Investigating lactate as an inflammatory early signal in ectopic lymphoid neogenesis and its translational impact in patients with autoimmune diseases

Lead Research Organisation: University of Birmingham
Department Name: Institute of Inflammation and Ageing

Abstract

Ectopic lymphoid-like structures (ELS) are defined as lymphomonocytic cell clusters forming at sites of chronic inflammation which acquire phenotypic (i.e. segregation of T and B lymphocytes in discrete areas with differentiation of networks of stromal-derived follicular dendritic cells) and functional (i.e germinal centres) features of secondary lymphoid organs. The formation of ELS has been described in organ-specific autoimmune conditions, solid tumorigenesis, in chronic infections and graft rejection. In the context of Sjogren's syndrome (SS), the second most common rheumatic autoimmune disease with a prevalence between 100,000-250,000 in UK and a predominance in women (female to male ration 9:1), ELS develop in the salivary glands in 30/40% of the patients. In SS, the presence of ELS with germinal centres is associated with a more severe disease and a 16-fold higher risk of B cell lymphoma, frequently arising in the affected glands.
The mechanisms of ELS formation are largely unknown but appear to be the result of the progressive accumulation and organization of T and B cells within chronically inflamed tissue whereby the activation of the lymphotoxin/lymphoid chemokine feedback loop is triggered by pro-inflammatory cytokines, most notably IL-17 and IL-17 related cytokines such as IL-22. These mediators are primarily released by CD4+ T cells which are critical also for the cognate interaction with B cells and the development of ectopic germinal centres. In previous work (as well as work currently under re-review at Cell Metabolism) we demonstrated that lactate, whose accumulation is a typical feature of chronically inflamed tissues, modulate CD4+ T cells via the specific lactate transporter SCL5A12 promoting an expansion of Th17 cells and their retention as site of inflammation.
Thus, in this application, which is a Partnership between 2 UK Universities, we plan to perform a series of comprehensive ex vivo, in vivo and in vitro studies with the aim to: 1) investigate whether salivary glands and peripheral SLC5A12 and IL-17 expression in CD4+ T-cells is differentially regulated in ELS+ vs ELS- SS patients and in responders vs non-responders to B cell depletion; 2) assess the impact of knocking out SLC5A12 (genetic approach) and the therapeutic potential of blocking SLC5A12 (pharmacological approach) in modulating ELS in an inducible murine model of salivary gland ELS and in SS salivary gland organ cultures; 3) dissect the metabolic pathways underlying the observed effects of lactate/SLC5A12 on CD4+ T-cells from SS patients.
Overall, this proposal will highlight the importance of a novel key pathway linking metabolism and immune cell function by addressing its impact on the development of immunopathology lesions which are extremely relevant to SS and common to many chronic autoimmune/inflammatory diseases. In the short term, it is envisaged that, if successful, this work will bring new knowledge to a poorly investigated field and to a condition, SS, which is considered an orphan disease with clear unmet knowledge and clinical needs. Importantly, our work has the potential to impact on other medical conditions characterised by the formation of ELS, including cancer, chronic infection and graft rejection. We also anticipate that by showing "clinical" and biological efficacy of blocking the lactate/SLC5A12 pathway with a novel anti-SLC512 monoclonal antibody - which we developed - in a murine model of ELS and in human organ cultures, in the long term we will pave the way towards translation of our research towards patient benefit.

Technical Summary

Based on our published and new data (revised manuscript resubmitted at Cell Metabolism), we hypothesize that T cells entering the inflamed tissue sense lactate via the expression of SLC5A12 with consequent loss of motility, increased retention at the site, and enhanced production of IL-17. Thus, we will test that:
i) CD4+ T lymphocytes infiltrating the inflamed tissue undergo a profound intracellular metabolic rewiring and activate biosynthetic pathways as a consequence of lactate influx through the carrier SLC5A12, which in turn causes the observed marked production of IL-17 and reduced migratory abilities by CD4+ T cells;
ii) This is a fundamental process in the control of ELS formation, maintenance and function in the inflamed tissue.
We plan to proceed in 3 progressive steps performing a comprehensive set of ex vivo, in vivo and in vitro experiments, as reflected in the plan of investigation:
Step 1) Investigate SLC5A12 and IL-17 expression on peripheral (blood) and salivary gland CD4+ T-cells in ELS+ versus ELS- SS patients and before/after Rituximab:
i) Multicolour confocal microscopy coupled to digital image analysis;
ii) Multicolour FACS analysis;
iii) Transcriptomic analysis.
Step 2) Assess the impact of knocking out SLC5A12 (slc5a12-/- mice) and the therapeutic potential of blocking SLC5A12 in modulating ELS formation, maintenance and function in an inducible murine model of salivary gland ELS that we have developed and in SS salivary gland organ cultures:
i) Murine model of adenovirus-induced salivary gland ELS;
ii) Ex vivo salivary gland organ cultures.
Step 3) Dissect the metabolic and intracellular pathways responsible for lactate/SLC5A12-mediated induction of IL-17 and other inflammatory cytokines and inhibition of migration in CD4+ T-cells from SS patients:
i) Mass spectrometry unsupervised metabolomics;
ii) Mass spectrometry stable isotope (13C-lactate) tracer analysis;
iii) Pathway/functional analysis

Planned Impact

The project will have a significant impact both on basic research and applied translational science.

1. Impact on basic research and knowledge acquisition within and beyond the field of SS and musculoskeletal disorders.
Both metabolic regulation of immune cell (dis)function and the mechanisms underlying the formation of ELS are high impact fields of very active research with repercussions spanning different fields of biomedical sciences. Thus, the impact of this proposal which combines via a series of mechanistic steps the investigation of the role of the lactate/SCL5A12 pathway on IL-17 dysregulation and CD4+ T cell entrapment at the site of inflammation with the formation of ELS in models of autoimmunity, is potentially extremely significant. At the basic science level a better understanding of the above key immunoregulatory pathways in the context of SS as a paradigm disease characterized by ELS formation in the salivary glands will broadly benefit the scientific community with considerable advance in knowledge not only in the specific field of SS but also in other autoimmune diseases, characterized by ELS (i.e. rheumatoid syndrome, lupus nephritis, inflammatory bowel diseases, multiple sclerosis, autoimmune thyroiditis, etc.) and in other infectious and neoplastic diseases where the formation of ELS has been described and associated with profound influences in disease evolution and prognosis.

2. Impact on translational and clinical research. Demonstration of the therapeutic potential of blocking the SLC5A12/lactate pathway in the preclinical models employed in this proposal (inducible ELS formation in mice and the human salivary glands organ cultures) could pave the way for clinical translation in to early phase clinical trials in collaborations with Industrial partners, which will be immediately sought. In this regard, Dr Mauro has already generated blocking anti-human SLC5A12 monoclonal antibodies with pump priming from Queen Mary Innovation (Patent application WO2016063037 A1), which, if proven effective in our proposal, will be further developed towards clinical application. A defined developmental programme will be considered based upon the findings of this research. Prof Bombardieri at QMUL and Dr Barone at UoB (see letter of collaboration) collaborate closely with several Industrial partners in order to develop novel early phase clinical trials with novel biologics targeting ELS with primary Sjogren's syndrome as main indication ensuring a rapid translation towards clinical development if supported by the scientific data proposed here. Both Academic partners are leading members of the NIHR NOCRI Translational Research Collaboration and have contributed to develop the anti-CD40 and the anti-ICOSL randomized clinical trials in SS patients.

3- Impact on patient health. It is hoped that the ultimate beneficiaries of this research will be in primis patients with autoimmune disorders characterized by ELS formation in their target organs including SS, a disease with prevalence in UK between 0.2-0.5% (100,000-250,000 patients) which currently lacks disease modifying drugs. As mentioned above, both Academic partners are very well positioned to move this research towards translation into clinical application once data in support of this strategy are generated in the preclinical models proposed.

Publications

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Certo M (2020) Lactate: Fueling the fire starter. in Wiley interdisciplinary reviews. Systems biology and medicine

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Pucino V (2020) Metabolic Checkpoints in Rheumatoid Arthritis. in Frontiers in physiology

 
Description DIF/Evotec
Amount £60,000 (GBP)
Organisation University of Birmingham 
Sector Academic/University
Country United Kingdom
Start 02/2021 
End 08/2021
 
Description Bi-lateral collaboration with the Maffia's group at University of Glasgow (2019 - Still Active) 
Organisation University of Glasgow
Country United Kingdom 
Sector Academic/University 
PI Contribution We have shared with them some of our Slc5a12KO mice to perform a PCSK9-induced athero model
Collaborator Contribution They have performed an athero model for us
Impact None
Start Year 2019
 
Description Bi-lateral collaboration with the Ruscica's group at University of Milan (2016 - Still Active) 
Organisation University of Milan
Country Italy 
Sector Academic/University 
PI Contribution Study of lactate and lactate transporters in the chronic inflammation associated to adipose tissue
Collaborator Contribution Study of the lactate transporter Slc16a1 (MCT1) KO in models of obesity
Impact Pucino et al Cell Metab 2019 is the main output of the collaboration so far
Start Year 2016
 
Description Collaboration Agreement with Lmito Thereapeutics Korea (2020 - Still Active 
Organisation Lmito Therapeutics Inc
Country Korea, Republic of 
Sector Private 
PI Contribution We will use their small molecules acting on lactate production pathway in the cell to investigate immune functions and ageing responses
Collaborator Contribution They provide the small molecules
Impact None
Start Year 2020
 
Title Inhibitors of lactate transporters for use in the treatment of inflammatory diseases 
Description The invention relates to the use of specific inhibitors of lactate transporters in the treatment of chronic inflammatory diseases. The invention also relates to methods of treatment of such diseases. The inventors have surprisingly found that extracellular sodium lactate and lactic acid inhibit the motility of CD4+ and CD8+ T-cells respectively and that this selective control of T-cell motility is mediated via subtype-specific transporters (Slc5a12 and Slc16a1) that are selectively expressed by CD4+ and CD8+ subsets, respectively. This is a previously unknown feature that differentiates these two subsets. The inventors have shown that inhibition of these lactate transporters promotes the release of T-cells from the inflamed tissue. Thus in a first aspect the invention provides a method of treating an inflammatory disease in a subject comprising administering to the subject a therapeutically effective amount of an inhibitor of Slc5a12 and/or Slc16a1. 
IP Reference WO2016063037 
Protection Patent application published
Year Protection Granted 2016
Licensed No
Impact None as yet