Does arginase mediate immune suppression in the brain?

This week I want to talk about an interesting enzyme: Arginase-I (Arg1) and its metabolic pathway in an immune microenvironment. Arg1 is a cytosolic protein that is involved in the urea cycle. Specifically, it catalyzes the hydrolysis of L-arginine to L-ornithine and urea. It has been shown that the expression of Arg1 by macrophages has an important role in tumor growth. Macrophages that are recruited into the tumor microenvironment (tumor associated macrophages) express high levels of Arg1 resulting in the depletion of arginine – an essential nutrient required for T cell metabolism. Cytotoxic T cells, therefore, can no longer function and inhibit the tumor cells from proliferating. Arg1 is implicated in several inflammatory diseases as well as in autoimmunity. Arg1 plays a significant role in mediating immune suppression and blocking its metabolism is a novel strategy in preventing tumor growth and other inflammation-related conditions.

T cell suppression by MDSC
Myeloid-Derived Suppressor Cell suppresses cytotoxic T cell function through Arg1 metabolism. (IFNγ, IL-4, and IL-13 are cytokines that induced MDSC activation)

One hypothesis is that the immune suppressive cells in other immune microenvironments in the body must be similar to the Myeloid-Derived Suppressor Cells (MDSCs) that induce T cell suppression in the cancer microenvironment. The question is, do such immunosuppressive cells express increased levels of Arg1 and act through the Arg1 metabolic pathway? Since I am interested in the brain and neurodegeneration, this hypothesis can be extended to the brain immune microenvironment. Microglial cells in the brain also upregulate Arg1 and are neuroprotective in nature (in a healthy brain). These cells are the resident macrophages of the central nervous system and function by phagocytosing cell debris and toxic misfolded proteins (that eventually form aggregates and lead to neuronal death as seen in Alzheimer’s disease) out of the brain environment. The question now is – do the microglial cells exhibit immunosuppressive behavior by altering their Arg1 metabolism?

Kan et al., 2015, recently showed that CD11c positive microglial cells are immunosuppressive in the CVN-AD mouse model and that immune suppression is caused due to the deprivation of arginine (increased levels of extracellular Arg1 causing decreased levels of total brain arginine). What isn’t explicitly mentioned in this study is that arginine is also the substrate for nitric oxide synthase (NOS) that makes nitric oxide (NO) in an alternate L-arginine metabolic pathway. L-arginine is a substrate for both Arg1 and NOS. The Arg1 pathway polarizes the macrophages to M2 phenotype and the NOS pathway polarizes the macrophages to the M1 phenotype (Rath et al., 2014). The current model of microglial activation in the CNS is limited to these two polarized states, where, the M1 microglia are neurotoxic and the M2 microglia are neuroprotective. Arg1 is upregulated in microglia in the healthy brain and aids in phagocytosis of misfolded proteins and other cell debris. The classical microglial activation is through the M2 phenotype wherein the induced nitric oxide synthase (iNOS) is upregulated thereby accelerating inflammation in the brain (neuroinflammation is one of the hallmark characteristics of several neurological diseases such as Alzheimer’s Parkinson’s, Multiple Sclerosis, Traumatic Brain Injury, etc).

M1 and M2 microglia
The current model of microglial activation is limited to the Arg1-mediated M1 and iNOS-mediated M2 polarized states.

So, if an immunosuppressive cell exists in the brain, is it possible that the immune suppression is regulated through the M2 activation and that M1 activation is absent? In other words, L-arginine is metabolized through the Arg1 pathway and not through the NOS pathway. Other questions to consider: MDSCs upregulate both Arg1 and iNOS – so how does that fit into the two-state polarization model? How does iNOS modulate MDSC activity? We know that increased NO expression by MDSCs increases T cell suppression in the tumor microenvironment. Currently, both Arg1 and iNOS inhibitors are being developed to block the immune suppressive activity of MDSCs in the cancer microenvironment. However, understanding immunosuppression in the brain is still a long way to go and the idea is not widely accepted within the neurobiology community (my understanding from the currently available literature or published studies). Investigating this mechanism in the brain will be useful in developing potential therapeutic strategies for treating neuroinflammation and neurodegeneration.

References:

  • Kan MJ, Lee JE, Wilson JG, et al. Arginine Deprivation and Immune Suppression in a Mouse Model of Alzheimer’s Disease. The Journal of Neuroscience. 2015;35(15):5969-5982. DOI:10.1523/JNEUROSCI.4668-14.2015.
  • Rath M, Müller I, et al. Metabolism via arginase or nitric oxide synthase: two competing arginine pathways in macrophages. Front. Immunol., 27 October 2014. DOI: https://doi.org/10.3389/fimmu.2014.00532

Metabolic interplay

fimmu-08-00248-g001
Renner K et al. Front Immunol. (2017)

I recently came across this figure that shows the key metabolic processes that dictates an immune cell behavior and function. Biochemists and pharmacologists sometimes focus on one or two key pathways in a disease model and forget that proteins don’t function in isolation. Protein networks are complex pathways with many overlays. A drug designed to inhibit or activate a specific protein can also affect other proteins in the connected pathways. This figure is focussed on an immune cell (natural killer cell) and its interaction with a tumor cell. The interplay between the different metabolic pathways applies to all kinds of cells in the body.

This figure is also quite interesting to me because I have been studying the arginase-1 (Arg1) pathway in microglial cells and this gives me a brief overview of where my study lies in the spectrum of key cellular metabolic pathways. Arg1 is an enzyme that metabolizes L-arginine to L-ornithine and urea in the urea cycle. With the help of ornithine decarboxylase (ODC), L-ornithine further makes polyamines that are important (? – it depends) for cell growth and survival (? – it depends). I think it is quite interesting to see how Arg1 and ODC would dictate the phenotypes of the microglial cells in the brain. Microglia are the brain’s resident immune cells – they chew up all the toxic stuff and get rid of them (this is known as phagocytosis). We have always studied these cells based on their two active states (M1 or M2). There has been evidence in the recent years to show that these cells in fact may exhibit multiple activated states (not just M1 and M2). Just like many immune cells in the body that exhibit a heterogenous phenotype, microglia in the brain may be no different. I’m curious if Arg1 and ODC may be involved in regulating a similar mechanism in microglial cells during neurodegeneration..

Source: Renner K., Singer K., et al. Metabolic Hallmarks of Tumor and Immune Cells in the Tumor Microenvironment. Front Immunol. 2017; 8: 248.

 

It’s brain awareness week!

Hello all!  I wanted to take a few minutes to write something for the brain awareness week. This is important to me because my research focusses on understanding the role of the immune system in the brain. For a very long time, the brain was thought to be an “immune privileged” organ i.e., it was thought that the brain is protected from all the peripheral insults and that it is “divorced” from the rest of the body. In 2015, it was shown that there exists certain lymphatic vessels that connect the CNS to the rest of the body (1). The lymphatic system carries immune cells through a network of vessels and tissues; it connects the bloodstream and tissues in order to remove dead cells and other debris. The discovery of the new “glymphatic system” has opened new avenues to study the connection between the brain and the rest of the body. This is especially helpful in understanding the role of the peripheral immune system on the CNS during infections, injury, and other disease insults.

glymphatic system
Old lymphatic system (left) and newly discovered lymphatic system in the CNS (right). Source: University of Virginia Health System

My work focusses on a specific cell type in the brain known as microglia which are are the resident macrophages of the CNS (they eat up and clear out the bad stuff in the brain like dead cells and mis-folded proteins). Microglia are the only known immune cells of the brain. Compared to all that’s known about the cells of our body’s immune system (B cells, T cells, NK cells, neutrophils, basophils, Treg cells, MDSCs, TH1, TH2, and many many more with several subtypes of each cell), it is safe to say that cells of the CNS are poorly understood. My efforts are focussed towards understanding the role of microglial cells in neurodegenerative diseases such as Alzheimer’s Diseases (AD) , Parkinson’s Disease (PD), Multiple Sclerosis (MS), etcetera. These diseases are characterized by mis-folded proteins that aggregate in the different regions of the brain tissues causing the neurons to degenerate and eventually die. The microglial cells in these disorders play a major role in disease progression by regulating many pathways involved in cell-cell communication, cell survival, and cell death. This is a relatively new and an exciting area of study with many missing links and questions to be answered. I will try my best to keep this space alive with updates and stories! In the meantime, here’s a fun read on Leonardo da Vinci’s contributions to neuroscience: http://www.sciencedirect.com/science/article/pii/S0166223600021214

And here’s a 1504-1506 drawing of the human brain by da Vinci:

Leonardo da Vinci's contributions to neuroscience
In the upper figure, the three ventricles are labeled imprensiva (anterior ventricle, corresponding to the paired lateral ventricles), senso comune (third ventricle), and memoria (posterior or fourth ventricle). Below the ventricles, seven pairs of cranial nerves are shown. The lower figure shows a human head in an exploded view, with the skull raised over the brain and from the head. Source: 

Sources:

  1. Louveau A, et al. Structural and functional features of central nervous system lymphatic vessels. Nature. 2015;523(7560):337–341. doi: 10.1038/nature14432.

The Biology of Vaccination

I was never too involved in the vaccination debate until I came to the United States. Back home in India, the majority of us seem to be grateful to science for being able to wipe out dreadful diseases like MMR (mumps-measles-rubella) and polio, and prevent the lifelong suffering of thousands of people. A friend recently mentioned that his mother refused to vaccinate him as a child when she observed escalated fever-like-symptoms every time he got an immunization shot. This is one small example of a widespread scientific ignorance that lures people into believing in absurd anti-vax propaganda.

Let’s talk about the biology of vaccination. A vaccine is a weakened form of a disease-causing agent that boosts the immune system and provides protection against natural infection. This “agent” may be an altered form of the infection or its less dangerous close relative. A vaccine is usually combined with an adjuvant – a chemical that enhances the immune response. Prior to vaccination, a process known as variolation remained popular in the 17th and 18th century. In this, scab material taken from a mild form of smallpox was inoculated through the skin to curb the disease. Variolation was in no way harmless and therefore ceased to be in use when safer alternatives were sought. The history of vaccination is one of the most interesting stories in the field of science and medicine. Edward Jenner (the father of Immunology) – after having observed that milkmaids exposed to cowpox were protected from smallpox disease,  treated the locals with cowpox scabs and successfully prevented the occurrence of smallpox.

So how does vaccination work? I have briefly talked about the two main kinds of immune responses in one of my earlier posts. Further, acquired immunity consists of antibody (humoral) response and cell-mediated response that involves various types of white blood cells (WBCs) like macrophages, dendritic cells, T-lymphocytes and B-lymphocytes. When an infectious agent enters the body, chemicals called chemokines and cytokines recruit WBCs to the area of infection. The pathogen is broken down into its constituent proteins by Antigen-Presenting Cells (APCs) and is then “presented” to the helper T-lymphocytes (CD4+ T cells). These lymphocytes actively mediate protective immunity.

In humoral immunity, the receptors on B-cells recognize specific antigenic proteins, get activated and multiply to make hundreds of identical cells. Upon maturation, these plasma cells release a large number of antibodies that are specific to the antigen. This rapid increase in the number of antibodies is sufficient to eliminate the pathogen. Apart from the B-cells, cytotoxic T-cells (CD8+ T cells) also induce an immune response by directly destroying antigens that are presented by the APCs.

Primary exposure to pathogen via vaccine and secondary exposure to pathogen via infection - Sequence of events.
Primary exposure to pathogen via vaccine and secondary exposure to pathogen via infection – Sequence of events with respect to humoral immunity. Cell-mediated immunity works similarly through cytotoxic T cells – Activated cytotoxic T cells directly destroys the antigen. (Not shown) — CLICK TO ENLARGE —

When the infection is cleared, the immune response reduces and so does the number of antibodies and cytotoxic T-cells. During this time, some of the T- and B-cells become memory cells and preserve their antigen-specific surface receptor. These cells stick around in our serum and wait for a subsequent attack by the same pathogen. This is the crux of vaccination.

When our body is invaded by the same pathogen again, these memory cells immediately proliferate and release surplus of specific antibodies against it. This secondary response is faster and involves a greater number of cells, and is therefore more effective than the primary response. Vaccination establishes a pool of memory cells that are specific to the antigen and prepares the body in case of future infection. Therefore, when a weakened form of the pathogen is intentionally administered to us, our body develops an “actively acquired immunity” for a quicker and a more efficient secondary response.

Antibody response during primary and secondary exposure
Antibody response during primary and secondary exposure

The milkmaids from Edward Jenner’s anecdote had acquired an active immunity for smallpox virus because they were previously infected by the cowpox virus (both poxviruses, members of the Poxviridae family) due to their occupation. Also, when my friends mother observed an escalated fever-like symptoms after the vaccine shot, it was merely the body’s primary immune response to the infection – completely normal and a sign of an actively functioning immune system.

Though the science of vaccination is pretty forthright, many concern arises regarding its safety, constituents, production and side-effects. It is important to understand that every immune system is unique due to which every person may respond differently to different vaccines. Many of the health and safety claims (with respect to autism, mercury, formaldehyde, and so on) have already been debunked extensively by reputed scientific sources. Also, parents choosing not to vaccinate their kids against the government’s decision are endangering the rest of the community. Herd immunity works when the larger part of the population is resistant to a pathogen providing protection to those without immunity thereby preventing an outbreak. And finally, if you’re against vaccination due to your religious beliefs, please pack up and leave.

Interestingness –

  1. How the anti-vaccine movement is endangering lives
  2. The dangerous consequences of anti-vaccine propaganda in one map
  3. Understanding Herd Immunity
  4. 9 vaccination myths busted. With science!

Memory response of Natural Killer cells

The beauty of our immune system lies in the diversity of the cells involved in the responses triggered by a range of pathogens. These responses can be broadly classified into two groups: innate immune response, which is mainly a non-specific first line of defence, and adaptive immune response, which is acquired over the lifetime of all vertebrates and contains highly differentiated cells that are specific to the antigen. These cells are capable of recognizing the antigens upon secondary exposure and therefore contribute to the memory of the immune system.

Until now, natural killer (NK) cells were considered to be a significant part of the innate immune system participating in non-specific responses. These cells play important functional roles against tumor cells and virus infected cells, through their cytolytic activity. Like T and B cells of the adaptive immunity, NK cells do not express RAG (recombination-activating gene) proteins that are required for assembling the antigen specific receptors on the cells. Based on this concept, RAG knockout mice are common model organism to study cell-mediated immune responses as they lack a functional adaptive system.

Figure 1 - Rag1/2 knockout mouse lacks memory
Figure 1 – Rag1/2 knockout mouse lacks memory

Contact hypersensitivity (CHS) is another significant experimental model used to study cell-mediated immune function in the context of memory.1 In this, epidermal cells that are exposed to haptens (compounds that modifies proteins and elicit an immune response) exhibit a delayed-type hypersensitivity reaction. It was first shown by O’Leary et al.2 that Rag2 knockout mice exhibited hapten induced CHS due to NK cells, thus confirming the role of NK cells in memory responses.

Figure 2 - Hapten induced Rag2 knockout mouse exhibited CHS
Figure 2 – Hapten induced Rag2 knockout mouse exhibited CHS

So how did O’Leary et al. confirm that it was indeed NK cells that was responsible for inducing hypersensitivity and not other cell types of the innate immune system? As shown in Fig. 2 above, Rag2 knockout mouse that is incapable of producing mature T and B cells still reacted to the hapten. However, Rag2/II2rg double knockout mice did not exhibit the hypersensitivity reaction when exposed to haptens. These double knockout mice lack NK cells along with T and B cells. Moreover, the Rag2 deficient mice that were treated with anti-NK 1.1 antibody (binds to receptors on NK cells and prevents activity) also showed no hypersensitivity response to the haptens. The wild type mice treated with anti-NK 1.1 antibody exhibited CHS due to the activity of T and B cells.

The study of immune responses is still a new and exciting field with many unanswered questions and unknown terrains yet to be discovered. This novel insight on NK cells possessing adaptive immune-like responses and its role in memory could be significant in designing next generation vaccines against a range of pathogens.

References:

  1. Gaspari AA, Katz SI. Contact hypersensitivity. Current Protocols in Immunology. 2001 Chapter 4, Unit 4 2.
  2. O’Leary, J.G. , Goodarzi, M. , Drayton, D.L. & von Andrian, U.H. T cell- and B cell- independent adaptive immunity mediated by natural killer cells. Nature Immunology. 7, 507–516 (2006).