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Immunometabolism – A therapeutic perspective

Immunometabolism – A therapeutic perspective

Introduction

Immunometabolism is an exponentially growing, multi-disciplinary field of research aiming at deciphering the dynamic cellular and molecular mechanisms that interweave metabolic and immunological processes together. Although the term “immunometabolism” first appeared in the literature in 2011, the first studies investigating the connection between immune and metabolic disorders date back to the late 19th century. Our expanding understanding of how different immune cell functions correlate with particular metabolic configurations during homeostasis and inflammation has opened new therapeutic possibilities where the modulation of immune responses might be achieved/facilitated by metabolic reprogramming of immune cells.

T cell metabolism at the steady-state and during an immune response

Among all immune cell subsets currently identified, T lymphocytes are generally considered of particular interest due to their crucial role in anti-tumor, anti-viral and autoimmune responses. At the steady-state, naïve T cells mainly operate under catabolic metabolic programs (oxidative phosphorylation) that maximize energy production (in the form of ATP) over biosynthesis, in order to ensure resting cell survival and basal antigenic immune surveillance. Upon activation (following proper antigenic and co-stimulation cues), T cells must rapidly acquire specific effector functions, change their migratory patterns and actively proliferate – all changes that are highly demanding in energy. The activation of naïve T cells is therefore concomitant with a metabolic reprogramming where oxidative phosphorylation is replaced by the anabolic process of aerobic glycolysis: effector T cells will preferentially ferment glucose to satisfy their energy requirements, even though sufficient oxygen is present to ensure mitochondrial oxidative phosphorylation which would have a better ATP yield (this phenomenon is known as the “Warburg effect”, and is incidentally also observed in cancer cells). Ultimately, once the antigen challenge is cleared, most effector T cells undergo apoptosis, while a pool of long-lasting memory T cells remain to provide enhanced defense against a potential subsequent antigen re-encounter. Memory T cells return to using oxidative pathways to support their long life-span and low turnover rate. Interestingly, memory T cells display a greater spare respiratory capacity in their mitochondria compared to effector T cells, which allows them to rapidly produce ATP in case of secondary antigen exposure, and to quickly and effectively respond to the challenge.

Naïve T cells can differentiate into a variety of subsets. For instance, CD4+ T cells can differentiate into T helper type 1 (characterized by interferon γ secretion), type 2 (IL-4 and IL-13 secretion), type 17 (IL-17 and IL-22 secretion) or regulatory T cells. Importantly, metabolic reprogramming events are not uniform among all T cell subsets and metabolic pathways are central in regulating T cell differentiation and functional responses. Understanding how specific immune functions are endorsed by distinct metabolic programs can help design therapies targeting particular metabolic pathways to precisely modulate T cell differentiation and effector functions. For example, metabolic interventions impacting the ratio between effector and regulatory T cells could help tip the balance between immunogenic and tolerogenic phenotypes, in order to boost or dampen immune responses in specific clinical applications such as cancers or autoimmune diseases.

Main methods used to assess metabolism in immune cells

The methods mentioned below summarize the main experimental assays which can be used (individually or in combination) to characterize metabolic processes in immune cells and their dynamic changes in health and disease.

Measure of glucose uptake: as glucose entry into the cells is one of the early steps necessary to ensure that energy requirements are met, quantifying the rates of glucose uptake can give insight into the metabolic status of immune cells. To perform glucose transport assays, cells are first incubated with a traceable form of glucose: historically, radio-labelled glucose (like 2-deoxy-D-[3H] glucose) was used but is now being replaced by fluorescent glucose analogs (like 2-NBDG or 6-NBDG) that are easier to handle. After a certain period of time, the amount of tracer that has been taken-up by the cells is quantified through scintillation counting or flow cytometry, and then normalized to cell number.

Measure of glucose transporter expression: in human cells, cell surface and/or intracellular expression of glucose transporters, which are mainly represented by members of the Glut family, can be easily assessed by flow cytometry with the use of fluorochrome-labelled antibodies. However, special care must be taken to confirm the staining specificity and efficiency of the detection antibody used, and changes in glucose transporter expression should always be compared with changes in glucose uptake or any other functional metabolic parameter.

Evaluation of mitochondrial function: a global picture of overall cellular metabolism can be drawn by assessing mitochondrial oxygen consumption and glycolysis. One of the most straightforward technology to do that is through the live-cell metabolic assay platform developed by Agilent with its line of Seahorse XF Bioanalyzers™ that can measure the real-time oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells in a multi-well plate, while simultaneously allowing compound addition. The measure of these two parameters, and how they evolve in response to various compounds targeting different mediators of mitochondrial function, can help the experimenter to elucidate the mechanisms involved in metabolic dysregulation.

Measure of metabolite flux via mass spectrometry: the improvement of mass spectrometry technologies over the years has led to the development of new mass spectrometry-based metabolic assays where the usage and flux individual metabolites can be measured ex vivo with the help of isotopically labeled tracers.

Assessment of metabolic inhibitors: a broad array of chemical inhibitors, targeting various sections of different metabolic pathways, can be used in combination with the assays described above to study dysregulated metabolic processes in immune cells, and determine how they impact their status and function. The table below provides a non-exhaustive list of small molecules that can be used to probe metabolic reprogramming events in immune cells.

Name Target Metabolic outcome
2-deoxyglucose Hexokinase ↓glycolysis
3-bromopyruvate Hexokinase ↓glycolysis
Ritonavir Glucose transporter 1 ↓glycolysis
Dichloroacetate Pyruvate dehydrogenase kinase ↓glycolysis
FX11 Lactate dehydrogenase ↓glycolysis
4-CIN Monocarboxylate transporters ↓glycolysis
DASA and TEPP46 Pyruvate kinase M2 ↓HIF1α
C75 Fatty acid synthase ↓fatty acid synthesis
Etomoxir Carnitine palmitoyl transferase I ↓fatty acid oxidation
AICAR AMP kinase ↑fatty acid oxidation
Metformin AMP kinase ↑fatty acid oxidation

↓Complex I

↓ mitochondrial reactive oxygen species.

Cerulenin Fatty acid synthase ↓fatty acid synthesis
Rotenone Complex I ↓OXPHOS
BPTES Glutaminase ↓glutaminolysis
Oligomycin ATP synthase ↓OXPHOS
TOFA Acetyl CoA carboxylase ↓fatty acid synthesis
UK5099 Pyruvate transporter ↓TCA cycle


Immunometabolism as a therapeutic target

As technologies to unravel immunometabolism continue to progress and enable us to learn more about the metabolic changes associated to specific immune cell polarization, activation and function, it also encourages researchers to consider metabolism in immune cells as a potential therapeutic target for diseases where pathogenesis is driven by dysregulated immune responses. This rational has been fueled by early clinical observations that metabolism-targeting drugs currently used for patients with metabolic disorders (diabetes, dyslipidemia…) also affect immune cell function, thereby opening a way to reposition these drugs as immunomodulators.

We will briefly describe here two clinical settings where immunometabolism has shown potential as a therapeutic target: cancers and autoimmune diseases. In both cases, pathogenesis is attributable to an imbalance between effector and regulatory T cell responses. In cancer, the aim is to enhance beneficial anti-tumoral effector T cell responses and attenuate regulatory processes; while on the contrary in autoimmune disease, the goal is to dampen deleterious overactivated autoreactive effector T cells and boost regulatory responses. For both conditions, an active line of therapeutic research revolves around cell-based immunotherapies that rely on the generation of robust long-lasting populations of antigen-specific T cells whose metabolic pathways can be targeted to enhance their function and persistence in vivo.

Targeting immunometabolism in cancers

A first line of thought when considering the modulation of metabolism in anti-tumoral T cell responses as a therapeutic intervention is to appreciate that the tumoral microenvironment itself (ie hypoxia, nutrient deprivation and local immunosuppression induced by growing cancer cells) profoundly impacts the phenotype and function of T cells in situ. Consequently, metabolism-targeting interventions must not only distinguish between T cells and cancer cells to limit undesirable off-target effects, but immunotherapy products – typically, anti-tumoral T cells isolated from the patient, expanded/modified in vitro and transferred back to the patient – must also be robust enough to resist metabolic changes by the tumoral microenvironment.

The table below recapitulates some of the molecules targeting metabolic pathways that can be used to improve anti-tumoral responses in vivo or as conditioning agents during the in vitro manufacturing of cell-based immunotherapeutic products.

Molecule Target Metabolic outcome Effects on T cells
2-deoxy-D-glucose Hexokinase ↓ glycolysis ↑ memory T cell generation.

↑ anti-tumoral T cell function.

Mdivi-1 Drp-1 ↓ Mitochondrial fission ↑ anti-tumoral response.
JQ1 c-Myc ↓ glycolysis ↑ T cell longevity and anti-tumor activity.
Rapamycin mTOR ↓ glutamine metabolism ↑memory T cell generation.
Metformin AMPK ↑ fatty acid oxidation ↑ memory T cell generation.
Fenofibrate PPARα ↑ fatty acid catabolism ↑ T cell activity.
Etomoxir CPT1A ↓ fatty acid oxidation ↓ regulatory T cell differentiation.
1-methyl-tryptophan IDO ↓ tryptophan catabolism Reactivation of exhausted or tolerant tumoral-specific T cells.
INCB024360 IDO ↓ tryptophan catabolism Reactivation of exhausted or tolerant tumoral-specific T cells.
Ipilimumab CTLA4 ↑ glucose metabolism ↓ CTLA4-mediated T cell inhibition.
Imatinib TK ↓ glucose uptake

↓ tryptophan catabolism

↓ Lck-mediated T cell receptor signaling.

↑ CD8 T cell activation.

↑ regulatory T cell apoptosis.


Targeting immunometabolism in autoimmune diseases

In autoimmunity, modulating metabolism to limit the activation of autoimmune effector cells and favor the emergence of immune cells with a regulatory phenotype could present an interesting therapeutic approach to control inflammatory autoimmune responses. In that perspective, some encouraging results obtained in experimental models of rheumatic diseases could potentially be translated to the clinic for the treatment of pathologies such as systemic lupus erythematosus, rheumatoid arthritis and osteoarthritis. Interestingly, some standard of care treatments in these diseases already impact metabolic processes (e.g: methotrexate, metformin).

The table below provides a few examples of metabolic drugs that could improve clinical outcome in rheumatic diseases and are currently under investigation.

Drug Target Metabolic outcome Effects on T cells
Methotrexate JAK-STAT ↓ One-carbon metabolism ↓cell growth

Redox balance modulation

Epigenetic modulation

Metformin mTOR ↓ glutamine metabolism ↓ cytokine production

↓ B cell differentiation

↓ Th17 differentiation

↑ Treg differentiation

2-deoxy-D-glucose Hexokinase ↓ Glycolysis ↓ proliferation

↓ activation

Bz-423 F0-F1-ATPase ↓ Mitochondrial oxidation ↑ lymphocyte apoptosis
Dichloroacetate Pyruvate Dehydrogenase ↓ Glycolysis ↑ ROS

↑ Treg differentiation

3-bromopyruvate Hexokinase 2 ↓ Glycolysis ↓ Th17 differentiation
C968 Glutaminase ↓ Glutaminolysis ↓ proliferation

↓ activation


Conclusion

As we garner more knowledge about the close relationship between T cell metabolism and T cell survival, differentiation and functionality, we also come to truly appreciate how deeply interconnected immune networks and systemic metabolism are. Therapeutic approaches aiming at modulating immune responses though metabolism, although promising, often report contradictory results in pre-clinical and clinical studies, and therefore warrant additional investigations to characterize their effects on different immune subsets in various microenvironments.

References

All the information provided above were extracted from the following publications, and the reader is advised to look further into them from a comprehensive and detailed overview of the subject.

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29th Aug 2019 Dr Celine Van Damme

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