"Metabolic immunomodulation" by natural products

Macrophages are cells of our immune system which fulfill a variety of functions, including the protection against infection, clearance of debris by phagocytosis, initiation and resolution of inflammation as well as antigen presentation to cells that then produce tailored antibodies. In order to meet these varying functions, macrophages must show a high degree of plasticity. For this they adopt a range of different polarization states, triggered by cytokines or tissue environment, with the pro-inflammatory M1 (kill) and the anti-inflammatory M2 (heal) phenotype constituting the extremes of a continuous spectrum. Each polarization state is characterized by a distinct set of marker proteins and is notably dependent on a distinct metabolic program that allows the cell to meet the requirements in energy, building blocks and reductive power needed for the respective polarization state. Secondary plant metabolites are often produced for protection (against UV, fungi etc) or in order to attract, repell or kill an animal. Thus, it is assumed that plant compounds co-evolved with their target in order to allow an optimal fit that privileges plant compounds for exerting bioactivity. Therefore it is not surprising that multiple natural products also show immunomodulatory effects (e.g. salicylic acid from willow bark to counter inflammation). However, the underlying molecular mechanism remains to be still uncovered for many of those compounds. In this project we want to systematically address or reassess the questions whether (i) selected natural products can influence macrophage polarization, (ii) they alter energy metabolism or metabolite levels in macrophages, (iii) whether and, if yes, (iv) how both processes are interconnected on the molecular level. We will seek answers in in vitro models using primary murine macrophages with a combination of state of the art techniques in protein-, metabolite- and bioenergetics analysis as well as molecular and cell biology. Relevance of in vitro findings will be underpinned in appropriate in vivo models. Concerning the natural products, we focus on urolithin A, a bioavailable conversion product of ellagitannins (compounds found in berries, nuts and pomegranate) produced by microbes in our gut, and on sulforaphane activating the stress responsive transcription factor Nrf2. Obtained results are expected to foster our understanding of to what extent cellular energy metabolism can be a driver for the pro-or anti-inflammatory activities of natural products. Moreover, they can give a possible molecular explanation for other observed consequences of urolithin A exposure/or Nrf2 activation and potential insights into how metabolic cues can be finally translated into the language of distinct macrophage responses.

The project uncovered previously unknown mechanisms underlying the anti-inflammatory effects of natural products, focusing on macrophage polarization and the bioactive compounds sulforaphane and urolithin A. Macrophages, key players in the innate immune system, serve diverse roles in inflammation-from its initiation to resolution. To fulfill these functions, macrophages adopt various polarization states along a spectrum, with M1 and M2 at the pro- and anti-inflammatory extremes, respectively. Notably, these polarization states are defined by distinct metabolic profiles: M1 macrophages predominantly utilize aerobic fermentation of glucose, whereas M2 macrophages go for fatty acid oxidation for their energy and biosynthetic needs. This connection between metabolism and immune function has given rise to the field of immunometabolism, which explores the potential of modulating immune responses by targeting specific metabolic pathways. Building on this concept, we explored the metabolic impact of sulforaphane-a compound readily available in broccoli-and urolithin A, a metabolite produced by gut bacteria from ellagitannins (found in foods like pomegranate), in their established anti-inflammatory effects on macrophages. Our findings demonstrated that both natural compounds prevent the polarization of macrophages towards the pro-inflammatory M1 phenotype while also inducing metabolic shifts in treated cells. Specifically, our subsequent experiments with the aim to challenge correlation for causality revealed the following: - Sulforaphane increased glycolytic activity (aerobic fermentation) in macrophages; however, this enhancement of glycolysis was not essential for the reduced M1 phenotype, but rather a byproduct of sulforaphane's other activities. - Sulforaphane exerted protective effects on mitochondria, and maintaining mitochondrial integrity (with an intact citric acid cycle, efficient respiratory chain function, minimal fission, and low superoxide production) was crucial for preventing M1 polarization. - Functional mitochondria were essential for restricting two-carbon units for fatty acid synthesis, histone acetylation, and chromatin remodeling, processes vital for pro-inflammatory macrophage polarization. - Urolithin A also increased glycolysis, which was necessary for its anti-inflammatory effect, as it provided energy to macrophages while their mitochondria underwent fission and autophagy. Notably, urolithin A's anti-inflammatory activity was dependent on supporting mitophagy; without this metabolic adaptation, its anti-inflammatory properties were lost. These insights reveal the metabolic underpinnings of sulforaphane and urolithin A's anti-inflammatory actions, highlighting the potential of targeting cellular metabolism to modulate macrophage behavior and inflammation.