Histamine Derived from Probiotic Lactobacillus reuteri Suppresses TNF via Modulation of PKA and ERK Signaling

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 H2 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

Conventional protein kinase C and atypical protein kinase Cζ differentially regulate macrophage production of tumour necrosis factor-α and interleukin-10

In chronic inflammatory diseases such as rheumatoid arthritis, joint macrophages/monocytes are the major source of pro- and anti-inflammatory cytokines. Little is understood regarding the signalling pathways which determine the production of the pro-inflammatory cytokine, tumour necrosis factor-α (TNF-α) and the anti-inflammatory cytokine, interleukin-10 (IL-10). Two pathways integral to macrophage function are the protein kinase C (PKC)- and the cAMP-dependent pathways. In this report, we have investigated the involvement of PKC and cAMP in the production of TNF-α and IL-10 by peripheral blood monocyte-derived macrophages. The utilization of the PKC inhibitors Go6983, Go6976 and RO-32-0432 demonstrated a role for conventional PKCs (α and β) in the production of TNF-α in response to stimulation by lipopolysaccharide and phorbol 12-myristate 13-acetate (PMA)/ionomycin. PKC stimulation resulted in the downstream activation of the p42/44 mitogen-activated protein kinase (MAPK) pathway which differentially regulates TNF-α and IL-10. The addition of cAMP however, suppressed activation of this MAPK and TNF-α production. Cyclic-AMP augmented IL-10 production and cAMP response element binding protein activation upon stimulation by PMA/ionomycin. In addition, cAMP activated PKCζ; inhibition of which, by a dominant negative adenovirus construct, selectively suppressed IL-10 production. These observations suggest that pro-inflammatory and anti-inflammatory cytokines are differentially regulated by PKC isoforms; TNF-α being dependent on conventional PKCs (α and β) whereas IL-10 is regulated by the cAMP-regulated atypical PKCζ