Response to peripheral immune stimulation within the brain: magnetic resonance imaging perspective of treatment success

In the last few decades, a large body of knowledge has been accumulated regarding bidirectional communication between the immune system and the brain (for review see [1–7], for historical overview of the discoveries see [8]). A new branch of research, psychoneuroimmunology has emerged primarily from the great interest in the role of the immune system in neuropsychiatric disorders, especially major depression. Much attention has been attracted following the discovery that increased peripheral inflammation is associated with depression and fatigue [3–6, 9–13]. Noteworthy is the frequent occurrence of depression in patients with rheumatoid arthritis (RA) and infectious and autoimmune diseases [14–16], and, inversely, there are elevated concentrations of inflammatory markers in medically healthy patients with major depression. These findings stimulated further investigation of mechanisms by which inflammatory information is transmitted from the periphery to the central nervous system (CNS). It is now known that proinflammatory cytokines, in particular interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF) alpha, are important transmitters of this information. IL-6 and TNFα appeared to be the most consistently elevated cytokines in the studies conducted on depressive patients (see meta-analysis [10]) as well as in animal models of depression [17]. Released when confronted with an immune challenge—infection, injury, or stress—or externally administered (e.g., interferon alpha in the case of cytokine therapy), cytokines not only trigger cascades of defensive responses on molecular and cellular levels, but also act on an organismic level. The cytokines instruct the CNS how it should adapt its behavior to the altered conditions or, in other words, how to conserve energy for the purpose of optimal recovery [18]. Furthermore, cytokines mediate genetic factors [19] and social factors such as stress [5, 6, 11, 20–23] or “social pain” [24], also promoting the development of depression.

Peripheral cytokine signals reach the brain via three principal pathways—humoral, neural, and cellular—via several mechanisms [4, 5] including (adapted from [5]): passage through leaky regions in the blood–brain barrier at circumventricular organs; active uptake across the blood–brain barrier; the “neural route” via local actions at peripheral vagal nerve afferents that relay cytokine signals to relevant brain regions, including the nucleus of the solitary tract and hypothalamus, and directly at sensory neurons; activation of endothelial cells and perivascular macrophages in the cerebral vasculature to produce local inflammatory mediators such as cytokines, chemokines, prostaglandin E2 (PGE2), and nitric oxide (NO); and recruitment of activated monocytes/macrophages and T cells from the periphery. A potential gateway for immune cells into the brain has been discovered recently: functional lymphatic vessels lining the dural sinuses [25]. Within the brain, peripheral cytokines act on a central network of microglia, astrocytes, and neurons, which in turn produce cytokines [26]. This can amplify cytokine signals. Cytokines activate the hypothalamic–pituitary–adrenal axis, stimulating the production of corticotropin-releasing factor, adrenocorticotropic hormone, and cortisol [1], and influence many other physiological processes in the CNS. The cytokines alter the metabolism of neurotransmitters serotonin [27, 28], dopamine [28–31], glutamate [32, 33], which in turn leads to decreased production of the trophic or growth factors necessary for neurogenesis and neuroplasticity [6, 34, 35], or norepinephrine [5, 6]. Many studies demonstrated reduction of brain matter, especially in hippocampus [19, 35–37], but also different alterations in other areas of the brain [38–43] under continuously elevated IL, or changes in functional connectivity [43–48]. The latter can best be encompassed by functional neuroimaging methods and will be discussed in a separate section.

Multiple studies on humans and on experimental animals with elevated levels of proinflammatory cytokines in the periphery report in their subjects the so-called “sickness behavior” [49, 50]. This phenomenon has been well described and implicates, depending on the severity of the disturbance, such symptoms as fatigue, psychomotor slowing [22, 29], anxiety, anhedonia [51], cognitive dysfunction [52] (for review of the role of cytokines in maintaining the normal cognitive function see [53]), social withdrawal [54], sleep alterations, and loss of appetite, in different combinations. Notably, the sickness behavior develops very rapidly. Frenois et al. [55], using a range of behavioral tests, distinguished two phases and characterized their time development in mice injected with lipopolysaccharide (LPS). The authors have shown that sickness behavior peaked after 6 h, followed by the depressive-like behavior 24 h post LPS. The latter was paralleled by a decrease in cellular activities, particularly within the extended amygdala, hippocampus, and hypothalamus as shown by immunohistochemistry. Stone et al. [56] revealed that the exploratory movement activity of mice injected with LPS was reduced a mere 2 h post treatment. Similar timing was described in studies on healthy humans using, for example, Salmonella typhi vaccine as an inflammatory challenge [22, 29, 57–59]. Vaccination with S. typhi appears to be an appropriate model for investigation of depressive symptoms not superimposed by actual disease burden; it does not induce sickness. In these studies, deterioration of mood was observed starting within 3 h and lasting for at least 6 h after injection.

Normally the release of proinflammatory cytokines is adaptive and temporary, and so are its consequences as already described. Quite a different situation arises if a cytokine challenge becomes chronic; for example, in cases of chronic inflammatory diseases or permanent stress [18]. In these cases, in addition to the known primary symptoms, we can expect the development of clinically relevant psychiatric disorders such as major depression.

RA is a chronic autoimmune inflammatory disease severely affecting the joints. As the disease progresses, RA rapidly leads to destruction of cartilage and bone tissue, which is associated with pain, swelling, stiffness, and even immobility of the joints. Quality of life for people suffering from RA is dramatically diminished, and pain is the prevailing symptom of this disease.

Treatment of RA was extremely challenging before the development and introduction of drugs inhibiting biological activity of TNF (anti-TNF therapies). While affected joints express a multitude of inflammatory mediators, systemic inhibition of TNFα turned out to be a particularly successful therapeutic strategy [60–62]. This success is based in part on the efficient alleviation of joint inflammation upon disruption of the inflammatory cytokine network in the affected joint. However, it has always been surprising how quickly the patient condition improves taking into account that RA causes irreversible structural damage to the bones, the cartilage, and the joint innervation pattern [61, 63]. Nevertheless, amelioration of pain and improvement of personal subjective feeling set in soon after the start of TNF neutralization, long before inflammation decline becomes identifiable using common clinical tests.

This observation led to the idea that there must be an independent, faster impact of TNF inhibition on the CNS. Do anti-TNF therapies elicit pain-reducing effects in the central pain pathway? Consequently, functional brain imaging studies have been conceived to investigate changes in brain function—pain processing in particular—under TNFα inhibition.

Neuroimaging methods have brought new opportunities for linking fields of medicine operating at different levels; that is, immunology and neurology [19, 21, 24, 29, 48, 54, 57, 58, 64, 65] (for review see [38]). In particular, neuroimaging has revealed individual brain structures, but also complex neurocircuits in the CNS that appear to be modulated by cytokine signaling from the periphery; for instance, the anterior cingulate cortex (ACC), the basal ganglia, including nucleus accumbens, striatum, and substantia nigra, and the insular cortex. Subgenual ACC plays an important role in depression, and dorsal ACC in anxiety and alarm. Basal ganglia are essential for motor activity and also for motivation. The insular cortex is strongly linked to the emotional state (particularly its anterior, limbic-related portion), self-awareness, and empathy, and plays an important role in the regulation of the body’s homeostasis.

Vaccination with S. typhi was paralleled by increased activation in subgenual and dorsal ACC, as demonstrated by functional magnetic resonance imaging (fMRI), and a deterioration in mood and anxiety [57]. In subjects vaccinated with S. typhi, perturbed activity in the substantia nigra was associated with an increase in the amount of time required to solve a cognitive task, consistent with the notion that cytokines influence dopamine transmission [30, 51], and also correlated with increased levels of IL-6 in the blood [29]. Persons injected with Escherichia coli endotoxin exhibited increases in depressed mood and anhedonia over time, correlated with a significant reduction of activity in the ventral striatum, part of the reward circuit, evoked by monetary reward cues [51]. Women exposed to E. coli endotoxin became more vulnerable to social exclusion and, in a similarly structured fMRI study, exhibited increased activity in dorsal ACC and anterior insula paralleled to an elevation of IL-6 in blood and to a deterioration in mood [24]. Functional connectivity of the insula has been shown to increase under inflammatory conditions [47].

General evidence from functional neuroimaging suggests that two main mechanisms may be largely responsible for clinical pain in rheumatic diseases: CNS sensitization/impairment of inhibition; and alterations in the medial pain system (for review see [38]), which is responsible for the affective-motivational pain component (Fig. 1).

Using fMRI, we [45] addressed the question of the rapid improvement of RA patients’ disease state following the start of TNF neutralization by anti-TNF. The rationale of the investigation was as follows. Given that TNFα also acts as a pain mediator, we hypothesized that during RA the cytokine constantly changes pain processing in the CNS. Systemic inhibition of TNFα should thus positively influence central pain processing, and this may happen long before it affects joint inflammation. Using blood oxygen level-dependent (BOLD) fMRI we measured the network of brain structures in RA patients activated in response to nociceptive stimulation of the affected joints before and at various points in time after intravenous infusion of infliximab, an anti-TNFα monoclonal antibody. Nociceptive activity in the brain pain matrix was significantly reduced as quickly as 24 h after TNF neutralization. This activity remained low until the end of the observation period 42 days after anti-TNF administration. BOLD activity decreases were observed in thalamus and in primary and secondary somatosensory cortices—structures responsible for the sensory-discriminative aspect of pain—but also to a large extent in parts of the limbic system, such as the cingulate and insular cortex, which are responsible for the affective-motivational or emotional pain component. Importantly, standard clinical measures of the disease activity—joint swelling and joint tenderness, composite disease activity scores (Disease Activity Score in 28 joints), and laboratory parameters such as the blood sedimentation rate and serum C-reactive protein and IL-6 levels—did not change within the first 24 h, but improved at a later stage of the treatment process. In contrast, subjective perception of pain in these patients, judged according to the visual analog scale (VAS), was ameliorated as early as 24 h after the first infusion, in parallel with the changes of nociceptive activity in the brain detected by fMRI.

These findings have been further substantiated by investigation into an animal model of arthritis: knockin mice overexpressing human TNFα (hTNFtg) [66]. Behavioral screening of these hTNFtg mice demonstrated that these animals develop characteristic signs of arthritis; for example, bone degeneration [61, 63] and decreased mobility [45]. Similar to humans, 24 h after treatment with infliximab arthritic mice showed significantly diminished sensitization to noxious stimuli, as has been established by von Frey as well as Hargreaves’ tests, which remained at the level of wild-type mice for at least 72 h. During the first 24 h following TNF neutralization, no apparent changes to clinic analog parameters or histopathologic signs of arthritis were observed. Remarkably, TNFα inhibition also completely restored motor activity (indicative of good mood), as demonstrated by the Rotarod test, within 24 h.

The fMRI part of the study in the hTNFtg mice was designed in a way which is possibly similar to the study of RA patients. As a response to nociceptive stimuli, before treatment, one could see significantly greater activation in the brain pain matrix of these mice compared with the wild-type mice. TNFα neutralization with infliximab led to a drastic reduction in this activity down to the level of wild-type mice (even lower in limbic areas) within 24 h. Again, there were profound decreases not only in the somatosensory cortex (sensory-discriminative aspect of pain), but also in expanded parts of the limbic system (emotional aspect).

Our research group [45] came to the conclusion that TNFα inhibition has a direct impact on central pain processing by far preceding its anti-inflammatory effects at the periphery. Suppression of neuronal activity in the limbic brain areas, in the cingulum and insula, could well explain the fast improvement of the subjective pain rating and overall feeling of RA patients following TNF neutralization. Therefore, one can speculate that anti-TNF also exhibits rapid antidepressant effects.

BOLD fMRI has been proven capable of issuing an early prediction of therapeutic success for TNF inhibition. This advantage of fMRI is especially valuable in light of the following dilemma: on the one hand, TNF blockers are expensive drugs with a potential risk for serious toxicity; and on the other, they have been shown to be highly effective, although not in all patients suffering from RA. To further investigate whether fMRI can also be used to differentiate between prospective responders and nonresponders to anti-TNF, we undertook a follow-up investigation [67]. In the follow-up investigation, RA patients received a standard injection of certolizumab-pegol, a subcutaneously administered anti-TNF, and fMRI measurements with the same stimulation paradigm as in the previous study [45] were performed before treatment and at several points in time after treatment. Clinical parameters were assessed at the same time points, as well as patients’ rating of global disease activity by VAS. In this study, similarly to clinical practice, one-half of the patients exhibited a significant clinically identifiable improvement after 28 days (responders), while the other half did not (nonresponders), although initial clinical baseline disease activity did not differ between the two groups. In accordance with the previous findings, the responder group demonstrated rapid (detectable on the third day) reduction of nociception-related activity in the brain pain matrix, which continued consistently over the following fMRI measurements. In the nonresponder group, in contrast, there was only a spurious decrease of the BOLD activity after 3 days, which then increased back to the initial levels after 7 and 28 days. A remarkable finding in this study was the striking difference in the number of significantly activated voxels under the nociceptive stimulation between the two groups before treatment: this number was high in the responders and low in the nonresponder group, particularly in the somatosensory, limbic, and associative brain areas. This study shed even more light on the predictive power of fMRI; the qualitative difference in the mean value of BOLD activation between the potential responders and nonresponders to anti-TNF was obvious even before treatment. As a next step, the quantitative determination of a threshold between responders and nonresponders is currently being performed in a clinical phase III multicenter study (PreCePRA NCT01864265).