Ibrahim T. Mughrabi, MD, PhD - Spleen neuro-immune interactions and inflammation

So good morning, everyone. Today I'm going to talk to you about a fundamental sociological mechanism in the human body that we think is relevant to endometriosis. These are my disclosures. Okay. So most of you are familiar with this organ, the spleen. It is the largest secondary lymphoid organ in our body, and it plays a center role in immune surveillance and systemic inflammation. It is anatomically divided into two regions, the white pulp where TMB cells are activated and antibody produced, and the red pulp where the blood is filtered from pathogens, immune complexes, and dying red blood cells. It is also a reservoir of circulating monocytes that are deployed to sites of inflammation and injury. Now, this is how we classically represent the spleen in textbooks and lectures. However, there is a crucial element missing in this schematic, and it actually reflects our previous understanding of how the immune system is regulated.

We have historically viewed the spleen as largely autonomous, regulated by chemokines, cytokines, and cell cell interaction. However, we now know that this view is incomplete. And the critical element missing in these schematic is innervation or neural integration. This is an optically cleared mouse spleen stained for tyrosine hydroxylase in green, which is the enzyme responsible for making the neurotransmitter and norepinephrine and epinephrine. And here it's used to stain the sympathetic motor neurons in the spleen. And as you can see, the spleen is densely innervated with sympathetic fibers that are expanding every corner of the organ and they actually arborize and branch into T and B cell zones. Recently, the spleen has also been shown to be innervated by sensory neurons as well. Now, this innervation of the spleen wires back to the autonomic nervous system via the celiac ganglion. Sorry. Via the celiac ganglion and the splenic nerve.

The splenic nerve originates from the celeganglion, travels along the splenic artery and then enters the spleen via the hilum and then branches into the different compartments of the spleen. This affords close proximity of immune cells to nerve fibers and allows for cross-communication and cross-influence.

So how does the autonomic nervous system control the immune response in the spleen? It does that via autonomic reflexes. Sensory inflammatory stimuli in the periphery are sensed by autonomic and spinal sensory neurons in the vagus and spinal nerves. And these inflammatory stimuli could be chemokines or cytokines or other immune mediators released by immune cells. And the reason why these are able to produce a sensory signal is because sensory fibers express receptors for these immune mediators. Sensory fibers can express receptors for interleukin-1 beta, TNF for IL-6. Now, in addition to these cytokines producing a signal in the sensory neurons, they can also hypersensitize these neurons and lower the thresholds of pain. Now, sometimes when these inflammatory mediators bind to receptors on syncine neurons, the sensory neurons release neuropeptides and factors that modulate the immune cells as well, depending on the context. They could either support inflammation or be anti-inflammatory.

Now, once the sensory signal is generated, it is sent up to the brain, brainstem and brain for integration. And then an infector signal is produced and sent down via the motor arm of the reflex to the cell ganglion, through the vagus and other sensitive neurons. Then the celiac ganglion relays that signal via the splenic nerve to the spleen. Norepinephrine is released. And once nurepinephrine is released in the spleen, it can be received by a specific subset of T-cells termed CHAP-positive T-cells. And these T-cells have this enzyme that makes acetylcholine, the choline cell transferase enzyme. Once neurepinephrine binds to these cells, these cells will make acetylcholine. Acetylcholine then can be received by macrophages, which express the receptor for acetylcholine, the alpha-7 nucotinicacoline receptor, and that drives inflammation down. So it actually switches the phenotype of the macrophage to a less pro- inflammatory response and actually a more anti-inflammatory response.

Acetylcholine can also bind to other cells on the spleen. For example, it can bind to B-cells and alter the B-cell response. And I'm going to tell you more about that in the coming slides. Neuropinephrine can also bind to other cells on the spleen. For example, it can bind to macrophages because macrophages express both alpha and beta-adrenergic receptors, and it can actually alter the phenotype both ways, anti or pro depending on the context and the amount of norepinephrine in the spleen. So this is summarized.

The pathway through the neuropinephrine down acetylcholine to macrophage is work pioneered by the TRACI group and it's termed the inflammatory reflex, but that concept expanded much more until recently and there are other pathways and other organs involved. Okay. So in our lab, we're interested in studying these neuroimmune regulatory pathways involving the slean. And in one of our studies, we wanted to measure the amount of nurepinephrine released if we target different nerves of the autonomic nervous system. So in a mouse model, we stimulated the vagus nerve, the splantic nerve, and the splenic nerve, and we measured the release of norepinephrine in real time. We borrowed a technique from chemistry that measures the amount of nopinephrine as electrical current. And what we found is that all the simulation of all three nerves produced norepinephrine. As you can see in these traces, they represent the amount of norepinephrine.

And when we quantified the amount from each nerve, we found that splenic nerve stimulation produces the largest amount of norepinephrine to the spleen, followed by splanknic nerve simulation, and vacant nerve simulation actually released the least amount of nupinephen in the spleen. We next wanted to see the effects of this real-time release, an LPS intertoxemia model. So we wanted to look at the TNF response after LPS introduction to the peritoneum while stimulating these nerves. And we found, like other people have found, that stimulating the splenic nerve reduced the amount of TNF released in these animals compared to sham stimulation. What's really interesting here is we found that the degree of TNF suppression correlated with the amount of norepinephrine released, and it was actually a reverse correlation. So the more nerbinephrine you had, the less suppression you have, which suggests that more simulation is not necessarily better.

And then these neuromodulatory approaches should be fine-tuned to produce an anti-inflammatory effect.

Now, in another set of experiments, we wanted to study how changing the level of activity of the autonomic nervous system chronically would affect the immune response on the spleen. And for that, we implanted animals mice with a vagus nerve stimulator, and we simulated the vagus nerve chronically for four weeks. And then after two weeks of simulation, we introduced an immune challenge in the form of immunization. We immunized with a material called NPCCG. And then two weeks after the immunization, we looked at titles of antibodies specific to that immunogen. And what we found is that chronic stimulation of the vagus nerve decreased the amounts of antibodies specific for this immunogen. So the high opinion antibodies against MP were reduced compared to Shamy simulation animal. And one of the reasons why this happened is that this response to happen in the spleen, for this B-cell response to happen in the spleen, a specific subset of normal cells of the spleen should form clusters.

These cells are called follicular degeneric cells or FDCs. They should form clusters, and these clusters would present the antigen to B-cells to produce antibodies. And we found that chronic VNS actually changes the organization of cells in the spleen and disperses these clusters. And the reason that these clusters were dispersed, and these are the red dots here, you can see that are dispersed here. The reason why they're dispersed is because they down-regulated their TNF receptor. These cells require TNF to cluster, and the source of the TNF is actually B-cells. We also found that these B-cells dowrated the TNF because of the chronic VNS. We actually found other findings in this model as well. For example, B-cells were more likely to die, so there was less B-cells in the spleen to actually produce the antibodies. So what we learned from this experiment is that the activity of the autonomic nervous system chronically can actually change the structure of the spleen and the immune response in the spleen.

And this is relevant to many chronic inflammatory diseases because many of these diseases are associated with dysautonomia.

Now, finally, we wanted to study whether changing the activity of intrasplenic nerves would affect diseases that have inflammation as a pathogenic component. And for that, we looked at two diseases, cardiovascular disease. We looked at pulmonary arterial hypertension or PAH, where there's chronic inflammation of the vessels of the lung that produces narrowing of the vessels and increases the pressure of the pulmonary artery leading to right ventricular systolic pressure increases. The other disease was HFEF. HFPEF also is a condition of systemic inflammation, and there's inflammation in the cardiac muscle that causes fibrosis, and eventually that sort of dysfunction. Now, to modulate the activity of the intersplenic innervation, we used focused ultrasound stimulation, which has been shown recently to increase the release of fumarpinephrine into the spleen and to have anti-inflammatory effects. We have previously published data showing that FUSA spleen actually improves PAH and it's published in circulation research.

An MD-PhD student in our lab, Alexander Picarido, expanded on these findings, and she looked at whether females also have that same response because PAH is actually more prevalent in females than males. And she found that similar to males, female rats with PAH that underwent FUSE stimulation had significantly lower RVSP or right ventricular systemic pressure. She also looked at the amount of inflammatory cells in the lungs, and she found that FUSE significantly decreased the amount of CD68 positive macrophages in the blood vessels of these animals. Now, in HFpEF, and this is preliminary data, FUSE of the spleen in this cardiovascular disease, we found that delivering the simulation of the spleen with focused ultrasound improved some of the echocardiographic findings in these animals. For example, they have a lower IVRT, which is an index of how fast the heart relaxes, and these animals did better on functional tests.

So usually FPEF animals cannot run for long distances, and delivering FUS to these animals allowed them to actually run longer distances. And these changes were not explained by the focused ultrasound blocking the risk factors that developed the disease because in both treated and untreated animals, there was no difference in obesity or hypertension. So in these two conditions are examples of chronic inflammatory conditions in a distant organ from the spleen where modulating the innervation of the spleen can change the inflammatory picture of the organs involved in the disease and can actually improve the disease.

Now, how is all of this relevant? Well, chronic inflammation in the peritoneum could involve chronically activated immune cells, including macrophages and other cells that are releasing inflammatory mediators. They're hypersensitizing pain fibers. The motor arm of the reflex could be trying to control inflammation, and the spleen could be contributing inflammatory cells to the peritoneum, including macrophage and other cells. So maybe modulating the different pathways and organs involved in the reflex could change the immune environment in endometriosis and change the course of the disease. SARVS will tell you more about some of the approaches that we can take in the next talk. Thank you very much.