**2. Axes of Neuroimmune Control and Regulation**

It has been shown that there is an important neural control of the immune system [17]. A well-known principle of the physiology in mammals is that the nervous system is responsible for

achieving homeostasis by modulating of the function of other systems in the body through the HPA axis, the inflammatory reflex, the enteric nervous system (ENS) and finally the brain-gut axis.

Microglia is the resident immune cell in the CNS which represents 5 to 20% of glial cells. It is a myeloid cell, phagocytic, and has the activity of an antigen presenting cell (APC). In addition, it releases cytokines and can activate inflammatory-type responses [18,19]. During the early development stage, the microglia "brand" and "clean" synapses through a process called "synaptic pruning", promotes the "wiring" of neuronal circuits and releases cytokines and chemokines that assist and guide the process of neuronal differentiation [18,20]. The microbiota has a direct influence on the maturation and function of the microglia. In germ-free (GF)-animals, the microglia display a longer development process and with more derivations, with high levels in the expression of receptor-1 of the colony stimulating factor (CSF1R), F4/80 and CD31, factors that decrease in expression during development. This suggests that there is an important effect of the microbiota on the microglia, which depends on the stage of development and/or the time of microbial colonization.

The microglia of adult GF-mice can be functionally damaged when there are alterations caused by lipopolysaccharide (LPS) or by lymphocytic choriomeningitis virus, which in turn causes alterations in the correct activation of the immune system, including an increase in the release of proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6. These functional deficits are consistent with the concept that the naive microglia of adult GF-mice has a significant decrease in the expression of several genes important to interferon (IFN)-mediated responses, in genes for innate immune responses and genes for viral defense response and effector processes [9,21].

The mechanisms through which intestinal microbes exert their influence on microglia in the brain are not clear but it seems that there is a "microglial modulation" according to a specific type of bacteria [9]. This has raised the question of whether the effects of microbiota on microglia are not regulated by bacteria in general, by the microbiome, or if very specific microbial species are required [9]. The alterations in the morphology of the microglia in GF-animals and the alteration in the expression of genes can be normalized thanks to the post-natal supplementation with short-chain fatty acids (SCFAs), which are products of bacterial fermentation [22,23], suggesting that the bacterial species producing SCFAs are able to restore the alterations that occur in the microglia in GF-mice or treated with antibiotics [9].

The coordination of information between neurons, microglia and the responses at the central level with the periphery is carried out through the different axes of regulation and control; the HPA axis, and the inflammatory reflex (Figure 1). The coordination of these defense responses is mediated by signaling pathways related to the hypothalamus, the pituitary gland and the adrenal glands (e.g., HPA-axis), which causes the release of chemical molecules capable of altering behavior, including glucocorticoids, mineralocorticoids, and catecholamines. The activity of the HPA-axis is regulated by multiple sympathetic, parasympathetic and limbic circuits (amygdala, hippocampus and medial prefrontal cortex) that will directly or indirectly activate the hypothalamic paraventricular nucleus (PVN) [24]. Under normal conditions HPA-axis activity exhibits continuous oscillatory activity synchronized with circadian as well as ultradian rhythms [25,26].

The sympathic nervous system (SNS) and HPA-axis activation are the main components of neurotransmitter release and of neuroendocrine molecules of the stress response [27]. To respond to stress, the SNS is responsible for increasing catecholamine levels in the systemic circulation and tissues, with the concomitant release of corticotropin releasing factor (CRF) from hypothalamic paraventricular neurons, then the release of the adrenocorticotropic hormone (ACTH) from the anterior pituitary gland is stimulated, and ACTH travels through the circulation systemic and induces the synthesis and release of glucocorticoids from adrenals, cortisol in humans and corticosterone in animals. The primary function of the SNS and HPA-axis activation is to prepare the body to respond to damage by increasing the level of glucose in the blood through gluconeogenesis, the suppression of the immune system (suppression of cytokines) and increasing the metabolism of fats and proteins [27,28].

**Figure 1.** Regulation and control of neuroimmune axes. The three systems of regulation and control of information between the central nervous system (CNS) and the periphery are the hypothalamic pituitary adrenal (HPA)-axis, the sympatho-adrenal medullary (SAM)-axis and the inflammatory reflex. These systems are permanently sensing through nociceptive receptors and send information in real time to the CNS. ACTH, adrenocorticotropic hormone; NST Nucleus of the solitary tract; LC Locus coeruleus; TNF, tumor necrosis factor; IL, interleukin.

The responses of the HPA-axis as well as other variables of the stress response are regulated by exposure to psychological and physical stressors, such as infections [29]. The response of the HPA-axis and the SAM axis to psychological stress is mediated by neurotransmitter systems such as serotonin (5-HT), norepinepherine (NE) and endorphins, which play an important inhibitory role [29].

In addition, the HPA axis is strongly regulated to react efficiently to pathogens such as *Escherichia coli*. This response is mediated by the synthesis of prostanoids induced by the enzyme cyclo-oxygenase (COX). The elevation in corticosterone levels correlates with the increase in prostaglandin (PG) E2 in the circulation [29].

Interestingly, prebiotic intake in early life has been associated with beneficial neurological effects in adulthood. To demonstrate this effect, William and co-workers [30] fed neonatal rats (3 days old) with B-GOS during 19 or 53 days and showed that levels of the *N*-methyl-d-aspartate receptor (NMDAR) N2A subunit, synaptophysin and the brain-derived neurotrophic factor (BDNF) in the hippocampus of adult rats were elevated as compared with control fed animals. As the expression of the microtubule-associated-protein-2 (MAP2) was not affected, authors propose that neonatal B-GOS feeding impacts on neurotransmission, but not on synaptic architecture. Similar results were reported by Oliveros group [31] supplementing rat pups with 2 -fucosyllactose (FL) during the lactation period. When animals were evaluated just after weaning there was no change in behavior, although 2'-FL-feed rats evoked more intense long-term potentiation (LTP) than control ones. Same animals were evaluated at the age of 1 year and they performed significantly better in behavioral tests and still evoked more intense and longer TLP than the control group. Taken together, these results show that prebiotic administration early in life improves cognitive abilities both in childhood and in adulthood. However, in a randomized controlled trial, no significant improvement in neurodevelopmental outcomes was observed in preterm infants fed with breast milk or preterm formula supplemented with short-chain GOS/long-chain FOS/pectin-derived acidic OS between days 3 and 30 of life when they were evaluated at one or two years of age [32,33].

The basic organizational unit of the nervous system is the reflex arc, which is composed of sensory neurons (afferent) that report information to the CNS and motor neurons (efferent) that send "regulatory" signals to "target" tissues in the periphery. Recent advances in both neuroscience and immunology have revealed that there are neural reflexes that can regulate immune function in a wide range of species through evolution, from *Caenorhabditis elegans* to more complex mammals [17,34,35].

Inflammation is a key process of mammals in order to fight against pathogenic microorganisms and in the mechanism of wound healing. The molecular products of a bacterial invasion and of damaged tissue are rapidly detected by pattern recognition receptors (PRRs), which activate the cells of the innate immune system. The early response of these cells starts a cascade of events whose main function is the exclusion of pathogens and the subsequent restoration of homeostasis. This process includes the synthesis and release of proinflammatory cytokines and leukocyte recruitment [17].

It is crucial for the host to regulate and control an inflammatory response. Different mechanisms of regulation and control of inflammatory mediators have been described; for example, the release of inhibitory cytokines and soluble receptors to cytokines, as well as the activation of different subtypes of regulatory lymphocytes [36]. It is interesting to note that PRRs (Toll-like receptors, TLRs, and Nucleotide-binding and oligomerization domain (NOD)-like receptors, NLRs), in addition to the receptors for cytokines and PG are also expressed by sensory neurons [37]. This provides a molecular mechanism by which the CNS acquires information from a process of inflammation localized in the periphery. In addition, sensory nerves can react to the presence of microbial products independently of the activation of the immune system [17]. These nerves form a dense network along the external surfaces of the organism, and it has been suggested that this type of innervation provides the anatomical basis for a very precise "sensing" by the CNS against a potential infection with a pathogen, a tissue damage or an inflammatory process [17,37]. Reciprocally, many of the cells of the immune system express receptors for neurotransmitters such as dopamine (DA), acetylcholine (Ach) and norepinephrine (NE), all of which in turn regulate the differentiation and activity of leukocytes [17,38–40].

In this phenomenon, afferent signals are transmitted through the "vagus nerve", which are processed at the central level (CNS) and return to the periphery via the vagus efferent nerve, a process by which the release of cytokines is regulated by splenic macrophages [17,41]. Spleen is the major organ where TNF-α is synthesized and released systematically during an endotoxic process (endotoxemia). It has been shown that electrical stimulation of the vagus nerve significantly reduces the release of TNF-α in the spleen. However, the vagus nerve does not directly supply the spleen, the signal travels to the celiac ganglion, where the splenic adrenergic nerve also flows. The electrical stimulation of the latter also reduces the synthesis of TNF-α in the spleen. For this inhibition to occur, activation of the α7 subunit of the nicotinic acetylcholine receptor (α7nAchR) in splenic macrophages is necessary. The adrenergic nerve terminals are very close to a sub-type of T lymphocytes in the spleen that expresses the enzyme choline acetyltransferase (AchT), which has the ability to synthesize Ach, a neurotransmitter that is necessary to inhibit the synthesis and release of TNF-α in the spleen [42].

The vagus nerve, in addition to the splenic and splanchnic nerves, provide an important line of communication with the HPA-axis [29]. For instances, 2 h after vagal stimulation in rodents there is an increase in the expression of the mRNA of CRF in the hypothalamus and corticotropin-releasing hormone (CRH) which in turn increases the levels of ACTH and plasma corticosterone levels. The clinical relevance is the fact that vagal stimulation is associated with clinical benefits (signs and symptoms) antidepressants, together with the "normalization" of HPA-axis parameters in patients with refractory depression [43].

The impact of other prebiotics on brain physiology and biochemistry has also been experimentally studied. Rats that received a diet for 5 weeks supplemented with 2'-FL, the most abundant HMO, experienced increased BNDF levels in the striatum and hippocampus [44]. The expression of other two brain functional markers involved in the LTP process, the postsynaptic density protein (PSD)-95 and phosphorylated calcium/calmodulin-dependent kinase II (pCaMKII), was also augmented at frontal cortex and hippocampus, and at hippocampus, respectively. In accordance, authors reported an enhancement of synaptic plasticity in rats with that feeding regimen and in mice with a 2'-FL long-term feeding (12 weeks) protocol. Both species improved input/output curves and LPT experimentally evoked at hippocampal synapses, with a better performance of the animals in different applied tests of learning behavior.

Finally, some studies have addressed the impact of prebiotics in experimental models of neural dysfunctions. The first one was focused on analyzing the effect of GOS intake in a mouse model of amyotrophic lateral sclerosis (ALS) [45]. Animals started to receive the prebiotic at the age of 8 weeks on a daily basis until the end of the protocol. Mice orally fed with GOS experienced delayed onset of the disease, extended lifespan, improved muscle atrophy, attenuated oxidative stress of skeletal muscles, suppressed astrocyte and microglia activation, inflammatory response and apoptosis in spinal cord tissue. Authors attributed neuroprotective effects of GOS on ALS-sick mice to the amelioration on homocysteine serum levels, an amino acid related to neurotoxic effects in the pathogenesis of ALS, and to the increases in the amount of VitB12 and folato, both of which are involved in homocysteine metabolism [46]. Besides, beneficial effects of GOS have been also described in neuropsychiatric disorders where anxiety and neuroinflammation are clinically involved [47]. In this sense, the supplementation of 8 weeks-old mice standard diet with B-GOS incorporated to drink water during 3 weeks reduced LPS-induced anxiety. B-GOS intake also decreased elevated cortical IL-1β and 5-HT2A receptor expression mediated by LPS in the frontal cortex, in the absence of altered 5-HT metabolism. Thus, the anti-inflammatory effect of the prebiotic is probably modulating its anxiolytic activity. A similar link between anti-inflammation and neuroprotection was reported for chitosan oligosaccharides (CHO) [48] and FOS [49] in a rat model of AD. When CHO was orally administered to amyloid-β1-42-induced rats during 2 weeks, the learning and memory deficits were reduced, and the hippocampal cell death decreased. At the same time, CHO treatment inhibited oxidative stress together with a reduction in proinflammatory cytokines expression at the hippocampus, particularly IL-1β and TNF-α [48]. In the case of a study done with FOS, it was orally and daily administered to amyloid-β1-42-induced rats for 4 weeks or to D-Galactose-induced rats for 8 weeks, with similar outcomes. FOS intake improves inflammation and oxidative stress disorder, ameliorates learning and memory difficulties, and regulates the synthesis and secretion of neurotransmitters such as NE, DA, 5-HT, and 5-hydroxyindole acetic acid (5-HIAA) [49]. All these FOS-induced effects on AD are mediated by the regulation of the gut microbiota.
