Mapping the immunological homunculus

During the first century, the Roman physician Cornelius Celsus defined four cardinal signs of inflammation: redness, swelling, heat, and pain. These signs and symptoms occur during infection by invasive pathogens or as a consequence of trauma. Today, we understand the molecular basis of these physiological responses as mediated by cytokines and other factors produced by cells of the innate immune system. Cytokines are both necessary and sufficient to cause pathophysiological alterations manifested as the four cardinal signs. Importantly, this knowledge has enabled the development of highly selective therapeutical agents that target individual cytokines to prevent or reverse inflammation. For example, selective inhibitors of TNF, a major inflammatory cytokine, have revolutionized the therapy of rheumatoid arthritis, inflammatory bowel disease, and other autoimmune and autoinflammatory diseases affecting millions worldwide. Now, in PNAS, Hess et al. (1) use functional MRI to monitor brain activity and report that patients with rheumatoid arthritis who receive anti-TNF develop significant changes in brain activity before resolution of inflammation in the affected joints.

To accomplish this, the authors measured blood oxygen level-dependent (BOLD) signals in the brain after compressing the metacarpal phalangeal joints of the arthritic hand. They observe enhanced activity in the brain regions associated with pain perception, including the thalamus, somatosensory cortex, and limbic system, regions known to process body sensations and emotions associated with the pain experience (1). Brain activity was significantly reduced within 24 h after treatment with TNF inhibitors, a time frame that preceded any observable evidence of reduced signs of inflammation in affected joints. Clinical composite scores, comprising measurements of C-reactive protein, a circulating marker of inflammation severity, were not improved until after 24 h. This suggests that selective inhibition of TNF has a primary early effect on the nervous system pain centers.

The authors further explore this result in genetically engineered transgenic mice that overexpress TNF and develop spontaneous arthritis. Administration of anti-TNF significantly improved the development of mechanical hyperalgesia in the standardized von Frey filament test system and also improved the response to thermal hyperalgesia in the Hargreaves test. This clinical improvement occurred within a short period after administration of anti-TNF, before any significant improvement in joint swelling or grip strength, and before histopathological improvement of synovitis. As in the human subjects, there was significant abolishment of BOLD signals in the somatosensory cortex. Brain activation was more extensive in the transgenic mice and affected more brain regions compared with WT mice, and this spreading of brain activation was down-modulated by administration of the TNF inhibitors. Administration of anti-TNF reversed the prolonged activation of BOLD activity that was

The nervous system is hardwired to monitor the presence of cytokines and molecular products of invaders.

observed during the intervals between stimulations, and was particularly pronounced in the somatosensory cortex and limbic system.

These intriguing findings provide a glimpse into the future, when it is likely that sensory and motor brain regions associated with immune responses will be mapped as an immunological homunculus (2). Of particular significance here is that this approach bridges two major fields of medicine, neurology and immunology.

Reflexes Regulate Immune Responses

Recent advances at the intersection of these fields have provided direct evidence that neural circuits reflexively control the onset and resolution of inflammation, and previously undescribed molecular mechanisms have been defined (3). The fundamental principle of neurological reflexes, as originally established by Sir Charles Scott Sherrington in the early 20th century, is that neural circuits cooperate to maintain the organism's homeostasis. He elucidated the role of the nervous system in integrating reflexes to provide stable physiological systems. He famously remarked that “Existence of an excited state is not a prerequisite for the production of inhibition; inhibition can exist apart from excitation no less than, when called forth against an excitation already in progress, it can suppress or moderate it.” These principles were first defined in relatively accessible organ systems, including cardiovascular, gastrointestinal, and respiratory, which enabled the mapping of reflex units. For example, by recording signals to the heart, it was possible to define a reflex that controls heart rate: increased heart rate activates afferent arcs that in turn elicit firing of efferent arcs in the vagus nerve, which slows heart rate.

More recently, these principles have been applied to mapping the somewhat more elusive immune system. The combined use of neurophysiological stimulating techniques and current molecular strategies to measure immune response established the “inflammatory reflex” (3). Cytokines activate afferent signals in the vagus nerve, which culminate on an efferent arc that inhibits further cytokine production. The molecular basis of this cytokine-inhibiting action is attributable to acetylcholine, the principal neurotransmitter of the vagus nerve, interacting with α7 nicotinic acetylcholine receptor subunits expressed by cytokine-producing cells. Signal transduction through this receptor–ligand interaction down-regulates the activity of NF-κB, a primary pathway for regulating cytokine transcription and synthesis. Accordingly, α7 KO mice rendered deficient in this pathway are exquisitely sensitive to inflammation caused by pathogen-associated molecules and experimental arthritis. The efferent arm of the inflammatory reflex, termed the cholinergic antiinflammatory pathway, has enabled the development of experimental nerve stimulators and highly selective pharmacological agents that suppress cytokine-mediated disease in standardized preclinical models.

Sensing Inflammation and Infection

Less is known about the mapping of the afferent or sensory arc of the inflammatory reflex, and there is significant interest in the question of how the nervous system monitors the state of inflammation. The seminal work of Watkins et al. (4) demonstrated that the fever-causing activities of the pyrogenic cytokine IL-1 required an intact vagus nerve, because administration of IL-1 into the abdomen failed to produce fever if the vagus nerve had been cut. These researchers proposed a critical role of the glomus cell, specialized to detect pH and other chemical changes in the internal milieu, in sensing IL-1 (5). Glomus cells express IL-1 receptors, and it is a plausible hypothesis that IL-1 binding to glomus cells stimulates the release of dopamine, which, in turn, triggers afferent signaling in the vagus nerve. Ascending action potentials culminate on the nucleus tractus solitarius and are then relayed to higher brain regions. Sensory neural circuits can also be activated by LPS, and Toll-like receptor 4 (TLR4), the principal endotoxin receptor, is expressed by neurons in the nodose ganglia (6). These neurons have been implicated in transmitting afferent signals in the vagus nerve. Another mechanism by which the nervous system can sense the presence of injury is dependent on the expression of TLR4 in microglial cells, which is required for the development of neuropathic pain in a murine model of nerve transection (7). This TLR4 pathway, which may be activated even in the absence of infection by HMGB1 released from injured cells (8), stimulates TNF release in the development of chronic pain states. Together, these results indicate that the nervous system is hardwired to monitor the presence of cytokines and molecular products of invaders.

The presence of TNF can also be sensed by sensory neurons, which express type I and type II TNF receptors (9). Local or regional administration of TNF elicits pain, partially by inciting tissue damage and activating release of other molecules capable of activating sensory neurons. TNF receptor–ligand interaction on sensory neurons may activate pain fibers directly, however, serving as a pathway that may activate the brain's pain centers as described here. Others have shown that administration of anti-TNF antibodies into the cerebrospinal fluid of rats subjected to experimental arthritis significantly attenuated pain-related behavior and the development of inflammation in the joints (10). Moreover, intrathecal administration of antibody was significantly more effective compared with systemic anti-TNF, suggesting that the principal effects of anti-TNF are mediated through direct interaction with neurons. Considered in light of the current paper, it is plausible that local accumulation of TNF in the region of sensory pain circuit neurons drives activation of the brain's pain centers. Thus, it is possible that the rapid early improvement from anti-TNF results from acutely neutralizing TNF and blocking its ability to stimulate pain fibers. Predictably, this circuit would not require any significant improvement in the severity of inflammation in the arthritic joints in order for benefit to be perceived.

The current study also raises another possibility, that anti-TNF antibodies traversed the blood–brain barrier and directly altered the activity of brain neurons in the pain centers. Until quite recently, it has been dogmatic that antibodies do not enter the brain, but this has been overturned by studies in a murine model of the autoimmune disease lupus (11). This work identified antibodies that bind to dsDNA (a common antibody phenotype in this syndrome) and to the NMDA receptor expressed on brain neurons. These antibodies readily access the brain, because the blood–brain barrier can be opened during relatively mild stress caused by administration of endotoxin, cytokines, or even epinephrine. On entering the brain, the interaction of these antibodies with neurons in the hippocampus and other regions mediates cell damage and cognitive or behavioral dysfunction (12).

In light of the present study, it is interesting to consider whether inflammatory mediators produced during arthritis might open the blood–brain barrier to enable anti-TNF direct access to brain neurons. TNF expressed by glial cells in brain regulates synaptic scaling, a mechanism that adjusts the strength of neuronal synapses under conditions of prolonged alterations of electrical activity (13). A testable hypothesis emerges from this reasoning: The systemic inflammation associated with rheumatoid arthritis opens the barrier to anti-TNF, which inhibits glial cell-derived TNF and alters the strength of synapses in the brain's pain regions. This might explain the observed reversal of prolonged activation of BOLD activity during intervals between stimulations in the arthritic mice.

Immunological Homunculus

Advances in brain imaging modalities and molecular biology are mapping brain networks and circuits with high resolution and precision. These maps reveal sensory and motor circuits that are somatotopically and functionally organized, and form the basis for understanding the neural circuits that coordinate physiological responses to the external and internal environment. Early advances were achieved by mapping the relatively accessible domains of body sensations and skeletal muscle motor function. Now, knowledge of the inflammatory reflex as a prototypical circuit that regulates inflammation, combined with the ability to visualize brain activity in patients and mice with inflammation, makes it possible to make additional advances. It is interesting to consider that in addition to defending the host from infection, the immune system functions as a sensory organ that transmits information in real time to the central nervous system about the tissue response to injury and infection. This presents important possibilities for understanding fundamental mechanisms that maintain physiological homeostasis. Progress in this field is all but certain to reveal the identity of other circuits that reflexively regulate the immune system and to produce maps that will guide understanding of the neurological basis of immunity and physiology.