Acute vs. Chronic Peripheral Immune Stimulation: Differences in Effects on Microglia and Inflammation

The concept of “immune memory” has long been an important, albeit, little understood part of our study of the immune system. For centuries, we have unknowingly utilized immune memory against foreign pathogens (1) that the body has seen before. Whenever a new virus or infection appeared, people who had been exposed to it seemed to suffer fewer symptoms, which we now know is due to this immune memory. We have even “hijacked” this endogenous process and sculpted it to fit our own needs by creating vaccines, which provide immunological memory against particular pathogens to adapt our response, resulting in less damage if we encounter the specific pathogen again. However, we still do not know exactly how immune memory works.

            There are two main elements of the immune system: the innate component, and the adaptive component.(2) The innate component is usually the first line of defense, and along with providing physical barriers against external pathogens (such as skin or mucous), it also attacks any pathogen that enters the body. It relies on large numbers of cells that are not extremely accurate or specific.(3) However, the adaptive immune system is the exact opposite. It is part of a delayed response, and produces T and B cells that specifically target the pathogen that has entered the body. Although it takes longer to initiate, it is much more accurate and helpful in eradication of a particular pathogen.(4)

            Until the last few years, scientists thought that immunological memory existed only in cells of the adaptive immune system.(5) However, a new paper by Wendeln et al. (2018) studied the function of microglia, a component of the innate immune system and located in the Central Nervous System (CNS), and their function after peripheral immune stimulation. Because the Blood Brain Barrier physically separates the brain from the rest of the body and restricts entry of most cells or pathogens into the brain, the brain is classified as “immunoprivileged”, and microglia, in addition to maintaining homeostasis, act as the main immune cell in the brain. However, microglia are not specific to certain pathogens, and are therefore part of the innate immune system. Wendeln et al. found that intraperitoneal application (injection into the abdomen) of a peripheral inflammatory stimulus “trained” microglia to alter their function and either promoted or inhibited inflammation in the brain for up to six months, depending on whether the inflammatory stimulus was acute or chronic. When the scientists tested their hypothesis in a mouse model of Alzheimer’s pathology, they found that the acute peripheral stimulus caused the microglia to decrease their clearance of Amyloid-Beta, which is one of the two main proteins that build up in Alzheimer’s Dementia. However, when the peripheral stimulus was induced chronically (four times), the microglia became “tolerized”, and released lower levels of proinflammatory cytokines (signaling molecules that are released by immune cells and can increase or decrease inflammation), resulting in less inflammation and lower amyloid-β buildup, compared to “trained” microglia.

            The authors injected the mice with four doses of lipopolysaccharide, LPS (an immune system stimulator (6)), over the course of four days. After the first dose, there was an increase in proinflammatory cytokines in the blood, but not the brain. After the second injection, levels of proinflammatory IL-1β, TNF-ɑ, IL-6, IL-12 and IFN-γ were all increased in the brain, showing how the brain-resident microglia had been “trained” to induce a proinflammatory response after peripheral immune stimulation. The third dose of LPS provided similar results to the second, but interestingly, the fourth dose eliminated most, if not all, proinflammatory secretion of TNF-ɑ, IL-1β and IL-6, while increasing anti-inflammatory IL-10. This indicates that the microglia had become “tolerized”.

Note: Anti-Iba1 stains Microglia

           To ensure that it was microglia that were responsible for the inflammation within the brain, the authors used genetic knockout mice whose microglia lacked Tak1, HDAC1 and HDAC2; without these genes, the microglia were unable to modify transcription of genes as part of immunological memory. The microglia in these knockout mice were unable to become “trained” or “tolerized”, and therefore levels of TNF-ɑ, IL-1β and IL-6 did not increase after the second dose of LPS before decreasing after the fourth dose, as they did in the normal wild-type mice. However, what I found most interesting about this aspect of the experiment was that no LPS was found in the brain; although it produced a peripheral immune response, somehow it was the peripheral response and not the LPS itself that signaled to microglia within the brain to release proinflammatory cytokines.

            The authors next examined how “training” and “tolerizing” microglia affected long-term brain immune responses, and therefore disease progression. They used APP23 mice, which are an animal model for Alzheimer’s Disease, and produced large plaques of amyloid-β, which activated the microglia.(7) They injected these mice with four doses of LPS and found that, again, acute peripheral inflammation increased the levels of proinflammatory cytokines while chronic inflammation resulted in fewer levels of these cytokines. However, they also found that the “trained” microglia, in addition to releasing IL-1β, IL-6 and IL-12, also caused greater buildup of amyloid plaque, while the “tolerized” microglia were able to decrease plaque load and total amyloid-β levels. This could have important clinical consequences; it implies that chronic peripheral immune stimulation, which happens in many diseases or autoimmune disorders, can “train” microglia to promote inflammation and amyloid buildup in the brain, accelerating the course of Alzheimer’s Disease.

            To determine how acute and chronic peripheral immune stimulation affected microglia, the authors analyzed the genetics of the microglia and determined which genes had different expression levels during acute vs. chronic exposure. They found that acute exposure (one or two doses of LPS) caused increased expression of hypoxia inducible factor-1ɑ, which resulted in inflammation and the release of proinflammatory cytokines via lactate secretion.(8) On the other hand, chronic exposure increased expression of genes involved in the “Rap1 signaling pathway”, which promoted phagocytosis and does not cause increased secretion of proinflammatory cytokines, therefore not resulting in inflammation.(9) Similar results were found in the APP23 mice, which explained why lower levels of amyloid-β were found in the chronically exposed mice, as their “tolerized” microglia had increased phagocytic capabilities.

            Future experiments should determine how exactly peripheral immune stimulation is able to pass through the Blood Brain Barrier and induce changes in microglia. As the authors found no LPS within the brain, it cannot be that the stimulant itself crosses into the brain to activate microglia. If scientists are able to determine exactly what in the periphery causes the microglia to become “trained” or “tolerized”, it can radically affect our treatment of neurological diseases, like Alzheimer’s Disease. If the signal is able to be blocked, we could decrease microglial activation, which would prevent inflammation within the brain and protect neurons, which would help to slow the course, or even stop the progression of these severe and widespread neurological diseases.

Bibliography

1.     Definition of a Pathogen: https://www.sciencedaily.com/terms/pathogen.htm

2.     PubMed Description of the Innate and Adaptive Immune Systems: https://www.ncbi.nlm.nih.gov/books/NBK279396/

3.     Definition of the Innate Immune System and its Functions from Molecular Biology of the Cell, 4th Edition: https://www.ncbi.nlm.nih.gov/books/NBK26846/

4.     Explanation of and Illustrations for the Adaptive Immune System:

http://library.open.oregonstate.edu/aandp/chapter/21-3-the-adaptive-immune-response-t-lymphocytes-and-their-functional-types/

5.     Netea MG, Latz E, Mills KHG, O'Neill LAJ. Innate immune memory: a paradigm shift in understanding host defense. Nature Immunology. 2015; 16:675–679.

6.     Description of the Structure and Function of LPS: https://www.sigmaaldrich.com/technical-documents/articles/biology/glycobiology/lipopolysaccharides.html

7.     Sturchler-Pierrat C, Staufenbiel M. Pathogenic mechanisms of Alzheimer's disease analyzed in the APP23 transgenic mouse model. Annals of the New York Academy of Sciences. 2000; 920:134– 139.

8.     Haas R, Smith J, Rocher-Ros V, Nadkarni S, Montero-Melendez T, D’Acquisto F, et al. (2015) Lactate Regulates Metabolic and Pro-inflammatory Circuits in Control of T Cell Migration and Effector Functions. PLoS Biol 13(7): e1002202.

9.   Chung, J., Serezani, C. H., Huang, S. K., Stern, J. N., Keskin, D. B., Jagirdar, R., Brock, T. G., Aronoff, D. M., … Peters-Golden, M. (2008). Rap1 activation is required for Fc gamma receptor-dependent phagocytosis. Journal of immunology (Baltimore, Md. : 1950), 181(8), 5501-9.

Link to the Article Here: Innate immune memory in the brain shapes neurological disease hallmarks

Peripherally Derived Macrophages Reduce Inflammation of CNS Lesion via Alterations of Local Microglial Functions

             

            Central Nervous System (CNS) lesions happen in an untold number of people every year, with numerous negative effects, including loss of senses, paralysis, and even death. They are often caused by physical injury, infections, or long-term diseases such as Multiple Sclerosis.(1) Most CNS lesions occur in the brain, and therefore can affect other parts of the body. Because of the broad range of symptomatology associated with them and the consequences they can cause, it is crucial that we increase our understanding of how the CNS repairs itself after lesion.

            Although it has been known that the immune system plays a role in recovery and repair of CNS lesions, it was previously unknown through exactly which mechanisms the immune system provided assistance. Because the CNS is protected by the Blood Brain Barrier (BBB), which is composed of astrocytic endfeet and acts as a physical barrier to only allow certain cells, substances and molecules into the CNS, scientists did not know exactly how peripheral monocyte-derived macrophages(2) (MDMs) regulated repair.

Previous studies had shown that the cellular response to CNS injuries and lesions was the rapid recruitment of tissue resident microglia (3) (4) and the later involvement of MDMs one to three days following injury (5). Although the arrival of MDMs decreased microglial proinflammatory functions, it was unclear exactly how this crosstalk occurred.

            A recent study from McGill (6) has examined the specific effects of MDMs on microglia in CNS lesions, explaining why their recruitment is a vital part of recovery. In the study, Greenhalgh et al. (2018) caused spinal cord injuries (SCIs) in mice, and used green fluorescent protein (GFP) to track both MDMs and resident microglia. They found that, in the lesions, the microglia often had beneficial effects initially in the first minutes-to-hours following injury by preventing lesion expansion; however, the microglia soon prove detrimental to recovery, and within a few days, caused a prolonged inflammatory response in which the microglia non-specifically phagocytized (ingested) cells within the lesion, promoted apoptosis (regulated death) of neighboring cells via release of inflammatory molecules, such as cytokines IL-1β, TNF, IL-6 and IL-10, and stimulated upregulation of microglial inflammatory phagocytosis and apoptosis-promoting genes.

         

  

However, the authors found that the recruitment of MDMs to sites of injury one-to-three days post-injury reverse many of these deleterious microglial functions. By co-culturing macrophages with adult microglia in vitro (outside the body), they were able to study the interactions between the two cell types, and found that macrophages suppressed microglial phagocytosis and inflammatory gene expression, via Prostaglandin E₂ binding to the EP2 receptor. Previous works found that Prostaglandin E₂ (PGE₂) signaling via the EP2 receptor, reduces IL-1β (a potent inflammatory cytokine) expression in microglia (7), and EP2 receptor activation inhibits phagocytosis (8).

By examining RNA transcript levels, the authors determined that the MDMs increased transcription of microsomal prostaglandin E synthase-1 (mPGES), which is required to make PGE₂, and resident microglia expressed high levels of the EP2 receptor. As well, the enzyme that degrades PGE₂ was found to have decreased expression in lesions, further implying that MDM PGE₂, via its binding to microglial EP2 receptors, inhibits microglial phagocytosis and proinflammatory gene expression.

To test their hypothesis in vitro, the authors treated microglia with an EP2 receptor antagonist, and examined microglial phagocytosis. They also performed an in vivo experiment (within a living animal) by genetically knocking out mPGES in macrophages, and inducing an SCI. Both experiments showed that when the PGE₂-EP2 pathway is disrupted, there is increased microglial phagocytosis; this supports their hypothesis that MDMs suppress microglial activity via this pathway.

        

    After determining the mechanisms for how MDMs decreased phagocytosis, the authors next examined what happened if infiltration of macrophages into the lesion was blocked. Because they knew that mice lacking CCR2 cannot recruit MDMs to CNS lesions (9), they studied CCR2 KO mice and found that these mice had, in addition to almost no MDMs, increased microglial phagocytic activity, and when they examined the genetic profiles of these microglia, they found high levels of MyD88, Cxd2 and NF-κB (which promote inflammation) and Trp53 and Bd2 (which are involved in stimulating apoptosis). As well, the CCR2 KO mice had greater microglial activation in lesions 28 days after SCI, which corresponded to increased myelin loss and worse locomotor recovery.

            These findings are important to both clinicians treating, and researchers studying, CNS lesions. Because many people suffer from these lesions and they have a variety of causes, it is imperative that we improve treatment of them. To do this, we need to better understand how CNS lesions heal. This article does that by examining the effects of the immune system on recovery, and how the MDMs are vital for healing and cessation of microglial inflammation. Although there is not much data available on CNS lesions because many can be asymptomatic and it is hard and expensive to identify a lesion even when a patient does present with symptoms, they undoubtedly take innumerable lives every year, can lead to serious diseases or symptoms, and cost us millions or billions every year. We must learn how to better treat them not only to save money, but, more importantly, prevent the pain, suffering and even death that can accompany them. Although this article was very helpful in explaining how MDMs are vital to healing CNS lesions, the authors did not explain if this would have any potential clinical relevance in the near future. One could hypothesize that if someone came into the Emergency Room with a CNS lesion, doctors could simply inject them with grafted MDMs to promote healing. However, this does not take into account the idea of immune rejection. If the body recognized foreign MDMs that were injected as “non-self”, it would create an immune response that would do much more harm than good. Therefore, I believe that future research should focus on how to increase MDM levels and circulation within the body. This would allow clinicians to promote healing without having to inject a patient with foreign MDMs, which brings with the risk of immune rejection.

Bibliography

1. WebMD Description of Brain/CNS Lesions: https://www.webmd.com/brain/brain-lesions-causes-symptoms-treatments#1

2. Description of MDMs: https://www.stemexpress.com/human-blood-products/peripheral-blood-monocyte derived-macrophages-frozen.html

3. Davalos D, Grutzendler J, Yang G, Kim J, Zuo Y, Jung S, et al. ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci. 2005; 6(8):752–8

4. Sevenich L (2018) Brain-Resident Microglia and Blood-Borne Macrophages Orchestrate Central Nervous System Inflammation in Neurodegenerative Disorders and Brain Cancer. Front. Immunol. 9:697.

5. Schwab JM, Zhang Y, Kopp MA, Brommer B, Popovich PG. The paradox of chronic neuroinflammation, systemic immune suppression, autoimmunity after traumatic chronic spinal cord injury. Experimental Neurology. 2014; 258(0):121–9.

6. Greenhalgh AD, Zarruk JG, Healy LM, Baskar Jesudasan SJ, Jhelum P, Salmon CK, et al. (2018) Peripherally derived macrophages modulate microglial function to reduce inflammation after CNS injury. PLoS Biol 16(10): e2005264.

7. Caggiano AO, Kraig RP. Prostaglandin E Receptor Subtypes in Cultured Rat Microglia and Their Role in Reducing Lipopolysaccharide-Induced Interleukin-1 β Production. Journal of Neurochemistry. 1999; 72(2):565–75.

8. Aronoff DM, Canetti C, Peters-Golden M. Prostaglandin E2 Inhibits Alveolar Macrophage Phagocytosis through an E-Prostanoid 2 Receptor-Mediated Increase in Intracellular Cyclic AMP. The Journal of Immunology. 2004; 173(1):559–65.

9. Ma M, Wei T, Boring L, Charo IF, Ransohoff RM, Jakeman LB. Monocyte recruitment and myelin removal are delayed following spinal cord injury in mice with CCR2 chemokine receptor deletion. Journal of Neuroscience Research. 2002; 68(6):691–702.