstream TNF production, demonstrating that ERK activation was necessary for production of TNF.

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