Research

 

Significant discoveries of the Murray Group  

How do immune cells sense and transfer information about metabolites into growth and activation decisions?

Overview: Immune cells require the importation of large quantities of amino acids to support proliferative expansion and activation to kill pathogens. Immune cells are auxotrophs for all the essential amino acids and most of the non-essential amino acids (e.g. glutamine, serine). However, the immunologic ‘checkpoints’ that control cell behaviour are devoted to arginine and tryptophan metabolism. Generally, one cell types consumes arginine or tryptophan (macrophages) depriving another cell of the necessary resources (activated T cells). The complex cross-talk between immune cells therefore extends beyond cell surface receptors, cytokines and chemokines into metabolites as signalling molecules.

These two papers, the result of a decade-long collaboration with Tom Wynn and the NIH, dissect the main immune pathways regulated by the arginine hydrolase Arginase-1 using the first conditional allele created to disrupt the gene. The papers collectively resolved long-standing issues in the field and set the stage for the genetic exploration of the biochemical control of arginine metabolism in immunity.

El Kasmi, K. C., …, Wynn, T. A. and Murray, P. J. (2008) Toll-like receptor-induced arginase 1 thwarts effective immunity against intracellular pathogens. Nature Immunol. 9: 1399-1406.

Pesce, J. T., … Murray, P. J.* and Wynn, T. A.* (2009) Macrophage/neutrophil specific arginase 1 functions as a negative regulator of Th2-driven inflammation and fibrosis. PLoS Pathogens 5(4): e1000371. *Equal corresponding authors

This paper established the mechanics of arginine utilization in activated macrophages. Contrary to prevailing expectations we found activated macrophages consume all arginine available until exhaustion, and at the same time, exclude the products of the reactions into the extracellular space (citrulline and nitric oxide). Once the arginine is consumed, citrulline is re-imported and recycled to arginine to continue NO synthesis. We concluded citrulline metabolism is a fail-safe mechanism to sustain NO output in intracellular infection.

Qualls, J. E., … Murray, P. J. (2012) Sustained generation of nitric oxide and control of mycobacterial infection requires argininosuccinate synthase 1. Cell Host & Microbe 12: 313-323.

These three papers describe the Group’s first attempts to decipher how T cells detect limiting amino acids, and how they translate this information into decisions about proliferation. Unexpectedly, we uncovered a key role for the mTOR complex mTORC2, which requires the structural protein Rictor, rather than the mTORC1 complex, which is linked to amino acid sensing in most other cell types.

Van de Velde, L., … and Murray, P. J. (2017) T cells encountering myeloid cells programmed for amino acid-dependent immunosuppression use Rictor/mTORC2 for proliferative checkpoint decisions. J. Biol. Chem. 292: 15-30.

Van de Velde, L. and Murray, P. J. (2016) Proliferating helper T cells require Rictor/mTORC2 to integrate signals from limiting environmental amino acids. J. Biol. Chem. 291: 25815-25822.

Van de Velde, L., … and Murray, P. J. (2016) Stress kinase GCN2 controls the proliferative fitness and trafficking of cytotoxic T cells independent of environmental amino acid sensing. Cell Reports 17: 2247-2258.


Macrophages: the immune system’s garbage disposal, killing machines and “stop or go” signal system for other immune cells, all in one cell type

Overview: Macrophages are found in all tissues of the body where they perform vital homeostatic functions. For example, microglia (macrophages of the brain) prune dendritic spines and remove dead neurons while macrophages in the spleen eliminate expired red cells and help recycle the body’s iron stores. In homeostasis, infection and all pathologic conditions, macrophages can be activated (‘polarized’) to different phenotypes by environmental and cytokine cues. Macrophage polarization is a very active area of research where we have made many contributions. The most significant concern how activated macrophages can be controlled.

This paper details the first elucidation of the central negative regulatory pathway that blocks M2 macrophages mediated by TNF. Although derived in cancer inflammation, we propose the TNF-mediated anti-M2 pathway will extend to any inflammatory scenario where TNF amounts are modulated or manipulated.

Kratochvill, F., … and Murray, P. J. (2015) TNF counterbalances the emergence of M2 tumor macrophages. Cell Reports 12: 1902-1914.

This paper describes an early attempt at a transcription wide level to understand how IL-10 negatively regulates inflammation. Although primitive by today’s high-resolution standards, this paper has stood the test of time because of its underlying experimental approach; by using IL-10 added back to IL-10-deficient macrophages in different inflammatory settings, we were able to provide the first ‘clean’ data set, and show IL-10 is a selective rather than general regulator of TLR-induced inflammation.

Lang, R., …. and Murray, P. J. (2002). Shaping gene expression in activated and resting primary macrophages by IL-10. J. Immunol. 169: 2253-2263.

Although the Lang et al. paper solved the problem of the general vs. specific nature of IL-10’s negative effects of inflammatory gene expression (the latter is true), and other groups including ourselves showed STAT3 is essential for the IL-10 signaling pathway, two unanswered questions were (i) whether transcription was the target, and (ii) whether STAT3 acted directly, or induced proteins to execute the inhibitory effect. I constructed a mouse where the 3’UTR of Tnf was replaced to bypass the confounding effects of mRNA stability. When combined with biochemical tests for mRNA transcriptional rate by measuring unsliced transcripts, I concluded IL-10 targets transcription, but required new protein synthesis. Downstream of IL-10 STAT3 does not act directly but induces the expression of other genes that control inflammation.

Murray, P. J. (2005) The primary mechanism of the IL-10-regulated anti-inflammatory response is to selectively inhibit transcription. Proc. Natl. Acad. Sci. USA. 102: 8686-8691.

SOCS proteins are inhibitors of cytokine receptors. In this key paper in the field we elucidated the mechanism of SOCS3-mediated inhibition of cytokine receptor signaling. Rather than inhibiting overall signaling output from gp130, we found SOCS3 also controls the types of signaling, and their amplitude. SOCS3 suppresses both STAT3 and the ectopic STAT1. Later work from us, and work from Aki Yoshimura’s group showed SOCS also suppresses the ability of gp130 to activate the IL-10-like STAT3 anti-inflammatory pathway.

Lang, R., … and Murray, P. J. (2003) SOCS3 regulates the plasticity of gp130 signaling. Nature Immunol. 4: 546-550.


Understanding the enigmatic ‘Myeloid-derived Suppressor”: immune cells that give the ‘stop’ signal

Overview: Chronic inflammation is also call ‘non-resolving’ inflammation and is found in all diseases where the insulting entity cannot be removed. Examples of non-resolving inflammation include chronic infection (tuberculosis, HIV), cancer (persistent malignant cells), diseases such as asbestosis where fibers cannot be removed, autoimmune diseases where the bodies own tissues provoke inflammation, and even situations such as tattooing, where ink particles persist in the skin and cannot be digested and destroyed by macrophages. In all these cases, the bone marrow increases the output of myeloid cells to try and combat the problem. In doing so, immature myeloid cells are also made, often in vast quantities. These cells are myeloid derived suppressors because they are potent blockers of T cells. A major goal of cancer research of to ‘suppress the suppressors’ to create environments where T cells can kill cancer. In other situations, myeloid suppressors could be useful to block aggressive immune responses (such as in transplantation).

This paper describes how cell death pathways can be used to define and ‘engineer’ myeloid-derived suppressors, an intrinsically heterogeneous population of immature myeloid cells arising in all forms of inflammation. We found monocytic MDSCs require suppression of the extrinsic death pathway (mediated by c-FLIP) and the intrinsic death pathway (mediated by MCL-1 or the MCL-1-like protein A1). Remarkably, granulocytic MDSCs are completely independent of the extrinsic pathway but have an absolute requirement for MCL-1. By manipulating the death pathways, we found monocytic MDSCs are the key MDSC sub-population required for suppression of T cell proliferation.

Haverkamp, J. M., … Murray, P. J. (2014) Myeloid-derived suppressor activity is mediated by monocytic lineages maintained by continuous inhibition of extrinsic and intrinsic death pathways. Immunity 41: 947-959.


 
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