Metabolite-sensing G protein-coupled receptors (msGPCRs) are GPCRs that are activated by metabolites originating from various sources. Several of the known msGPCRs are expressed on immune cells and adipocytes as well as the gut epithelium and metabolic tissues like the pancreas. Some of their known agonists are produced endogenously while others are of exogenous origin. Examples for agonists of exogenous origin are metabolites produced by (intestinal) bacteria. The expression profile and the nature of their agonists link msGPCRs to functions in the regulation of metabolic processes and immune cell responses.
Both receptors investigated in my thesis project, hydroxycarboxylic acid receptor 3 (HCA3) and GPR84, are msGPCRs highly expressed on cells of the innate immune system such as neutrophils, monocytes, and macrophages. HCA3 is a member of the HCA family consisting of three receptors, which show high sequence homology. Especially HCA2 and HCA3 only differ in a few positions resulting in an amino acid sequence differing in 17 amino acids and the extended C-terminus of HCA3. While HCA1 and HCA2 are present in the genome of all mammals, HCA3 is only present in higher primates such as chimpanzee, orangutan, and human. As a result, there is a lack of accessible animal models and the receptor is still insufficiently studied. 3 hydroxyoctanoic acid (3HO), 3 hydroxydecanoic acid (3HDec) and aromatic D-amino acids D-phenylalanine (D-Phe) and D-tryptophan (D-Trp) are previously reported agonists of HCA3 with 3HO being its endogenous agonist. Regarding its physiological function, it is known that the receptor is involved in a negative feedback loop in the regulation of lipolysis and fatty acid oxidation under prolonged fasting conditions in adipocytes. Further, it has been shown that aromatic D-amino acids induce chemotaxis in human neutrophils.
GPR84 is a receptor for medium chain fatty acids (MCFAs) with a chain length of 9 to 14 carbon atoms (C9 - C14) and their hydroxylated derivatives. Thus, GPR84 and HCA3 share 3HDec as a common agonist. Further, both receptors couple to Gαi proteins resulting in the inhibition of adenylyl cyclase and subsequent decrease of intracellular cyclic AMP (cAMP) levels, and induce the phosphorylation and activation of extracellular signal regulated kinase1/2 (ERK1/2). As opposed to HCA3, GPR84 is present in the genome of most mammals and various studies have linked GPR84 to pro-inflammatory functions and processes like phagocytosis, chemotaxis and upregulation of pro-inflammatory cytokine release. Most of these studies on GPR84 were performed using surrogate agonists.
Because HCA3 is still poorly understood, the aim of my project was to shed some light on its function by evolutionary and functional analyses of HCA3 orthologs. Moreover, detailed analyses of its signal transduction and components involved in receptor-mediated downstream signaling events were performed as part of the present dissertation. Since HCA3 and GPR84 share at least one agonist and are co-expressed in different types of immune cells, we studied signaling of both receptors simultaneously.
Our functional analyses of human and great ape HCA3 orthologs using cAMP inhibition assays revealed the evolutionary conservation of the endogenous agonist 3HO.
By further functionally analyzing the primate HCA3 orthologs, we found both aromatic D-amino acids, D-Phe and D-Trp, to activate human HCA3 with the highest potency. Although D-Phe and D-Trp were previously described to induce HCA3-mediated chemotaxis in neutrophils a link to where the two D-amino acids would originate from in sufficiently high concentrations in a physiological context was missing. After extensive review of literature, we found that some intestinal bacteria and bacteria used to ferment food and beverages produce and secrete D-amino acids. This led us to investigate whether other structurally related D-amino acid metabolites produced by bacteria also activate HCA3. These investigations resulted in the discovery of lactic acid bacteria (LAB)-derived metabolites as highly potent agonists of HCA3. We tested both the Phe-metabolites D-phenyllactic acid (D-PLA) and L-PLA as well as the racemic mixture of the Trp-metabolite indole-3-lactic acid (ILA). All three compounds specifically induced activation of HCA3, but we found D-PLA to be a 35-fold more potent agonist than the L-enantiomer. Further, D-PLA proved to be 10-fold more potent than 3HO and 240-fold more potent than D Phe.
Since D-PLA is known to be present in LAB-fermented foods such as Sauerkraut, we investigated whether D-PLA is absorbed and enters the blood circulation after oral ingestion of 100 mg D-PLA or Sauerkraut (5-6 g per kg body weight), respectively. Both, ingestion of the pure compound and of Sauerkraut resulted in a significant increase of D-PLA in the plasma post-prandial resulting in concentrations sufficiently high to activate HCA3.
To examine HCA3 signaling and the involved components in more detail, we used several inhibitors of internalization and signaling components. The goal was to examine whether there are differences in the activation of signaling pathways, recruitment of signaling components, internalization behavior and endocytic routes of HCA3 and GPR84 in response to 3HO vs 3HDec and decanoic acid (C10) vs 3HDec respectively.
Initial analyses of the signaling kinetics of the two receptors, using dynamic mass redistribution (DMR) measurements, indicated differences between the two respective agonists for both HCA3 and GPR84. DMR measurements allow for a time-resolved recording of the activated signaling cascades, independent of the activated pathways. Additional use of pertussis toxin to inhibit Gαi proteins and dynasore to block dynamin-2 function revealed that signaling of both receptors induced by both respective agonists was completely dependent on G protein activation, but differentially dependent on dynamin-2 function suggesting differences in desensitization and internalization mechanisms.
Using cAMP inhibition and ERK1/2 activation assays, we further investigated the role of internalization for HCA3 and GPR84 signal transduction. Interestingly, ERK1/2 activation downstream of both receptors was strongly reduced when internalization was inhibited, while cAMP inhibitory signaling of HCA3 induced by both 3HO and 3HDec and GPR84 signaling induced by C10 were significantly reduced by blocking dynamin-2 function but not internalization in general. 3HDec-induced cAMP inhibition downstream of GPR84 was completely insensitive to inhibition of both.
Further experiments using dominant negative dynamin-2 mutants verified dependence of HCA3-mediated Gαi and ERK1/2 signaling on dynamin-2 function. Moreover, qualitative analysis of confocal images revealed that subcellular distribution of HCA3 but not GPR84 is altered when dynamin-2 function is impaired.
Interestingly, analysis of β-arrestin 2 recruitment by a luminescence-based assay (DiscoverX PathHunter) and imaging of HEK293T cells expressing mRuby-tagged receptor and YFP-tagged β-arrestin 2, showed that only 3HO- but not 3HDec-induced activation of HCA3 leads to recruitment of β-arrestin 2 and subsequent co-localization in intracellular vesicles. GPR84 data suggests that the receptor does not interact with β-arrestin 2 at all.
Finally, we also analyzed HCA3 signaling kinetics and β-arrestin 2 recruitment in response to the newly identified agonists D-PLA and L-PLA and the previously known agonist D-Phe. We found signaling kinetics of L-PLA and D-Phe to be similar to 3HDec-induced kinetics, while D-PLA appears to be similar to 3HO. This was further supported by the fact that D-PLA also induced β-arrestin 2 recruitment, while L-PLA and D-Phe did not.
Taken together, these findings advanced our understanding of HCA3 and GPR84. The work provides evidence that HCA3 evolved as a signaling system to communicate uptake of fermented food (together with fermenting bacteria) to the immune system. Our data in combination with literature reporting positive, anti-inflammatory properties of D-PLA and LAB in general suggests that at least some of the described positive effects are mediated by HCA3.
Furthermore, we showed for the first time biased signaling of these two receptors in response to their natural agonists. Our work increases the knowledge about specific signaling components involved in downstream signaling of the respective receptor in response to the different agonists, potentially linking e.g. activation of HCA3 by 3HO and D-PLA, but not 3HDec and D-Phe, to the inhibition of pro-inflammatory cytokine release through β-arrestin 2 dependent mechanisms. Moreover, 3HDec-induced signaling downstream of GPR84 is very different from that downstream of HCA3. This suggests that 3HDec also triggers different physiological responses in immune cells depending on its local concentration and the expression levels of the two receptors. However, these findings still need to be validated in cells of innate immunity like neutrophils and macrophages that endogenously express both receptors. At last, the physiological consequences such as increased ROS-production, pro-/anti-inflammatory cytokine release, migration, and phagocytosis need to be addressed in future studies to get a better understanding of the function as well as interplay of HCA3 and GPR84 in innate immunity and their suitability as drug targets.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:73284 |
| Date | 08 January 2021 |
| Creators | Peters, Anna |
| Contributors | Universität Leipzig |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | https://doi.org/10.1371/journal.pgen.1008145, https://doi.org/10.1186/s12964-020-0516-2 |
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