HdcA+L. reuteri 6475 inhibits H1R downstream signaling via dagK and causes immunosuppression of intestinal epithelium in gnotobiotic mice

Abstract

IntroductionProbiotics may beneficially affect the disease course of patients with chronic disorders like inflammatory bowel disease and colorectal cancer (CRC) via modulation of the host immune responses. A recent pangenomic study showed that human‐derived clade II L. reuteri strains contained a complete chromosomal histidine decarboxylase (hdc) gene cluster (genes hdcA, hdcB, hdcP) and have the genetic capacity to convert histidine to histamine. We found L. reuteri 6475 (clade II) derived‐histamine suppressed TNF production in human myeloid cells. In addition, administration of L. reuteri to HDC knock‐out mice bearing inflammation‐associated colon cancer (IaCC) showed suppression of inflammatory cytokines IL‐6, IL‐22, IL‐1α and TNF. Reduced inflammation was correlated with reduced CD11b+Gr‐1+ immature myeloid cells (IMCs) in spleen and bone marrow of hdcA+ L. reuteri HDC KO mice bearing IaCC compared to hdcA−L. reuteri HDC KO IaCC mice. However, signaling mechanisms of L. reuteri 6475 derived histamine and its ability to suppress inflammation are not well understood. So, we hypothesized that L. reuteri derived‐histamine down‐regulates histamine receptor 1(H1R) and allows H2R activation, thereby suppressing inflammation.MethodsWe used Swiss‐Webster WT and BALB/c WT germ‐free (GF) mice mono‐associated with L. reuteri WT, L. reuteri hdcA mutant or L. reuteri dagK mutant strain. Inflammatory biomarkers in blood plasma were analyzed with MAGPIX multiplexing. Gene expression profiling of cytokines and hdcA or dagK in the intestinal mucosa was achieved by qRT‐PCR. H1R and H2R protein expression was analyzed by fluorescence immunohistochemistry.ResultsThe results showed significant diminution of IL‐6 expression in the GF mice receiving L. reuteri WT and hdcA mutant strain. In addition, we found that both WT and hdcA mutant strain can produce Diacylglycerol kinase (dagK). dagK is known to inhibit DAG signaling by converting DAG to phosphatidic acid. Therefore, we speculate that IL‐6 is suppressed due to DAG inhibition of H1R downstream signaling and regulates the H2R signaling by L. reuteri histamine. We then created dagK mutant L. reuteri and colonized BALB/c mice with the mutant strain. Interestingly, dagK−L. reuteri colonized BALB/c mice showed significant increase in IL‐6 mRNA and IL‐1α protein expression levels. In addition, CD11b+Gr‐1+ IMC's are reduced in BALB/c mice colonized with WT but not in either hdcA−or dagK−L. reuteri colonized mice. In addition, mucus fucosylation was increased in mice that were colonized with WT L. reuteri bacterium compared to other groups.ConclusionIn conclusion, GF mice mono‐associated with either WT or mutants (hdcA/dagK) L. reuteri clearly shows that intact hdcA gene cluster is important for immunomodulatory effect of L. reuteri 6475 by synthesizing luminal histamine. An important finding of this study is that, in addition to hdcA, dagK expression by L. reuteri 6475 is equally important to exert immunosuppressive effects. This is achieved possibly by inhibiting DAG, suppressing phosphorylation of PKC via dagK synthesis, and thereby interrupting H1R downstream signaling in the intestinal epithelium. Altogether, L. reuteri 6475 represents an immunomodulatory probiotic and might be used to treat patients with IBD pathogenesis.Support or Funding InformationThe work was supported by National Institutes of Health (R01 AT004326, R01 DK065075, U01 CA 170930 and UH3 DK 083990).