Neurotrophins play a critical role in the control of neuronal survival, migration, and differentiation and modulate immune cell functions (Tabakman et al., 2004). Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) can inhibit MHCII expression in microglia in hippocampal slice cultures via the low affinity p75 neurotrophin receptor (Neumann et al., 1998). NGF also down-regulates the co-stimulatory molecules CD40 and CD86 in microglia (Wei and Jonakait, 1999), is increased in cerebral spinal fluid of MS patients (Laudiero et al., 1992), and NGF treatment delays EAE onset and clinical severity (Arredondo et al., 2001). In addition, NGF arrests astrocyte cell cycle possibly restricting glial scar formation after CNS injury via the p75 neurotrophin receptor, attenuating cyclins D1 and E and preventing the degradation of cyclin-dependent kinase inhibitors p15INK and p27kip1 (Cragnolini et al., 2012). Interestingly, NT-3 has anti-inflammatory properties by diminishing microglial inducible form of NO synthase, NO, IL1-β, and TNFα levels, and phagocytic activity after LPS stimulation. NT-3 exerts its effects mainly through the TrkC receptor leading to the activation of MAPK and PI3K cascades and decreasing the NFκB-p65 activity (Tzeng and Huang, 2003; Tzeng et al., 2005).

IL10 and TGFβ cytokines have anti-inflammatory and suppressive properties that importantly regulate CNS inflammatory responses and resident cells survival (Pratt and McPherson, 1997; Strle et al., 2001). Both cytokines and their receptors are expressed by neurons and glial cells throughout the CNS (Szelényi, 2001). These regulatory molecules down-regulate microglia inhibiting the expression of MHCII, pro-inflammatory cytokines such as TNFα and IL1β, as well as NO synthesis after LPS activation (Suzumura et al., 1993; Sawada et al., 1999; Heyen et al., 2000). Furthermore, IL10 and TGFβ have an important role on Tregs and keep autoimmune T-cells under the steady state (Saenz et al., 2010).

Among the neuropeptides and neurotransmitters with modulatory properties that inhibit microglial LPS-induced proinflammatory factors like IL1β, IL6, TNFα, and NO, are

#### **REFERENCES**

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vasoactive intestinal peptide (VIP), dopamine, norepinephrine (NE), and γ-aminobutyric acid (GABA; Färber et al., 2005; Bjurstöm et al., 2008; Delgado et al., 2008). VIP exerts its anti-inflammatory effects through the VPAC1 and VPAC2 receptors inhibiting p38 and p42/p44 MAPK and NFκB signaling cascades (Delgado et al., 2008). Also, VIP treatment avoids beta-amyloid neurodegeneration and MPTP-induced dopaminergic neuronal loss (Delgado and Ganea, 2003; Delgado et al., 2008). Additionally; VIP induces protective TH2 cells by upregulation of macrophage B7.2 expression and Tregs in a EAE model (Delgado et al., 2004; Fernandez-Martin et al., 2006). Physiological concentrations of GABA activate functional GABAA channels on encephalitogenic T-cell decreasing cell proliferation, while GABAB channels activation on microglia attenuates IL6 and IL 12p40 levels after LPS stimulation (Kuhn et al., 2004; Bjurstöm et al., 2008). Functional dopamine receptors D1 and D2 are expressed by microglia and their activation lead to attenuate NO production after LPS stimulation (Kuhn et al., 2004). CNS NE levels are relevant in order to maintain tissue homeostasis since NE loss contributes to neuroinflammatory processes that lead to neurodegenerative diseases, for instance depressed mice with low NE levels respond with higher TNFα production after LPS stimulation while increasing NE levels are necessary to reduce EAE severity (Szelényi and Vizi, 2007; Simonini et al., 2010). Moreover, NE regulates microglia morphology and motility by microglial processes retraction; in this dynamic process the β2 and α2A receptors are involved in resting cells and activated microglia cells, respectively (Gyoneva and Traynelis, 2013).

#### **CONCLUSIONS**

Traditionally glial cells are considered to be responsible for the regulation of immune processes in the CNS. Nevertheless neurons contribute to immune modulation through contact-dependent and -independent mechanisms (**Figure 1**). Several neuronal secreted as well-membrane associated molecules (**Table 1**) are implicated in the control of glial and T-cell functions, thus contributing to CNS immune privilege.

#### **ACKNOWLEDGMENTS**

Funding was provided by the grant IN217612 from Dirección General de Asuntos del Personal Académico (DGAPA), National Autonomous University of Mexico. The authors wish to acknowledge Bernardo Pohlenz for his artwork assistance.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 07 May 2013; accepted: 07 August 2013; published online: 02 September 2013.*

*Citation: Chavarría A and Cárdenas G (2013) Neuronal influence behind the central nervous system regulation of the immune cells. Front. Integr. Neurosci. 7:64. doi: 10.3389/fnint.2013.00064*

*This article was submitted to the journal Frontiers in Integrative Neuroscience.*

*Copyright © 2013 Chavarría and Cárdenas. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

### Hypothalamic neurohormones and immune responses

#### *J. Luis Quintanar\* and Irene Guzmán-Soto*

*Laboratory of Neurophysiology, Department of Physiology and Pharmacology, Centro de Ciencias Básicas, Universidad Autónoma de Aguascalientes, Aguascalientes, México*

#### *Edited by:*

*Beatriz Gomez-Gonzalez, Universidad Autonoma Metropolitana, México*

#### *Reviewed by:*

*Dumitru A. Iacobas, Albert Einstein College of Medicine of Yeshiva University, USA Fatih Tanriverdi, Erciyes University Medical School Endocrinology Department, Turkey*

#### *\*Correspondence:*

*J. Luis Quintanar, Department of Physiology and Pharmacology, Centro de Ciencias Básicas, Universidad Autónoma de Aguascalientes, Av. Universidad 940, C.P. 20131, Aguascalientes Ags., México e-mail<EMAIL_ADDRESS>
The aim of this review is to provide a comprehensive examination of the current literature describing the neural-immune interactions, with emphasis on the most recent findings of the effects of neurohormones on immune system. Particularly, the role of hypothalamic hormones such as Thyrotropin-releasing hormone (TRH), Corticotropin-releasing hormone (CRH) and Gonadotropin-releasing hormone (GnRH). In the past few years, interest has been raised in extrapituitary actions of these neurohormones due to their receptors have been found in many non-pituitary tissues. Also, the receptors are present in immune cells, suggesting an autocrine or paracrine role within the immune system. In general, these neurohormones have been reported to exert immunomodulatory effects on cell proliferation, immune mediators release and cell function. The implications of these findings in understanding the network of hypothalamic neuropeptides and immune system are discussed.

**Keywords: TRH, CRH, CRF, GnRH, LHRH, immune system, extrapituitary, receptors**

#### **INTRODUCTION**

The neurosecretion in the hypothalamus can be traced back to the work of Scharrer and Scharrer (1940). Posterior studies by Harris specified that the hypothalamic substances secreted into the portal vessels were pituitary specific and propose the concept of "releasing factors" whose purpose was to initiate a cascade of events resulting in the release of peripherally active hormones (Harris, 1948).

The discovery and chemical characterization of the first identified hypothalamic releasing factor by Burgus et al. (1969) and Boler et al. (1969) provided ultimate confirmation for the founding principles of neuroendocrinology and was followed by the discovery of other peptide-releasing factors (Nillni and Sevarino, 1999).

#### **THYROTROPIN-RELEASING HORMONE (TRH)**

TRH, the smallest known peptide hypophyseotropic hormone, is a tripeptide (pGlu-His-Pro-NH2; where pGlu stands for pyroglutamic acid) that is released from the hypothalamus and transported via the portal vascular system to the anterior pituitary where it stimulates the release of thyroid-stimulating hormone (TSH) and prolactin from the anterior pituitary (Matre et al., 2003).

TRH it is also known as thyrotropin-releasing factor (TRF), thyroliberin or protirelin (Khomane et al., 2011) and was the first chemically defined hypophyseotropic hormone (Bílek et al., 2011).

#### **SYNTHESIS**

This small tripeptide (362.42 Da) is indeed processed from a larger precursor protein, prepro-TRH (ppTRH), through now classic cleavage mechanisms, C-terminal amidation, and cyclization of the N-terminal Gln to pGlu (Guillemin, 2005).

The rat ppTRH is a 29 kDa polypeptide composed of 255 amino acids. The rat precursor contains an N-terminal 25-aminoacid leader sequence, five copies of the TRH progenitor sequence Gln-His-Pro-Gly flanked by paired basic amino acids (Lys-Arg or Arg-Arg), four non-TRH peptides lying between the TRH progenitors, an N-terminal flanking peptide, and a C-terminal flanking peptide. Rats and mice have five Gln-His-Pro-Gly TRH progenitor sequences, whereas humans have six TRH sequences (Chiamolera and Wondisford, 2009).

#### **TRH EXPRESSION REGULATION**

The maintenance of euthyroidism is dependent on a highly regulated balance of neuropeptides and neurotransmitters, where the dominant positive hypothalamic control for TSH is TRH, and the principal feedback control is through thyroid hormones (THs) (Nillni and Sevarino, 1999).

THs exert powerful feedback inhibition over the TRH response system by inhibiting TRH synthesis and processing in TRH neurons in the paraventricular region of the hypothalamus and decreasing TRH receptors (TRHR) and responses in the pituitary gland (Hinkle et al., 2012).

TRH gene expression is also regulated by temperature, food intake, and stress. Thus the TRH neuron is well positioned to integrate information about the environment as well as circulating TH levels and ultimately affect metabolism in response to these physiological changes (Chiamolera and Wondisford, 2009).

#### **HALF-LIFE**

The short half-life (4–5 mins) of TRH is most likely due to rapid degradation of the TRH at both the carboxy (COOH) and amino (NH2) termini. Cleavage of the pyroglutamyl moiety of TRH by enzymes like aminopeptidases and deamidases causes formation of the metabolite histidyl proline diketopiperazine (ClycoHistine-Proline, CHP) while its deamidation results in the formation of the free acid, that is, TRH-OH (Khomane et al., 2011).

#### **TRHR**

The TRHR is a member of a large family of transmembrane proteins (G-protein-coupled receptors, GPCRs) for which an interaction with an intracellular G protein is a critical part of the signal transduction pathway mediated by the receptor. It is thought that all GPCRs have a common tertiary structure composed of seven transmembrane helices. The topology of these membrane-bound proteins is defined by an extracellular amino terminus and an intracellular COOH terminus. Consequently, the helices are connected by intracellular-extracellular loops. Colson et al. (1998) suggested that TRH initially interacts with residues within the extracellular loops and then moves into the transmembrane binding pocket, aided by the motion of the loops.

A single TRHR gene has been found in humans and higher mammals and two genes encoding homologous receptors, TRHR1 and TRHR2, in rodents (Sun et al., 2003). TRHR1 predominates in the anterior pituitary gland while both TRHR1 and TRHR2 are found in rodent central nervous system (CNS) (Hinkle et al., 2012).

#### **EXTRAHYPOTHALAMIC AND EXTRAPITUITARY LOCATION OF TRH AND TRHR RESPECTIVELY**

Originally discovered in the hypothalamus, consistent with its classical role as a hypothalamic hypophysiotrophic factor, TRH and TRHR are now known to be distributed throughout the central and peripheral nervous system (PNS) and are found extensively in extrahypothalamic brain structures and in other organs and tissues (Sun et al., 2003; Kamath et al., 2009).

#### **TRH OUTSIDE THE PITUITARY ZONE**

TRH has been located in several brain areas other than the paraventricular nucleus as well as in non-CNS tissues:

#### **TRHR OUTSIDE THE HYPOTHALAMIC ZONE**

Besides the well-known TRHR location in the pituitary zone, TRH binding sites have been described throughout the central and PNS and in non-CNS tissues including:

Furthermore, TRHR expression in organs and cells of the immune system has been confirmed:

The widespread distribution of TRH and the TRHR within and outside the CNS supports a diverse range of roles for this molecule, roles likely to involve many functions outside of the traditional hypothalamic-pituitary-thyroid (HPT) axis (Nillni and Sevarino, 1999).

#### **TRH FUNCTIONS**

TRH indirectly stimulates TH biosynthesis and release. Thus, TRH is central in regulating the HPT axis. TRH influences the release of other hormones, including prolactin, growth hormone, vasopressin, insulin, and the classic neurotransmitters noradrenaline and adrenaline (Nillni and Sevarino, 1999).

#### **EXTRAHYPOPHYSIOTROPIC FUNCTIONS OF TRH**

More than two-thirds of immunoreactive-TRH in the brain is found outside of the traditional "thyrotrophic zone" of the hypothalamus. This extrahypophysiotropic TRH is believed to function as a neuromodulator of known neurotransmitters. Indeed, it might act as a neurotransmitter itself; it is present in secretory granules whose exocytosis is responsive to membrane depolarization, it acts through specific receptors that are widely distributed throughout the CNS, and it is rapidly cleared through specific catabolic pathways. In several areas of the brain, TRH is colocalized with other neurotransmitters and/or neuromodulators (Nillni and Sevarino, 1999).

These effects of TRH have been demonstrated using TRH and/or TRH analogs. Available TRH analogs have higher affinities for the TRHR, longer half-lives, etc. (Engel et al., 2006; Khomane et al., 2011; Monga et al., 2011).

Beside endocrine functions, TRH also possesses several neuropharmacological effects like cerebral nerve activation (stimulation of motor function), effects on sympathetic nervous system (elevation of blood pressure and stimulation of respiratory), effects on spinal function (stimulation of spinal motor neuron), effects on CNS (antidepressant activity) and peripheral actions (suppression of gastric acid secretion, stimulation of glucagon secretion). These CNS-mediated effects provide the rationale for the use of TRH in the treatment of brain/spinal injury and certain CNS disorders, including schizophrenia, Alzheimer's disease, epilepsy, amyotrophic lateral sclerosis, Parkinson's disease and depression (Khomane et al., 2011).

#### **TRH AND IMMUNE SYSTEM**

Two-way communication between the immune and neuroendocrine systems is now well recognized. These interactions are mediated by cytokines, neuropeptides, hormones, other soluble factors and their receptors (Grasso et al., 1998).

As mentioned above, cells of the immune system have been found to contain receptors for neuroendocrine hormones and can also be considered a source of pituitary and hypothalamic peptides (Matre et al., 2003).

#### **IMMUNE RESPONSE TO TRH ADMINISTRATION** *Proliferation*

*Induction.* Acute and chronic administration of TRH enhances the proliferation of splenic and thymic lymphocytes in rats (Pawlikowski et al., 1992; Raiden et al., 1995; Winczyk and Pawlikowski, 2000). In fact, it has been reported that TRH promotes thymic reconstitution in mice with anterior hypothalamic area lesions (Lesnikov et al., 1991). According with these data, it was also reported that TRH enhances the *in vitro* plaque-forming cell response (Kruger et al., 1989), antagonizes the dexometasone-induced thymocyte and splenocyte suppression (Winczyk and Pawlikowski, 2000) and administration in humans led to increased secretion of IL-2 into the blood (Komorowski et al., 1993).

*Suppression.* It has also been reported that TRH significantly inhibits monocyte activity (Lersch et al., 1989). The experimental work of Kunert-Radek et al. (1991) addressed the proliferation of murine splenocytes *in vitro* considering the 3H-thymidine incorporation into splenocyte DNA as an index of proliferation. They found that TRH suppressed the proliferation of splenocytes.

TRH has the capability to modulate the natural cell-mediated cytotoxicity. To try it out, TRH was added to the feed of White Leghorn male chicks and peripheral blood lymphocytes were cultured *in vitro* with or without different mitogens (Phytohaemagglutinin-A (PHA), ConA, or Lipopolysaccharide (LPS)), and the culture supernatants were tested for the presence of lymphokines. Results showed that the supernatant from *in vivo* 5 ppm TRH-treated lymphocytes significantly suppressed the natural cell-mediated cytotoxicity (Haddad and Mashaly, 1992).

Recently was published that oral administration of TRH in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, reduces the spinal cord inflammatory foci without increased frequency of regulatory T cells (Treg) in spleen (Brod and Bauer, 2013).

#### *Induces TSH production by immune cells*

At very low concentrations, TRH induces splenocyte production of TSH *in vitro* (Kruger et al., 1989).

Peripheral blood mononuclear cells (PBMC), rat splenocytes and transformed T cell lines produce TSH in response to TRH (Harbour et al., 1989; Raiden et al., 1995).

#### *Induces immune mediators production*

PBMC, rat splenocytes and transformed T cell lines can enhance or modulate the *in vitro* antibody response in response to TRH (Kruger et al., 1989; Hart et al., 1990).

Koshida and Kotake (1993) investigated the role of TRH on the superoxide anion (O2 −) production of rabbit peritoneal macrophages. Their results showed that TRH acts on macrophages and suggest that TRH possesses the priming action of O2 − release in response to the chemotactic peptide Nformyl-methionyl-leucyl-phenylalanine, and opsonized zymosan. Authors suggest that if this type of priming exists *in vivo* there should be a role to play in an inflammation process.

*In vitro* studies have shown that a TRH fixation occurs at the level of the human polymorphonuclear neutrophil, which suggests putative membrane receptor (s) for the hypothalamic hormone. The correspondent *in vivo* analyses demonstrated that after TRH administration, enzyme modifications (myeloperoxydase, alkaline phosphatase) and metabolism changes (PAS, Sudan black) happen, showing a functional activation of that blood cell (Blum et al., 1976). These *in vivo* effects could be due to TRH is being directly attached to its receptor on neutrophils or an indirect effect is being mediated by TSH or THs.

Intravenous bolus of TRH in normoprolactinemic women increases plasma gamma interferon (IFN-γ ) levels 45 mins after administration. In order to investigate a possible direct action of TRH on immune cells, the authors also examined the effects of this hypothalamic peptide on IFN-γ production by PBMCs stimulated *in vitro* with suboptimal concentration of bacterial superantigen staphylococcal enterotoxin A or concanavalin A (ConA), collected before the intravenous administration of TRH. The *in vitro* findings showed a directly enhanced IFN-γ production by activated lymphocytes (Grasso et al., 1998).

Furthermore, splenic and CNS lymphocytes showed significant decrease in levels of profile 17 helper t cells (T*H*17) and T*H*1 cytokines: Interleukin-17 (IL-17), tumor necrosis factor (TNF-α) and IL-2 respectively, after oral administration of TRH in EAE mice. This treatment also increased CNS lymphocyte production of T*H*2 cytokines, in particular IL-13 (Brod and Bauer, 2013). Thus, oral TRH shows a unique pattern of immunomodulation for an ingested neuropeptide although the poor oral bioavailability associated with TRH and some of its analogs (Khomane et al., 2011).

#### *TRH expression response against immune system challenges*

It is known that high concentrations of TRH seem to decrease the production of antibodies against sheep red blood cells (SRBC) (Kukain et al., 1982).

Pérez Castro et al. (1999) investigated whether TRH mRNA levels in the hypothalamus are altered after SRBC (a T celldependent antigen) immunization. An increased level of TRH mRNA detected by Northern blot autoradiography was observed at 4, 6, and 24 h post immunization in SRBC-injected animals.

The SRBC-induced effects on TRH mRNA levels are opposite to those induced by LPS, a T cell-independent response through macrophage and B lymphocyte activation. LPS induces a decrease in hypothalamic TRH mRNA levels that is observed at 24 h and more pronounced at 4 h (Pérez Castro et al., 1999).

As we can see, TRH effects could seem controversial but is important to consider that interactions between the CNS, the endocrine system and the immune system are mediated at multiple levels. These mediators include secreted chemical messengers such as hormones, cytokines, neurotransmitters and neuropeptides acting directly or via the nervous system.

Evidence indicates interactions at the level of receptors (e.g., the presence of neuroendocrine peptide receptors on immune cells), at the level of secretory function (e.g., the synthesis and secretion of neuroendocrine peptides by immune cells), and at the level of signal transduction (Kamath et al., 2009). Integration of these complex interactions in a single route would be inaccurate, for this reason, the most wise conclusion could be consider the TRH-immune system homeostatic hypothesis proposed by Gary et al. (2003) and Kamath et al. (2009), which states that TRH mediated mechanisms respond to many elements of the immune system and affect them in ways that tend to maintain or restore homeostasis.

The effects of TRH administration on immune system are summarized in **Figure 1**.

#### **CLINICAL RELEVANCE AND FUTURE DIRECTIONS**

Studies on the effects of TRH *in vitro* hardly can be extrapolated to *in vivo* conditions. The direct effect of TRH on the immune system provides a modulatory physiological role. However, pharmacological administration in humans and in experimental animals involves two pathways impact (1) through stimulation of TSH secretion and therefore of THs and (2) the direct effect on the immune system. TRH administration may affect these two ways and with addition or synergy effects. One possibility to explore the direct mode might be using hypophysectomized or thyroidectomized animals to avoid endocrine influence. According to the studies described above, it is possible to consider the possibility that clinical treatment with TRH may improve the immune response in patients with immune deficiencies or reduced response in autoimmune diseases such as multiple sclerosis or lupus erythematosus.

#### **CORTICOTROPIN-RELEASING HORMONE (CRH)**

CRH or Corticotropin-releasing factor (CRF) was characterized, purified and synthesized in 1981 by Vale. The composition of this 41 amino-acid peptide is different from one species to another. Its principal origin in the brain are the neurons from paraventricular nucleus of the hypothalamus. By the portal hypothalamicpituitary vessels, it reaches the anterior pituitary lobe where it stimulates the corticotroph cells (Raux-Demay and Girard, 1985; Emeric-Sauval, 1986). The plasma half-life of CRH in humans is 4 mins (Schürmeyer et al., 1984). The action of CRH on adrenocorticotrophic hormone (ACTH) secretion is predominant, but it acts in relation with arginine-vasopressin and potentially with angiotensin and catecholamines. Adrenal steroids (glucocorticoids) act on feedback control of CRH secretion in the hypothalamus and also on pituitary corticotrophs. CRH has shown to be an important regulator of the endocrine stress response, and it is now known to play a role in diverse functions in the homeostatic balance, important in mobilization of resources and behaviors during stress (Bale and Vale, 2004), it is involved in the regulation of food intake and satiety, as well as gastrointestinal tract motility, vascular tone, and development, and also acoustic and cardiac function (Heinrichs et al., 1992; Koob and Heinrichs, 1999; Maillot et al., 2000; Inoue et al., 2003; Janssen and Kozicz, 2013).

#### **CRH RECEPTOR (CRHR)**

High-affinity CRHRs which mediate the actions of the hypothalamic peptide on ACTH release, have been principal identified in the anterior pituitary gland. Interaction of the pituitary receptor by CRH agonists stimulates ACTH release. CRH exerts its effects by binding to specific cell surface receptors, of which two receptor subtypes have been reported, CRH-R1 and CRH-R2. These receptors share high sequence homology and belong to the family of seven transmembrane GPCRs (Vaughan et al., 1995; Lewis et al., 2001).

Actions of this peptide are initiated by binding and activating GPCRs and subsequently the activation of adenylate cyclase and cyclic adenosine monophosphate dependent protein kinase (Grammatopoulos and Chrousos, 2002). In the regulation of ACTH secretion, the effects of CRH on the corticotroph are integrated with the stimulatory actions of cyclic adenosine monophosphate and the inhibitory effects of glucocorticoids. Following adrenalectomy, the progressive elevation of plasma ACTH levels is accompanied by a concomitant decrease in pituitary CRHRs (down-regulation) (Wynn et al., 1985; Aguilera et al., 1986).

#### *Extrapituitary expression of CRHR*

CRH-R1 is a 415 amino acid peptide and has a widespread expression in stress-related areas located in the CNS (Justice et al., 2008; Kuhne et al., 2012). Outside the CNS, the CRH-R1 is expressed in the adrenal gland, gastrointestinal tissue, ovary, testis, skin, thymus and spleen (Dufau et al., 1993; Nappi and Rivest, 1995; Slominski et al., 1995; Baigent and Lowry, 2000; Müller et al., 2001; Chatzaki et al., 2004). CRH-R2 is a 397–437 amino acid protein and it is abundantly expressed in the CNS (Korosi et al., 2007; Justice et al., 2008; Kuhne et al., 2012). In the periphery, the receptor has been identified in the retina, heart, skeletal muscle, vasculature, adrenal gland, and gastrointestinal tissue (Lovenberg et al., 1995; Müller et al., 2001; Chatzaki et al., 2004).

Binding sites for CRH have been reported on adherent splenocytes (Webster et al., 1990), monocyte-macrophages (Audhya et al., 1991) and T lymphocytes (Singh and Fudenberg, 1988). CRH-R1 and CRH-R2 expression was described in granulocytes and lymphocytes in inflamed tissues (Mousa et al., 2003). CRH-R1 mRNA expression had been reported in the human leukemic mast cell line (Theoharides et al., 1998) and mast cells were reported to express immunoreactive CRH-R1 in inflamed human synovial tissue (McEvoy et al., 2001).

#### **CRH EFFECTS ON CNS AND PNS**

Independently of anterior pituitary, CRH has effects within of nervous system. The central administration of CRH produces hypertension, tachycardia and an elevated oxygen consumption. CRF acts within the brain to increase plasma concentrations of noradrenaline and adrenaline resulting in increased plasma concentrations of glucagon and in hyperglycemia. Likewise, also acts increasing the plasma concentrations of vasopressin. CRH induces a reduction in food intake and increases grooming behavior and locomotor activity. The physiological significance of the peripheral actions of CRH on various organ systems is unknown. Intravenous administration of CRH reduces gastric acid secretion, gastric emptying and blood pressure but increases heart rate, venous return to the heart, mesenteric and aortic blood flow and pancreatic bicarbonate and protein secretions (Lenz, 1987; Chen et al., 2007).

#### **CRH AND IMMUNE SYSTEM**

The cellular components of the immune-inflammatory response include leukocytes, such as monocytes–macrophages, polymorphonuclear neutrophils, eosinophils and basophils, platelets, dentritic cells, mast cells, epithelial and endothelial cells, and fibroblasts which belong to the innate immune system, as well as lymphocytes T, B and natural killer (NK) which are included in the adaptive immune system. These cells cooperate using molecular signals, including ILs, colony-stimulating factors, TNF, IFNs, transforming growth factor, and chemokines, vasoactive amines (histamine and serotonine), plasma proteases (kinine and complement systems), arachidonic acid metabolites (prostaglandins, leukotrienes, and lipoxins), platelet-activating factor, nitric oxide, and neuropeptides (Mastorakos et al., 2006).

The majority of *in vivo* studies suggest that direct stimulation of hypothalamic CRH by cytokines is the principal route by which immune activation stimulates the hypothalamic–pituitary– adrenal (HPA) axis (Besedovsky et al., 1986; Harbuz et al., 1992) and this process is prostaglandin-dependent. Thus, experimental evidence suggests that CRH may modulate the immune and inflammatory responses via two pathways: an anti-inflammatory one operated by centrally released CRH, most likely through stimulation of glucocorticoid and catecholamine release, and one pro-inflammatory, through direct action of peripherally secreted CRH (Karalis et al., 1997).

#### *CRH in immune cells*

*Mast cells.* Mast cells are critical for allergic reactions due to their secretion of numerous vasoactive molecules and cytokines (Kobayashi et al., 2000). Evidence indicates that mast cells are also involved in innate and acquired immunity (Marone et al., 2002), as well as in neuroinflammatory conditions such as those affected by stress (Theoharides and Cochrane, 2004). Mast cells secrete numerous pro-inflammatory cytokines, such as IL-6, IL-8, and TNF-α (Kobayashi et al., 2000; Marone et al., 2002), and secretion of vascular endothelial growth factor (VEGF) by CRH stimulation (Cao et al., 2005). Interestingly, human mast cells were shown to synthesize and secrete large amounts of CRH, suggesting that this peptide could regulate mast cells through CRH receptor in an autocrine manner. Secretion of CRH from mast cells could also be triggered by pro-inflammatory molecules that are released during the initial phases of inflammation (Kempuraj et al., 2004). CRH causes mast cell degranulation in human skin, releasing great amounts of histamine, which appears to be the principal mediator of the vasodilatory effects of CRH (Wright, 2003).

*Mononuclear cells.* In mononuclear cells, it has been reported that CRH stimulates immune functions, proliferation of lymphocytes both in the absence and presence of T cell mitogens, and the expression of IL-2R antigen on T cells (Singh, 1989; Singh et al., 1990). It has also been reported the presence of binding sites for CRH on monocytes, T and B lymphocytes and thymus (Singh and Fudenberg, 1988). The CRH is also a stimulator of production of two interleukins, IL-1 and IL-2. IL-1 has been shown to stimulate the secretion of CRH by hypothalamic cells (Sapolsky et al., 1987) and CRH stimulates the production of IL-1 and IL-2, suggesting the presence of a complete network between the neuroendocrine system and the immune system (Singh and Leu, 1990).

The release of CRH plays a role in modulating NK activity following stress also implies that CRH in the brain may be involved in the functional population of lymphocytes. Studies have been conducted to analyze the effect of CRF on the reduction of several cellular immune measures such as splenic and peripheral blood NK activity and lymphocyte responses to mitogenic stimulation (Strausbaug and Irwin, 1992; Boyadjieva et al., 2006). Other observations also indicate that central doses of CRH slow the kinetics and reduce the level of IgG response to a specific T-cell-dependent antigen (Irwin, 1993). The administration of central CRH antagonist induces suppression of NK cytotoxicity. In contrast, peripheral application has no effect on CRH modulation of immunity (Irwin et al., 1987).

Other studies have shown CRH to inhibit IFN-γ secretion (Angioni et al., 1993). IL-2-induced splenocyte proliferation as well as LPS-mediated IL-1 and IL-6 production by peripheral blood monocytes (Hagan et al., 1992).

The effects of CRH were almost exclusively examined in the mononuclear cells, however in other studies has been reported that polymorphonuclear cells (neutrophils), express higher levels of CRH-R1 than mononuclear immune cells in mouse (Radulovic et al., 1999). It has been shown that CRH inhibits the secretion of IL-1β from mature neutrophils purified from spleens of mice injected with LPS (Radulovic and Spiess, 2001).

It has been described the CRH presence in macrophages (Brouxhon et al., 1998). Results showed that CRH could be detected in normal peritoneal macrophages and the levels increased significantly after stimulation with LPS (Wang et al., 2012). The role of CRH may be immunomodulatory, however the effects remains to be studied.

On other hand, studies shown that cluster of differentiation (CD14+) group of cell surface marker proteins, the CRH can activate CD14+ cells to produce TNF-α cells and compromise endothelial barrier function by inducing apoptosis of the endothelial cells (Song et al., 2013).

#### *CRH and its relationship to immune therapies*

Drugs have been proposed to either block CRHR-1 and CRHR-2 receptors as CRH inhibitors with therapeutic properties through modifying the immune response as tool for both CNS and inflammatory disorders associated with central and peripheral CRH. CRH-R1 antagonist could be considered for the treatment of allergic conditions, such as urticaria, asthma, eczema, or disorders that increase blood–brain barrier permeability (Theoharides et al., 1998; Esposito et al., 2002), chronic inflammatory bowel syndromes, irritable bowel disease, and ulcerative colitis and other pathologies (Kawahito et al., 1995; Gravanis and Margioris, 2005).

The effects of CRH administration on immune system are summarized in **Figure 2**.

#### *Future directions*

There are two major problems associated with the HPA axis. The inflammatory process and the reduction in the immune response. Chronic exposure to antiinflammatory steroids have effects such as immunosuppression which under certain conditions can cause undesired effect. Identifying specific receptor subtypes to CRH, or applying interleukin receptor blockers may provide a viable alternative to maintaining of the homeostatic mechanisms.

Emotional stress causes somatic alterations including the immune response and is perhaps, one of the most pressing problems in global public health that needs to be addressed.

#### **GONADOTROPIN-RELEASING HORMONE (GnRH)**

GnRH, also known as luteinizing hormone releasing hormone (LHRH), is a small peptide hormone (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) of 1.2 kDa, with an established role as central regulator of the neuroendocrine reproductive axis in both males and females (Cheung and Wong, 2008).

This neuropeptide is mainly synthesized in hypothalamic neurons of the medial preoptic area. It is well established that adenohypophysial gonadotropic cells stimulate the synthesis and secretion of gonadotropic hormones such as follicle stimulating hormone (FSH) and luteinizing hormone (LH) (Cheng and Leung, 2005). LH and FSH are released into the systemic circulation in a pulsatile pattern, which is dependent upon the GnRH pulse frequency, and act to affect gonadal steroidogenesis and gametogenesis (Sasaki and Norwitz, 2011).

#### **GnRH ISOFORMS**

At the present, it is known that, in addition to the hypothalamic GnRH, several other isoforms of the decapeptide exist in vertebrates. All these isoforms are characterized by the conservation of the length of the peptide as well as by the amino acid sequences of the N-terminal (Glp-His-Trp-Ser) and C-terminal (Pro-Gly-NH2) domains, indicating that these molecular features are critical for the binding to, and the activation of, the receptor (Limonta and Manea, 2013). The classical hypophysiotropic GnRH located in the anterior hypothalamus/preoptic area is also designed as GnRH-I (Stevenson et al., 2012).

A second isoform of GnRH, originally isolated from the chicken hypothalamus and referred to as chicken GnRH or cGnRH, has been isolated in most vertebrate species—including humans—is now referred to as GnRH-II and is 70% homologous to GnRH-I (Sasaki and Norwitz, 2011). GnRH-II is expressed at a higher level outside of the brain (Tan and Bukulmez, 2011).

A third GnRH isoform, GnRH-III, was isolated from sea lamprey and was also detected in the brain of mammals (Tan and Bukulmez, 2011). This isoform has 60% homology with GnRH-I. In the lamprey, GnRH-III is localized in brain areas involved in the control of reproductive functions and it has been reported to stimulate steroidogenesis and gametogenesis, but it has 500–1000 times less potency in LH and FSH secretion both *in vitro* and *in vivo* (Limonta and Manea, 2013).

GnRH is small and has good thermal and chemical stability and no internal Cys bridges (minimal fixed structure). Its size and stability has led to the availability of thousands of analogs (Conn and Janovick, 2009).

#### **SYNTHESIS AND SECRETION**

GnRH is synthesized as a prohormone, human preproGnRH that contains 92 amino acids (Harrison et al., 2004). The gene encoding preproGnRH1 gene has been cloned in number of species and is approximately 4300-bp with four relatively short exons separated by three large introns (Clarke and Pompolo, 2005).

Neurons that synthesize and release GnRH (GnRHergic neurons) have their origin in embryonic olfactory placodes. These embryonic cells, migrate and colonize the basal forebrain, preoptic area around the basal and medium hypothalamus. The median eminence in mammals contains a large amount of GnRH, so is the area in which the peptide is stored in neural terminals before release into the hypothalamus-pituitary portal system (Clarke and Pompolo, 2005).

GnRH is released as synchronized pulses from the nerve endings of about 1000 neurons into the hypophyseal portal system every 30–120 mins to stimulate the biosynthesis and secretion of LH and FSH from gonadotrophs (Millar, 2005). In adult rodents, GnRH is released from the median eminence at a frequency of about one pulse every 30 mins. A slightly slower frequency of release (approximately 50–60 mins intervals) is observed in primates (Yin and Gore, 2006). On the other hand, continuous (non-pulsatile) GnRH administration or long-lasting GnRH-a desensitize GnRHR, thereby decreasing/inhibiting the release of LH and FSH by the pituitary (Wilson et al., 2007).

#### **HALF-LIFE**

GnRH half-life normally is about 2–4 mins (Walters et al., 2008). Its short half-life is because of the rapid cleavage of the bonds between amino acids at positions 5–6, 6–7, and 9–10. Substitution at position 6 in GnRH agonists (GnRH-a) yields them resistant to degradation and increases its half-life and the time of receptor occupancy (Magon, 2011).

#### **GnRH RECEPTOR (GnRHR)**

The human pituitary GnRHR belongs to the family of GPCRs and is one of the smallest GPCRs (328 amino acid protein in the human); it may contain only the essentials required for ligand binding and signal transduction (Conn and Janovick, 2009).

Distinct forms of the GnRHR can be broadly divided into two groups:

#### **EXTRAHYPOTHALAMIC AND EXTRAPITUITARY LOCATION OF GnRH AND GnRHR RESPECTIVELY**

#### *GnRH outside the pituitary zone*

GnRH has been located in several extrahypothalamic cells and tissues: GnRH-like substances have been reported to be secreted from tissues outside the CNS of rats and humans. Particularly, a (GnRH)-like protein has been detected in human ovaries (Aten et al., 1987; Dong et al., 1993; Peng et al., 1994). Also, Oikawa demonstrated by PCR amplification of cDNA from rat hypothalamus, granulosa cells, and whole ovary, a 241-bp product identical to the authentic GnRH sequence based on analysis on both strands (Oikawa et al., 1990).

RT-PCR analysis together with Southern blot analysis demonstrated the presence of GnRH mRNA in human testes and mammary gland (Dong et al., 1993).

Furthermore, the gene coding for GnRH is expressed in immune tissue and immunoreactive GnRH is measurable in immune cells. Analysis of mononuclear cells by RT-PCR coupled to Southern hybridization of total RNA confirmed that GnRH genes are expressed in human peripheral blood lymphocytes. The GnRH transcripts in mononuclear cells were identical to the hypothalamic GnRH and PRL release inhibiting factor cDNA, at least within this 380-bp fragment (Chen et al., 1999).

Rat splenocytes contain an immunoactive and bioactive GnRH (Emanuele et al., 1990; Azad et al., 1991) that has also been found in lymphocytes from rat thymus (Marchetti et al., 1990; Maier et al., 1992). Moreover, it has been reported that normal and leukemic human T cells produce GnRH-II and GnRH-I and that the incubation of those cells with these neuropeptides triggered *de novo* gene transcription and cell-surface expression of a 67-kDa non-integrin laminin receptor that is involved in cellular adhesion and migration and in tumor invasion and metastasis (Chen et al., 2002).

The fact that recent evidence shows the existence of diverse isoforms of GnRH, with high sequence homology and a core variable region raises the issue that previous GnRH distribution studies may have identified a variety of isoforms. To confirm the distribution of GnRH-I only, Khan et al. (2003), reported that intense GnRH-I immunoreactivity was found in Kupffer cells, and less intense in spleen lymphocytes and follicular dendritic cells in rat.

Other cells that have been reported to express GnRH and its mRNA are mast cells from the nervous system and the peritoneal cavity, as detected by immunohistochemistry and RT-PCR, respectively (Silverman et al., 1994; Gill and Rissman, 1998; Khalil et al., 2003).

#### *Extrapituitary GnRHR*

GnRH binding sites have been described throughout the CNS and PNS as in several non-CNS cells and tissues:

Human ovarian granulosa-luteal cells also express the GnRHR mRNA (Chen et al., 1999).

GnRHR expression in organs and cells of the immune system has been confirmed: GnRH binding sites have been reported in rat spleen (Batticane et al., 1991) and thymus, thus potentially enabling GnRH-a to have direct stimulatory effects that might contribute to improved T-cell function (Marchetti et al., 1989; Batticane et al., 1991; Morale et al., 1991).

RT-PCR coupled to Southern hybridization of total RNA obtained from mononuclear cells confirmed that the GnRHR is expressed in human peripheral blood lymphocytes (Chen et al., 1999) as in porcine lymphocytes (Standaert et al., 1992).

Chen and colleagues reported that the GnRHR transcript expressed in mononuclear cells has an identical nucleotide sequence as the pituitary GnRHR cDNA. In this work, a time-course study of the effect of GnRH on mononuclear cells was performed and it was found that GnRHR mRNA increased gradually after *in vitro* culture, and this increase was further augmented by treatment with GnRH for up to 24h. In contrast, the expression of GnRH mRNA was decreased by GnRH treatment in a dose-dependent manner and the IL-2Rγ subunit gene was highly expressed in mononuclear cells. At this point we can speculate that these GnRH-binding sites in lymphocytes are probably functional, as many studies have demonstrated that GnRHa caused a dose-dependent increase in LH production, whereas GnRHR antibodies blocked this action (Chen et al., 1999).

It has also been shown that blockade of central and peripheral GnRHR with a potent GnRH antagonist (GnRH-ant) during a critical period of maturation can impair the morphological development of thymus and the cellular and humoral immune responses in rats (Morale et al., 1991) and monkeys (Mann et al., 1994).

Particularly, neonatal exposure of male primates to a GnRHant alters early postnatal programming of immune function by reducing the number of B cells and T cells in the thymic medulla and T cells in the periarterial lymphatic sheaths of the spleen. GnRH-ant treatment increased the frequency of clinical problems, lowered circulating levels of lymphocytes, total T cells, CD8+ T cells and B cells, and altered the proliferative index of lymphocytes to mitogens. As adults, the cell- and humorallymediated immune responses remained impaired (Mann et al., 2000).

To evaluate regions of GnRH-I binding activity, a biotinylated GnRH-I sequence was used with avidin-labelled HRP, and results showed GnRH-I binding activity present in Kupffer cells, spleen lymphocytes and follicular dendritic cells, as well as in thymocytes and peripheral blood lymphocytes and neutrophils (Khan et al., 2003).

#### **GnRH AND IMMUNE SYSTEM**

GnRH and sex steroids seem to be important components of immune system modulation and may play a role in the regulation of immune mediated diseases. A comprehensive description of such interactions has been reviewed by Tanriverdi et al. (2003) and Quintanar (2011).

#### *Immune response to GnRH administration*

#### *Proliferation*

*Induction.* When splenocytes and thymocytes are preincubated with GnRH-a or GnRH peptide, an augmentation of the proliferative response is obtained. Marchetti et al. (1989) showed a significant dose-dependent increase on proliferative response to the mitogen ConA in rat thymocytes. This response was abolished with simultaneous addition of a GnRH-ant. Also, splenocytes and thymocytes from proestrus female rats incubated with GnRH or its analogs, significantly increased the basal proliferative activity which was accompanied by a specific increase in IL-2 receptorpositive cells (Batticane et al., 1991).

Furthermore, *in vivo* GnRH-a treatment prevents thymus atrophy and markedly stimulates thymocyte blastogenic activity in hypophysectomized rats, thus suggesting that GnRH and its agonistic analogs are may capable to exert a direct modulation of immune system function (Marchetti et al., 1989).

Injection of GnRH-a increases CD8+ T-cell levels and proliferation to a variety of mitogens (Mann et al., 1994). In another study, it has been reported that GnRH immunization in rats induced the presence of eosinophils in intertubular tissue of testes whereas in unimmunized controls these cells were not present (Duckett et al., 1997).

Moreover, in a rat model of pregnancy-induced thymic involution, GnRH-a infusions markedly attenuated pregnancy-induced thymic involution resulting in significant increases in thymic weight and thymocyte numbers (Dixit et al., 2003a).

In the same way, Sutherland et al. (2008) made a pilot study in patients, where a GnRH-a (goserelin) was given prior to allogeneic or autologous hematopoietic stem cell transplantation. GnRH-a administration significantly increased neutrophil and lymphocyte numbers within the first month of posttransplantation indicating that either directly or indirectly, GnRH induced an increase in circulating neutrophils.

It has been reported that treatment with the GnRH-a, leuprolide acetate (50 μg/mouse, s.c.) prior to restraint stress, significantly prevented its effect on cell-mediated immunity, antibody titer levels, total leukocyte count and relative thymus weight, showing a significant increase of these immunological parameters (Umathe et al., 2008).

*Supression.* To test whether a GnRH-a could alter *in vivo* human immune cells, Ho et al. (1995) treated a group of infertile patients under a protocol of buserelin acetate administration and peripheral B cells, NK cells, CD4+ and CD8+ T cells, and the expression of CD69, CD25, human leucocyte antigen-DR (HLA-DR), and CD71 antigens on the T cells were serially examined by dual-color flow cytometry. They found that the GnRH-a had a transiently immunosuppressive effect on CD4+ and CD25+ T cells, but CD69+, CD25+, and HLA-DR+ T cells were activated during and after successful implantation. Regarding to the B cells, NK cells, CD8+ T cells, and CD71+ T lymphocyte subpopulations there were no changes throughout the whole course of treatment.

On the other hand, several groups have provided evidence that the *in vitro* proliferation of a variety of human ovarian, endometrial, and breast cancer cell lines can be inhibited by agonistic and/or antagonistic analogs of GnRH in a dose- and time-dependent manner (Eidne et al., 1987; Thompson et al., 1991; Emons et al., 1993a,b; Emons and Schally, 1994).

In ovarian cancer cells, GnRH seems to have two opposite activities:

Furthermore, biopsy specimens collected from lesions, myometria and corresponding endometria of women with ovarian endometrioma, adenomyosis and uterine myoma treated with the GnRH-a, leuprolide acetate, for a variable period of 3–6 months before surgery, showed a significantly decrease in the infiltration of CD68-positive Mφ, micro-vessel density and tissue levels of monocyte chemotactic protein I (MCP-I). Furthermore, when compared with the non-treated group, a significant increase in apoptotic index and quantitative-histogram scores of activated caspase-3 after GnRH-a therapy were observed in the eutopic endometria, lesions and myometria of these diseases (Khan et al., 2010).

Tanriverdi et al. (2005) demonstrated that GnRH-I and GnRH-II have differential modulatory effects on human PBMC proliferation and IL-2Rγ -chain mRNA expression in healthy males. Treatment of *in vitro* PMBCs with GnRH-I and with IL-2 resulted in a significant increase in cell proliferation as well as in an increased expression of IL-2Rγ mRNA in a dose dependent manner compared with the untreated control. In contrast, GnRH-II administration did not affect the proliferation of PMBCs alone, and IL-2Rγ expression was significantly decreased in all concentrations. This study clearly indicates the potential stimulatory effects of GnRH-I in PBMCs and reduction of these effects by GnRH-II.

*Induces GnRH production by immune cells.* Numerous interesting and solid studies show that T-cells (and other immune cells) can in fact produce and secrete various endogenous neurotransmitters, either spontaneously or after induction by external stimuli. Among neurotransmitters produced by T-cells are GnRH-I and GnRH-II (Levite, 2008).

Immunoactive and bioactive GnRH peptide is present and the GnRH message is expressed in human peripheral blood T-cells. Both CD4+ and CD8+ subsets of T-cells produce GnRH and it is significantly increased when cells are activated by either phytohemagglutinin or anti-CD3 antibody. This GnRH production in activated T-cells is an early activation event that is independent of IL-2 system activation and DNA synthesis (Azad et al., 1993).

*Induces immune mediators production.* To investigate the role of GnRH in the modulation of T helper cytokines in pregnant rats undergoing termination of pregnancy, Dixit et al. (2003b) administrated a GnRH-a to evaluate effects on Th-1 and Th-2 cytokines. A marked increase in IFN-γ , inhibition of IL-4 production and an IL-2 and IL-10 absent response was observed. It is possible that IL-2 levels are unchanged because it might has been consumed by activated T cells (Quintanar, 2011).

Recently, Goldberg et al. (2009) demonstrated in a clinically relevant model of allogeneic bone marrow transplantation, that after leuprolide acetate treatment, the enhanced peripheral T cell reconstitution is due to both increases in lymphoid-committed precursors as well as enhanced thymic regeneration.

GnRH has also been reported to regulate four angiogenic chemokines expression (CXCL2, CXCL3, CXCL6, and CXCL8) in human placenta, where trophoblasts were subsequently shown to recruit lymphocytes. Additionally, in Jurkat T cells and primary peripheral blood T and uterine NK cells; chemokine release was detected in chemotaxis assays and this was shown to be GnRH dependent (Cavanagh et al., 2009).

There are two works evaluating the efficacy of the GnRH-a, leuprolide acetate, on NK cells activity in endometriosis. Results are contradictory since the first one suggests an increased NK cell activity in peripheral blood samples determined by 51Cr release assay (Umesaki et al., 1999); and the second one reported that NK cell cytotoxicity from control and patients was significantly decreased with leuprolide acetate (Wong and Simon, 2004). These findings suggest a direct immunomodulatory role of GnRH on NK cell activity.

In an attempt to know the possible immunomodulatory effects of GnRH on LPS activated human peripheral blood monocytes cultured *in vitro*, Komorowski and Stepien (1995) found that GnRH did not affect the secretion of the cytokines IL-1β and IL-6.

On the other hand, there is a report where the immunomodulation exerted by GnRH on freshly isolated primary peritoneal macrophages is clearly observed. In this work the authors found that the production of nitric oxide, co-stimulated with LPS and IFNγ , and the activity of NF-kB was suppressed by GnRH exposure. Taken together, these results demonstrate that GnRH participates in macrophage function and indicate that the NF-kB signaling pathway may be responsible for GnRH-mediated immune modulation (Min et al., 2009).

#### *GnRH expression response against immune system challenges*

There is a work that reported that when lymphocyte lysates were applied to rat anterior pituitary cells in monolayer culture, significant stimulation of GnRH secretion was seen. This response evoked by lymphocyte lysates was found to be dose dependent and could be significantly inhibited by a GnRH-ant (Emanuele et al., 1990).

Otherwise, TNF has a number of regulatory actions on gonadotrophic functions: TNF and other cytokines, notably IL-1β, have powerful LH suppressing abilities on pituitary activity (Kalra et al., 1998).

#### *GnRH and autoimmunity*

Given that GnRH possesses direct immunostimulatory actions, it has been hypothesized that GnRH might play a role in the development of autoimmune disease.

Jacobson et al. (1994) demonstrated that the administration of GnRH antagonists in a mouse model of systemic lupus erythematosus (SLE), led to a reduction in autoantibody levels, total immunoglobulin levels, renal disease, and significantly improved survival, independently of gonadal steroids.

Based on the above mentioned findings and considering that (1) in the nonobese mouse model of autoimmune diabetes (NOD mouse), castration of male NOD mice leads to increased incidence of diabetes, and that (2) castration of male mice leads to an increase in GnRH action, Ansari et al. (2004) determined the effect of GnRH agonists and antagonists on expression of autoimmune diabetes in this mouse model. Their results showed that Antide administration prevented the increased incidence of diabetes, reduced total serum IgG levels, IL-6 cytokine expression in cultured splenocytes, and the lymphocytic infiltration of islets. GnRH administration exerted reciprocal effects, leading to earlier timing of onset of diabetes and increased serum total IgG levels.

In addition, due to that has been reported that motoneurons of spinal cord of rat from embryonic until adult stage, express GnRHRs which respond to GnRH (Quintanar et al., 2009) a possible clinical use in spinal cord injury (Calderón-Vallejo and Quintanar, 2012) and CNS autoimmune diseases has been proposed. Specifically, treatment with GnRH (Quintanar, 2011) or a synthetic analog (Guzmán-Soto et al., 2012) induces an improvement of clinical signs in the EAE model, where they remained significantly lower in EAE rats with GnRH administration compared to animals without treatment. In these experiments, also a significantly increased expression of neurofilaments (NFs) and myelin basic protein as well as more axons of larger areas in the spinal cord of EAE animals were found.

A great advantage of GnRH and its analog leuprolide acetate, is that they are capable to cross the blood brain barrier (Kastin et al., 1980; Barrera et al., 1991) and reach their targets into the CNS. Taking into account this advantage of leuprolide acetate and its effect on recovery in locomotion and increased expression of biological markers, the use of this agonist as a potential therapeutic approach for traumatic or neurodegenerative diseases such as multiple sclerosis should be considered.

The effects of GnRH on immune system are summarized in **Figure 3**.

responses (↑ = increase, ↓ = decrease).

#### *Clinical relevance and future directions*

GnRH induces the secretion of FSH-LH and consequently of sex hormones, which are involved in the immune response. Controversial is the idea that sex hormones are immunoprotective because depending on the type of hormone and experimental conditions can be obtained opposite effects.

The presence of GnRH receptors and GnRH secretion by cells of the immune system, opening the spectrum of possibilities for interaction of autocrine or paracrine regulation. However, the pharmacological use of GnRH or its analogues, independently of the direct effect of sex hormones, raise the possibility that can be used as a inflammatory regulator in autoimmune diseases. Retrospective studies can be analyzed to establish whether treatment with GnRH analogues have improved immunological conditions of patients with autoimmune pathology. This option can be explored in future works.

#### **CONCLUSION**

Hypothalamic hormones are closely related to the immune system. Their direct and indirect effects establish a bidirectional network to establish the required mechanisms to maintain homeostasis.

#### **REFERENCES**

TRH, CRH and GnRH exhibit common patterns of specific receptors in immune cells and tissues. It is hard to think in the action of a single independent factor within the neuroimmunoendocrine network. When any of these elements destabilize, the system is altered and is difficult to define which factor of all may have been responsible for the alteration. Perhaps because of the complexity of the network, experiments to demonstrate such events become complicated to study. Using *in vitro* models allow clarify in a specific manner some of the elements involved in the homeostasis, however, extrapolate it to *in vivo* models could lead to unreliable interpretations.

In the evolution of the knowledge of the involvement of neurohormones on the immune system, we can find that many of the investigations are related to their pharmacological application, however, few of them concern to the physiological mechanism of its immunomodulation either autocrine or paracrine. If more is known about the physiology of these neurohormones, expectations about their applications at therapeutic level or to the understanding of our complex responses to internal or external challenges to maintain the survival of the individual could be proposed.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 31 May 2013; paper pending published: 20 June 2013; accepted: 16 July 2013; published online: 13 August 2013.*

*Citation: Quintanar JL and Guzmán-Soto I (2013) Hypothalamic neurohormones and immune responses. Front. Integr. Neurosci. 7:56. doi: 10.3389/fnint.2013. 00056*

*Copyright © 2013 Quintanar and Guzmán-Soto. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Hormonal influences on neuroimmune responses in the CNS of females

#### *Nela Monasterio1, Edgar Vergara2 and Teresa Morales1\**

<sup>1</sup> Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Santiago de Querétaro, México <sup>2</sup> Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad de México, México

#### *Edited by:*

Beatriz Gomez-Gonzalez, Universidad Autónoma Metropolitana – Unidad Iztapalapa, Mexico

#### *Reviewed by:*

Beatriz Gomez-Gonzalez, Universidad Autónoma Metropolitana – Unidad Iztapalapa, Mexico George Chrousos, National and Kapodistrian University of Athens, Greece

#### *\*Correspondence:*

Teresa Morales, Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Boulevard Juriquilla 3001, 76230, Santiago de Querétaro, México e-mail<EMAIL_ADDRESS>
**INTRODUCTION**

Reproduction is one of the most significant events in the life of a mammalian female. It can be described as a rich social and hormonal experience that begins by interacting and mating with a male, followed by pregnancy, parturition, and ultimately lactation, interaction with pups, and weaning (Mann and Bridges, 2001; Serafim and Felicio, 2002). Striking behavioral and neuroendocrine alterations due to motherhood have been reported in several mammalian species including mice, rats, rabbits, sheep, and humans. They are reflected by changes at almost all brain levels and are essential to protect the developing embryo, and for successful delivery, maternal behavior, and pup survival (Kinsley and Lambert, 2008). These neuroanatomical and functional changes observed during pregnancy and lactation are necessary to cope with the demands of reproduction and to protect the maternal organism against dramatic hormonal variations (Mann and Bridges, 2001; Kinsley and Lambert, 2008). They include a marked adaptation of the hypothalamus-pituitary-adrenal (HPA) axis, which results in hypo-responsiveness to stress, diminished inflammatory responses (Walker et al., 1992; Windle et al., 1997a; Lightman et al., 1998; Tilbrook and Clarke, 2006), and diminished sensitivity of the brain of the mother against excitotoxic damage (Morales, 2011).

#### **MECHANISMS GOVERNING THE HYPO-RESPONSIVENESS TO STRESS DURING PREGNANCY AND LACTATION**

One of the best-known examples of naturally attenuated stress response is seen in the female rat during late pregnancy and lactation (Tilbrook and Clarke, 2006). During these reproductive

Particular reproductive stages such as lactation impose demands on the female. To cope with these demands, her physiology goes through numerous adaptations, for example, attenuation of immune and stress responses. Hormonal fluctuation during lactation exerts a strong influence, inducing neuroplasticity in the hypothalamus and extrahypothalamic regions, and diminishing the stress and inflammatory responses. Thus, hormones confer decreased vulnerability to the female brain. This mini-review focuses on the adaptations of the immune and stress response during maternity, and on the neuroprotective actions of progesterone and prolactin and their effects on inflammation. The importance of pregnancy and lactation as experimental models to study immune responses and disease is also highlighted.

**Keywords: stress, progesterone, prolactin, lactation, neuroimmune, HPA axis, female**

phases, the HPA axis maintains minimal responses necessary to cope with any situation that may threaten the homeostasis of the female. This axis comprises corticotropin-releasing hormone (CRH)- and vasopressin- neurons in the paraventricular nucleus of the hypothalamus (PVN), which when stimulated, release these peptides into the median eminence to stimulate pituitary cell production of adrenocorticotropic hormone (ACTH) that reaches the adrenal cortex to release cortisol or corticosterone. HPA axis activity is regulated by glucocorticoid negative feedback on the pituitary, the PVN, and higher brain centers.

The attenuation of the stress response during pregnancy and lactation has been documented in various different stress models (Lightman et al., 2001; Russell et al., 2008). Pregnancy in the rat is accompanied by a progressive decrease in HPA axis responses to a range of psychological (Neumann et al., 1998) and physical (Brunton et al., 2005; Douglas et al., 2005) stressors particularly in the last week of pregnancy, reflected by reduced ACTH and corticosterone secretion. This hypo-responsiveness persists through parturition (Wigger et al., 1999; Neumann et al., 2003) and lactation until weaning (Windle et al., 1997b). Suppressed responses to stress in pregnancy can be explained by adaptations in both the anterior pituitary and the hypothalamus (Brunton et al., 2005; Russell et al., 2008). Corticotrophs in the pituitary are less sensitive to secretagogs (Shanks et al., 1997; Neumann et al., 1998), and CRH mRNA expression induced by stress is attenuated (Brunton et al., 2005). Moreover, HPA axis responses to immune stress in early mid pregnancy are strong and similar to that in virgins, although activation of hypothalamic vasopressin neurons, rather than CRH neurons, may be more important in

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the stress response in pregnancy (Ma et al., 2005; Parker et al., 2011).

Studies in lactating rats have shown a flattening of the diurnal rhythm of corticosterone secretion (Walker et al., 1992; Atkinson and Waddell, 1995) during this phase, such that the nadir levels of corticosterone rise (Stern et al., 1973; Fischer et al., 1995) and the peak evening levels decrease (Windle et al., 2013). During lactation, basal plasma concentrations of both ACTH and corticosterone increased in lactating animals compared with those in virgin rats (Shanks et al., 1997, 1999). There is also an increased basal expression of CRH mRNA in the PVN in early lactation (day 3; da Costa et al., 1996), but low basal expression of CRH mRNA by the middle phase of the lactation period (days 7–10; Windle et al., 1997a; Walker et al., 2001). However, CRH expression in response to stress is diminished at either stage (Lightman and Young, 1989; Windle et al., 1997b), similar to findings in late pregnancy (Douglas et al., 2005).

The mechanisms for this altered neuroendocrine responses include diminished CNS excitatory signaling within stressresponsive systems like the catecholaminergic brainstem and limbic circuitry (Hoffman et al., 1994; da Costa et al., 1996; Wintrip et al., 1997) as well as an altered hypothalamic and adrenomedullary catecholamine function (Patel et al., 1993; Toufexis and Walker, 1996; Windle et al., 1997a; Toufexis et al., 1998). Attenuated noradrenergic tone also underlies stress hyporesponsiveness in lactation, and is clearly dependent upon suckling (Hoffman et al., 1994; Toufexis and Walker, 1996; Toufexis et al., 1998). Moreover, opioids switch to having a net inhibitory effect on HPA activity such that naloxone treatment enhances the ACTH response to IL-1β in late pregnant rats (Brunton et al., 2009). Growing evidence for an integrated participation of many other factors besides noradrenaline and opioids that underlie altered responses to stress, include CRH (Johnstone et al., 2000; da Costa et al., 2001), oxytocin (Neumann et al., 2003; Windle et al., 2004), prolactin (PRL; Torner and Neumann, 2002), steroids and neurosteroids (Douglas et al., 2000; Brunton and Russell, 2008, 2011; **Figure 1**).

Another mechanism for the attenuation of the stress response involves nitric oxide (NO) regulation of CRH expression within the PVN. NO is present in the PVN of female rats during lactation (Popeski et al., 1999; Otukonyong et al., 2000; Monasterio and Morales, 2011), as indicated by the NO markers NADPHdiaphorase and neuronal synthase (nNOS), and its synthesis dependent on suckling (Otukonyong et al., 2000) and PRL (Vega et al., 2010). These markers increase within the PVN in response to stress in male rats, but paradoxically, basal level of NO markers are present in the PVN of lactating females whose response to stress is attenuated. The activational c-fos response and an increase in NADPH-diaphorase- and nNOS-positive cells are clearly detected in the PVN of diestrus rats after an immune challenge, but not in lactating rats. Furthermore, the total concentration of nitrates in the hypothalamus and the circulating level of corticosterone and IL-6 increase significantly after stress in diestrus, but not in lactating rats, compared to their corresponding controls. Intracerebral administration of L-NAME, a general NOS inhibitor, reverses the attenuation of the activational response to stress in the lactating rats and increases CRH expression, suggesting that sustained NO production in the PVN during lactation contributes to attenuate the neuroendocrine response to stress (Monasterio and Morales, 2011; **Figure 1**).

#### **HPA AXIS HORMONES, PROGESTERONE, AND PROLACTIN IN THE REGULATION OF THE IMMUNE RESPONSES DURING PREGNANCY AND LACTATION HPA AXIS AND IMMUNE RESPONSES**

Cytokines, peptide hormones, neurotransmitters, and their receptors are endogenous elements of the nervous, endocrine, and immune systems. These systems share ligands and receptors that serve to communicate (Haddad et al., 2002; Elenkov and

**FIGURE 1 | Diagram illustrating the regulation of CRH expression in the parvocellular neurons of paraventricular nucleus of the hypothalamus.** During late pregnancy and lactation, there is a diminished noradrenergic tone reaching CRH neurons, and progesterone, allopregnanolone, prolactin, and nitric oxide will contribute to the inhibitory regulation of the CRH neurons. The cascade of neuroendocrine

responses, synthesis of ACTH and corticosteroids will also be modified. Neuroimmune interactions are represented by effects of glucocorticoids on cells of the immune system, and which molecules will affect CNS functions. ACTH, adrenocorticotropic hormone; AP, allopregnanolone; CRH, corticotropin-releasing hormone; CBG, corticosteroid-binding globulin; POMC, proopiomelanocortin.

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Chrousos, 2006). In addition to altered endocrine responses during lactation, stress also changes immune function in comparison to non-lactating animals (Monasterio and Morales, 2011), suggesting that bi-directional communication between the immune and endocrine systems is also altered during lactation. Pregnancy and lactation represent interesting experimental models that illustrate how the elevated basal plasma level of corticosterone and the diminished stress responses, affect the immune system. It is established that dampened HPA responses are associated with increased vulnerability to inflammation (Shanks et al., 1997, 1999) and therefore, alterations in endocrine regulation of immune responses during lactation may predispose the animal to inflammatory disease. However, not all of the immune responses are dampened during pregnancy and lactation (Jaedicke et al., 2009).

Several reports have documented motherhood-induced adaptations of the immune system in female rats, showing that this is a not always an immune suppression condition (Jaedicke et al., 2009). Early pregnancy has been associated with an increased cell density in the thymus and spleen, whereas late pregnancy and lactation have been associated with a decreased cellularity despite the expansion of the thymus medulla (Kendall and Clarke, 2000). Lactation delays the return of the thymus medulla to the original pre-pregnancy state. In addition, the changes in the thymus can be attributed to the adaptation of the maternal immune system to the semi-allogeneic fetus (Kendall and Clarke, 2000). Hormonal and neural changes can partially explain these modifications. Progesterone, PRL, and estradiol levels in plasma fluctuate in early, mid, and late pregnancy (Neville et al., 2002; Brusco et al., 2008). Nerve growth factor, nearly absent in the medulla of the adult thymus, is high in the medulla of the thymus during late pregnancy (Aloe et al., 1997). Also, lactation is associated with an increased susceptibility to parasitic infections (Barger, 1993), but much controversy exists in the effects of pregnancy and lactation on the progression of autoimmune diseases as documented by studies in rodents and in humans (Elenkov et al., 2001; Buchel et al., 2002; Vukusic et al., 2004; Gregg, 2009).

The immune system of lactating rodents has been the focus of only a few studies, which have shown that some immune functions become suppressed in this phase, while others remain unaffected or are enhanced (Jaedicke et al., 2009). For example, antibody production after immunization, and IL-6 (Monasterio and Morales, 2011) and IL-2 production in the spleen (Shanks et al., 1997) were suppressed during lactation in rodents. Conversely, evidence of increased concentrations of plasma IL-6 or an enhanced proliferative response of lymphocytes from mesenteric lymph nodes suggests activation of other immune responses (Shanks et al., 1997). In male rats, activation of the HPA axis and glucocorticoid release occurs during bacterial lipopolysaccharide (LPS) exposure, triggering an elaborate inflammatory response that involves the release of the pro-inflammatory mediators IL-1β, IL-6, and tumor necrosis factor (TNF)-α (Grinevich et al., 2001), which stimulate cells to upregulate the inflammatory reaction, and systemically they activate the HPA axis (Chrousos, 1995). However, in lactating rats, LPS significantly elevates circulating levels of ACTH and corticosterone, but the magnitude of hormonal and hypothalamic responses to LPS are significantly reduced in lactating animals relative to virgin controls (Shanks et al., 1999). Despite this attenuated stress response, systemic immune responses to stressors are modulated during lactation indicating that the immune system is not generally suppressed but rather adjusted in this stage (Jaedicke et al., 2009).

In humans, the early postpartum period has been associated with up-regulated inflammatory responses and a relapse of autoimmune disorders such as rheumatoid arthritis and multiple sclerosis, often interpreted as a flare-up due to the rebound of the immune system after pregnancy (Elenkov et al., 2001; Buchel et al., 2002). However, long term-studies have shown that relapse rate of multiple sclerosis remains similar to pre-pregnancy level, after an increase in the first trimester postpartum (Vukusic et al., 2004). A broad state of immune activation is also characteristic of the early postpartum period, as measured by levels of neopterin, soluble IL-2 receptor, and soluble CD8 antigen (Burns et al., 1999). This may help women recover from the biological stress of parturition, but more studies about the magnitude and length of such a state are necessary. Immune and inflammatory activation in postpartum women may be factors leading to anxiety and depression in the early days after delivery (Maes et al., 2000). CD4 cell counts are reported to rise during postpartum, primarily due to γδ T cells (Watanabe et al., 1996). Natural killer (NK) subsets with weak cytotoxic activity (CD16+, CD57+) were found to increase during months 1–4 postpartum (Watanabe et al., 1997), and lymphocyte proliferation was higher than non-postpartum controls. Furthermore, the postpartum period is also associated with the onset of autoimmune thyroid syndrome (Muller et al., 2001). Thus, immune responses linked to reproduction can either be dampened or enhanced depending on the stimulus and hormonal status (De Bellis et al., 2005; Hughes, 2012; Shelly et al., 2012).

In summary, during both pregnancy and lactation the immune responses are altered, but instead of considering them to be immune-suppressed stages, they represent reproductive conditions in which either stress or immune responses can vary depending on the hormonal status (Mor and Cardenas, 2010). The immunology of reproduction is the result of the combination of signals and responses originating from the fetal-placental and the maternal immune systems. This last is under the regulation of the CNS through the hormonal (glucocorticoids) stress response, pituitary responses, and the autonomic nervous system.

#### **PROGESTERONE ACTIONS ON CNS IMMUNE RESPONSES**

Among the hormones that have been considered candidate inducers of pregnancy- and lactation-related adaptations in HPA axis and immune responses is progesterone. Progesterone is a steroid hormone synthesized by the corpus luteum in cycling females and by the placenta during pregnancy; it can enter the brain from the circulation and can also be synthesized in the brain by oligodendrocytes and excitatory neurons (Stein, 2011). During pregnancy, progesterone levels in plasma and brain are increased and are accompanied by elevated levels of its metabolite allopregnanolone (Brunton and Russell, 2008; Mostallino et al., 2009).

Progesterone (and its metabolite allopregnanolone) has important actions in the female's brain, such as the expression of

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maternal behavior (Bridges, 1984). During pregnancy, high levels of allopregnanolone suppress HPA -axis responses to stress: allopregnanolone may enhance the action of GABA in the PVN or on afferent inputs to the CRH neurons to suppress stress responses, and it also induces and maintains the endogenous inhibitory opioid mechanism in the nucleus of the solitary tract (Brunton et al., 2009). Blocking allopregnanolone restores HPA axis responses to systemically administered IL-1β in late pregnant rats (Brunton and Russell, 2008). Allopregnanolone acts as an agonist on the GABA receptor, exerting anxiolytic, sedative, and antiepileptic effects, and it enhances the myelination/remyelination process in the central and peripheral nervous system (Wang et al., 2008; Gangisetty and Reddy, 2010). These steroid hormones have been described as potent regulators of growth factor expression during pregnancy: epidermal growth factor (EGF), insulin-like growth factor (IGF), and transforming growth factor (TGF-β1) in particular are all up-regulated, promoting neural proliferation (Wang et al., 2008). Moreover, they increase neurogenesis within the subgranular zone of the dentate gyrus and subventricular zone (Pawluski et al., 2009), and induce regenerative responses in a mouse model of Alzheimer's disease (Pike et al., 2009; Borowicz et al., 2011). During lactation, progesterone participates in glial changes in brain areas such as the cingulate cortex (Salmaso et al., 2009) and the dentate gyrus of the hippocampus (Cabrera et al., 2013), and this steroid is part of the hormonal cocktail responsible for diminished responses of astrocyte and microglial cells in the hippocampus of lactating rats to damage induced by excitotoxic insults (Cabrera et al., 2013).

Progesterone and allopregnanolone attenuate traumatic brain injury, and diminish the elevation of pro-inflammatory cytokines in a time-dependent manner, suggesting that the protection occurs by limiting the overexpression of cytokines, when they peak at 3 h after a brain injury, rather than inhibiting their expression later in the post-injury cascade of toxic events (He et al., 2004). Both steroids prevent breakdown of the blood–brain barrier in edema and stroke (Ishrat et al., 2010). Progesterone exerts some of its actions through the intracellular, membrane-bound progesterone receptor, while allopregnanolone does not bind to the progesterone receptor (Ishrat et al., 2010). Also, progesterone can reduce the excessive excitotoxicity and inflammation by stimulating activation of the neuroprotective mitogen-activated protein kinase (MAPK) and PI3-K pathways (Kaur et al., 2007). Through use of calcium imaging, electrophysiology, and the measurement of changes in activity-dependent gene expression, progesterone was found to block calcium entry through voltage-gated calcium channels, leading to alterations in the signaling of the activitydependent transcription factors nuclear factor of activated T-cells (NFAT) and cAMP response element-binding protein (CREB; Luoma et al., 2012). This effect of progesterone on calcium signaling provides a putative mechanism for its neuroprotective actions (Luoma et al., 2012). Furthermore, allopregnanolone is an allosteric modifier of the GABAA receptors expressed by oxytocin neurons (Koksma et al., 2003). Electrophysiological studies of hypothalamic oxytocin neurons showed that allopregnanolone acts via a G-protein mechanism involving protein kinase C to delay the closure of the Cl− channel after activation (Brussaard and Koksma, 2003), enhancing both the tonic and phasic actions of GABA in oxytocin neurons. This modulatory action on GABA transmission represents an alternative candidate pathway for the protective actions of this steroid.

Progesterone also plays an important role in the periphery. Progesterone-dependent immunomodulation is one of the mechanisms that enable pregnancy to proceed to term, because it protects the fetus from immunological rejection. Recent evidence suggests that autocrine/paracrine factors such as cytokines play a crucial role, possible as effectors of steroid hormones (Agrawal et al., 2011). A growing body of evidence implicates progesterone in the establishment of an adequate immune response during pregnancy (Hirsch and Muhle, 2002; Aisemberg et al., 2013).

Thus, progesterone and its metabolite allopregnanolone are important modulators of the immune responses in the periphery and within the CNS. Such effects are aimed to diminish the impact of an acute lesion or degenerative process in the nervous system while in the periphery contributes to the regulation of immune response against the fetus (Aisemberg et al., 2013).

#### **PROLACTIN ACTIONS ON CNS AND IMMUNE SYSTEM**

Prolactin is a peptide hormone secreted from the anterior pituitary into the circulation; it is thought to cross the blood–brain barrier and is known to regulate a wide variety of physiological process (Bole-feysot et al., 1998; Grattan and Kokay, 2008). PRL is also produced in a broad spectrum of extrapituitary sites including cells of the nervous and the immune system (Montgomery, 2001; Torner et al., 2002; Ignacak et al., 2012), and it is an important mediator of the immunoneuroendocrine network. However, effects of PRL on the immune system are complex. Removal of PRL by hypophysectomy impairs thymus growth (Nagy and Berczi, 1978) and immune reaction to immunogenic factors (Bernton et al., 1988) in rats. On the other hand, hyperprolactinemia (HP), in mice injected with *Listeria monocytogenes* increases mortality associated with impaired lymphocyte proliferation and decreased macrophage-activating factor production by T lymphocytes (Bernton et al., 1988). Furthermore, a PRL-like mRNA and a secreted product have been detected in human B-lymphoblastoid cell lines (Bernton et al., 1988;Baglia et al., 1991). Peripheral blood mononuclear cells also secrete a PRL-like protein, suggesting that it binds to PRL receptors and migrates to the nucleus, where it serves as a co-mitogen and autocrine regulator of cell growth. But, there are several reports showing that PRL is not essential for the proper development and function of the mouse immune system. Using PRL-deficient animals it was shown that PRL is not required for normal hematopoiesis (Horseman et al., 1997), and that PRL receptor signaling is not required for normal immunity (Bouchard et al., 1999). PRL is known to have other contradictory actions on the immune system that depends upon the concentration: it inhibits lymphocyte proliferation at high concentrations, while having enhancing effects at lower concentrations (Matera et al., 1992).

During pregnancy in rats, PRL plasma levels are high in the first half, they decrease until term, and then rise again at postpartum and throughout lactation (Neville et al., 2002). PRL actions in early gestation are crucial to prepare the mammary gland for lactation and in the CNS to establish the appropriate adaptive behavioral responses of the mother (Grattan and Kokay, 2008)

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toward the pups. PRL has been related to behavioral and neuronal effects, such as maternal behavior (Mann and Bridges, 2001), attenuation of anxiety and hormonal and neuronal responses to various stressors (Lightman et al., 2001; Torner and Neumann, 2002; Donner et al., 2007), neurogenesis (Shingo et al., 2003; Torner et al., 2009; Larsen and Grattan, 2010; Walker et al., 2012), and neuroprotection (Torner et al., 2009; Tejadilla et al., 2010).

Inflammatory response after a brain injury, such as proliferation and activation of glia is enhanced by PRL (Möderscheim et al., 2007), and elevated levels of PRL stimulate mitogenesis in astrocyte and oligodendrocyte populations of the subventricular zone (Larsen and Grattan, 2010). PRL also increases oligodendrocyte precursor cell proliferation, which in turn enhances the capacity to generate new oligodendrocytes and myelination. This process is associated with the capacity to repair white matter damage in the maternal CNS by increasing myelin basic protein expression (Gregg, 2009). During lactation, a physiological hyperprolactinemic state, there is a reduced sensitivity to kainic acid-induced cell damage in the dorsal hippocampus of the dam, showing that lactation is a natural model of neuroprotection (Vanoye-Carlo et al., 2008; Cabrera et al., 2009) in which PRL can participate. PRL systemic administration has been reported to protect the dorsal hippocampus of female rats against excitotoxicity induced by kainic acid administration, blocking cell loss and neurodegeneration, and diminishing the progression of kainategenerated behavioral manifestation of seizures (Tejadilla et al., 2010). Overall, these studies on protective effects of PRL within the CNS draw attention to the importance of studying females, and the relevance of neuroendocrine–immune interactions when investigating effects of this hormone.

An interesting example of the neuroendocrine–immune interactions is seen in the HP occurring in patients with autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus, and with organ-specific autoimmune diseases, as celiac disease, type 1 diabetes mellitus, Addison's disease, autoimmune thyroid diseases (reviewed by, De Bellis et al., 2005). In these diseases PRL increases the synthesis of IFNγ and IL-2 by Th1 lymphocytes. Moreover, PRL activates Th2 lymphocytes with autoantibody production. The inhibitory effects of IL-1ß on the tuberoinfundibular dopaminergic neurons that inhibit PRL secretion could explain this HP. González et al. (2004), showed that i.c.v. injection of LPS in rats, produces a decrease in tyrosine hydroxylase (TH) activity in the medial eminence, an increase in the serum levels of PRL, and a decrease in the number of TH- and TH mRNA-positive cells in the arcuate nucleus, indicating that dopamine neurons of the hypothalamus are functionally susceptible to local inflammatory stimuli. Additionally, treatment with the dopamine-agonist, bromocriptine, inhibits both PRL secretion and the severity of acute experimental encephalomyelitis (Riskind et al., 1991). This is an example of how molecules of the immune system could affect the neurons in the hypothalamus increasing the secretion of PRL, which in its turn will enhance peripheral inflammatory responses.

#### **CONCLUSION**

In summary, during pregnancy and lactation, responses of the HPA axis and the immune system are altered and clearly regulated by suckling and hormone fluctuation. Pregnancy and lactation are the most important periodsfor the conservation of the species, and they represent fundamental stages at which both mother and offspring must be protected. The immune system is crucial to protect the mother and the product against the environment and there is evidence supporting the notion that immunity is suppressed during motherhood. In this sense, the immune responses of the mother should be adjusted to conserve defenses, but should be tuned to preserve the developing offspring (Mor and Cardenas, 2010). Therefore, pregnancy and lactation are unique conditions in which the immune system is modulated or adjusted, but not fully suppressed.

#### **AUTHOR CONTRIBUTIONS**

Nela Monasterio conceived the main premises that are the focus of this review and wrote the largest portion of the paper; Edgar Vergara wrote important subsections of the paper; and Teresa Morales conceived the main premises that are the focus of this review, designed the structure of the paper, wrote a large portion of the paper, and edited the final version.

#### **ACKNOWLEDGMENTS**

The authors thank Dr. Dorothy Plessfor grammatical review. Cited work from our group was supported by PAPIIT-DGAPA, UNAM, and CONACYT, México. We are grateful to the reviewers whose comments help to improve this work.

#### **REFERENCES**

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 30 May 2013; paper pending published: 19 August 2013; accepted: 25 December 2013; published online: 17 January 2014.*

*Citation: Monasterio N, Vergara E and Morales T (2014) Hormonal influences on neuroimmune responses in the CNS of females. Front. Integr. Neurosci. 7:110. doi: 10.3389/fnint.2013.00110*

*This article was submitted to the journal Frontiers in Integrative Neuroscience.*

*Copyright © 2014 Monasterio, Vergara and Morales. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited andthatthe original publication inthis journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

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## The microbiota–gut–brain axis: neurobehavioral correlates, health and sociality

#### *Augusto J. Montiel-Castro1,2\*, Rina M. González-Cervantes 2,3, Gabriela Bravo-Ruiseco2 and Gustavo Pacheco-López <sup>2</sup>*

<sup>1</sup> Centro Darwin de Pensamiento Evolucionista and Philosophy Department, Social Sciences and Humanities Division, Universidad Autonoma Metropolitana Iztapalapa, Mexico City, Mexico

<sup>2</sup> Health Sciences Department, Biological and Health Sciences Division, Universidad Autonoma Metropolitana Lerma, Lerma, Mexico

<sup>3</sup> Biological Systems Department, Biological and Health Sciences Division, Universidad Autonoma Metropolitana Xochimilco, Mexico City, Mexico

#### *Edited by:*

Javier Velázquez-Moctezuma, Universidad Autonoma Metropolitana, Mexico

#### *Reviewed by:*

Toemme Noesselt, Otto-von-Guericke-University, Germany Richard Stevenson, Macquarie University, Australia

#### *\*Correspondence:*

Augusto J. Montiel-Castro, Health Sciences Department, Biological and Health Sciences Division, Lerma Campus, Universidad Autónoma Metropolitana, Av. de las Garzas No. 10, Col. El Panteón Lerma de Villada, Municipio de Lerma, Estado de México, C. P. 52005, Mexico e-mail: a.j.montiel@ centrodarwin-uam.mx

Recent data suggest that the human body is not such a neatly self-sufficient island after all.  It has recently become evident that such microbiota, specifically within the gut, can greatly influence many physiological parameters, including cognitive functions, such as learning, memory and decision making processes. Human microbiota is a diverse and dynamic ecosystem, which has evolved in a mutualistic relationship with its host. Ontogenetically, it is vertically inoculated from the mother during birth, established during the first year of life and during lifespan, horizontally transferred among relatives, mates or close community members. This micro-ecosystem serves the host by protecting it against pathogens, metabolizing complex lipids and polysaccharides that otherwise would be inaccessible nutrients, neutralizing drugs and carcinogens, modulating intestinal motility, and making visceral perception possible. It is now evident that the bidirectional signaling between the gastrointestinal tract and the brain, mainly through the vagus nerve, the so called "microbiota–gut–vagus–brain axis," is vital for maintaining homeostasis and it may be also involved in the etiology of several metabolic and mental dysfunctions/disorders. Here we review evidence on the ability of the gut microbiota to communicate with the brain and thus modulate behavior, and also elaborate on the ethological and cultural strategies of human and non-human primates to select, transfer and eliminate microorganisms for selecting the commensal profile.

**Keywords: microbiota–gut–brain axis, neurobiology, psychoneuroimmunology, evolutionary psychology, social bonds, kissing**

#### **INTRODUCTION**

There is hardly a place on earth without bacteria. They are found in every habitat imaginable: in every leaf in the lush Amazon forests; below scorching deserts' sands; within the coldest ice of the Antarctica; and even in the inhospitable environment of the ocean depths, under crushing pressures and in streams of boiling water. Not surprisingly, their realm includes the body surfaces and interior of animals, from minute crawlers to those with exceptional cognitive capacities like the human being. Recent genome sequencing projects suggest that most life forms share up to a third of their genes, and that those in humans show up to a 37% homology with those found in Bacteria and Archaea (McFall-Ngai et al., 2013). The sheer quantity of microorganisms inhabiting human bodies is enormous: more than 1000 different species have been found in a single sample (Relman, 2012). Such data have impacted our self-perception; From a viewpoint of the human body as a self-sufficient individual, to a perception of our bodies as super-complex ecosystems. This change of perspective has included a reappraisal of the role of microorganisms within our bodies (i.e., *endosymbionts*). While the popularly-held belief is that any microorganism found within the human body must have a detrimental effect on its health, emerging research has renewed an emphasis on the fact that many microorganisms have mutually beneficial relationships with their hosts (Archie and Theis, 2011), acting as a *probiotic*: a live microbe with a beneficial effect on the host via modifications of host-associated microbial communities, enhancing the host's response toward disease, its nutrient-exploitation capacity, or improving its environment (Verschuere et al., 2000). Recent research suggests how *microbiota*, i.e., a microbial community occupying a particular habitat (e.g., the *gut* microbiota), can serve its host by protecting it against pathogens, metabolizing complex lipids and polysaccharides that otherwise would be inaccessible nutrients, neutralizing drugs and carcinogens, modulating intestinal motility, and affecting visceral perception. Across evolution, endosymbionts have established important feedback channels with the central nervous system (CNS), some of which are crucial for maintaining homeostasis. For example, as microbial life was increasingly tolerated across generations of organisms, its presence has shaped the evolution of the immune system (Kelly and Mulder, 2012). The recognition that the gut microbiota influences several signaling pathways led to the suggestion of the concept of a microbiota–gut–brain

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(MGB) axis, a topic covered by extensive reviews (Rhee et al., 2009; Cryan and Dinan, 2012; Forsythe et al., 2012). The proposal of a MGB axis suggests that through a dynamic alignment, microbiota inhabiting the intestinal lumen affects its host's CNS activity (including vegetative and cognitive functions), and *vice versa* brain activity impacts microbiota development and composition. Clinical and experimental evidence indicate that this is also the case of human subjects, with such relationship playing a pivotal role in the development of metabolic and mental diseases. According to the World Health Organization, metabolic and mental disorders lead the global burden of disease, urging researchers and clinicians to set research priorities, and to governments, public agencies and private funds to apply urgent actions and investment (Mathers et al., 2008). In this regard, understanding the bidirectional signaling between the microbiota, gut and brain, underlie potential and significant impacts on global health, opening new preventive and therapeutic opportunities. Based on the above, the first section of our work provides an overview of the neurobiology supporting such interactions, focusing on key experimental and clinical data of the MGB axis and its potential impact on relevant metabolic and mental human disorders.

While recent years have witnessed an increasing interest in proximate questions regarding different aspects of microbiota, the coevolutionary interactions of animals and bacteria have been relatively unattended. This, in spite of the possibility that a focus on the evolution of the MGB axis could provide new theoretical frameworks for understanding complex evolutionary relationships involved in mutualisms between hosts and commensal bacteria. This includes the possibility that such mutualisms could influence the evolution of immunological systems, shape higher cognitive functions at the individual level, and work as a selective force promoting socialization and social structures, with an influence on the psychobiological basis of gregariousness, social perception, mate choice, and sexual behavior (Archie and Theis, 2011; Neuberg et al., 2011; Schaller, 2011). Therefore, our review is also aimed at understanding the relationship between the exchange of microbial-life among individuals and sociality. Clear suggestions in this regard have been advanced by Troyer (1984a) and Lombardo (2008). They have championed the hypothesis that social interactions may "tip" the precarious but crucial balance between the costs and benefits of group-living by providing an important but surreptitious benefit in the form of an exchange of mutualistic endosymbiotic microbes (e.g., as a defense against pathogens: Dillon et al., 2005). Thus, the next section, on the relationship between microbial-life and sociality, assumes a comparative viewpoint for evaluating such hypothesis. First, it begins by providing an overview of the evidence of symbioses across different animal taxa. It includes studies that either: (a) focus on the social aspect of endosymbiont-transmission or (b) describe whether an experimental intervention was used to clarify the degree by which the impairment of the vertical or horizontal transmission of endosymbionts may affect an organism's survival and/or reproductive success. Second, we suggest that the premises of Lombardo (2008) and Troyer (1984a) can be tested by means of the hypothesis that *the similarity of microbial communities across individuals is an index of the strength of their social bonds*. In our opinion, testing this hypothesis may add an important analytical tool to research focused on how social bonds (a social relationship defined by the degree upon which the exchange of any kind of information -or lack of it- has the potential to affect the survival and/or reproductive success of the individuals involved) translate into cooperation and cohesion at the group-level, an approach that could ultimately shed light on the origin and evolution of sociality (Dunbar and Shultz, 2010). We do this via a focus on the association between sociality and direct and indirect means of microbial-transmission in primates, with particular attention to mouth-to-mouth interactions. Finally, by means of an integrative perspective, the last section provides an overview of the different topics covered.

#### **MICROBIOTA–GUT–BRAIN AXIS AND ITS HEALTH IMPACT**

Multiple direct and indirect pathways maintain intensive and extensive bidirectional interactions between the gut microbiota and the CNS; involving endocrine, immune and neural pathways (Grenham et al., 2011), and form the basis of the so called MGB axis (**Figure 1**). For instance, under stress, the brain may influence the composition of the gut microbiota (Bailey and Coe, 1999) via the hypothalamus–pituitary–adrenal (HPA) axis, which regulates cortisol secretion, affecting immune cells activity; both locally in the gut and systemically. When the organism suffers an injury, the first immunological reaction is characterized by redness, pain and heat. These responses are constrained by neuronal regulation of inflammation process, carried out by the HPA axis via catecholamine (Sternberg, 2006) production. The necessary communication processes are based on neurotransmitters, neuropeptides, cytokines, hormones, growth factors (among others), which mediate the relationship between the immune system and the CNS. A feedback process leading to homeostasis (Downing and Miyan, 2000). Yet, disorders like stress (Glaser and Kiecolt-Glaser, 2005)can impact such equilibrium, leading to disease, allergic reactions, inflammatory disease and predisposition to infection. Additionally, cortisol can alter gut permeability and barrier function, and thus contribute to variations in gut microbiota composition (O'Mahony et al., 2011). Vice versa, experimental evidence indicates that the gut microbiota, and pre- and probiotic agents can alter the levels of circulating cytokines, which in turn can have a marked effect on several brain functions (Duerkop et al., 2009; Forsythe and Bienenstock, 2010). Additionally, both the afferent branch of the vagus nerve (Bercik et al., 2011a; Bravo et al., 2011) and modulation of systemic tryptophan, precursor of the neurotransmitter serotonin (Desbonnet et al., 2009), are strongly implicated in relaying the influence of the gut microbiota to the brain.

Experimental approaches on elucidating the MGB axis have so far included, the use of germ-free animals, animals with pathogenic bacterial infections, and animals exposed to probiotic agents or to antibiotics. For instance, germ-free mice have been used to assess neurodevelopmental effects of microbiota loss. Additionally, the administration of probiotic bacteria strains in adult animals or humans has been used to assess the effects of these bacteria on the host. On the other hand, infection studies have been used to assess the effects of pathogenic bacteria on brain and behavior, which are mediated largely through activation of the immune system. Finally, administration of antibiotics can disturb

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microbiota composition in a temporally controlled and clinically realistic manner and has therefore been a powerful tool to assess the role of the gut microbiota on behavior.

To date, studies investigating the effects of intestinal microbiota composition on brain function predominantly involve animal models of behavioral disorders such as anxiety, depression and cognitive dysfunction; however, accumulating evidence suggests that the composition of the gut microbiota may also have a role in several other metabolic conditions that involve the CNS (**Figure 1**). In addition, recent data have revealed that MGB axis has multiple effects on emotions, motivation and other higher and complex cognitive functions; reviewed elsewhere (Mayer, 2011). Such evidence suggests that various forms of subliminal interoceptive inputs from the gut, including those generated by intestinal microbiota, may even influence memory formation, emotional arousal, affective behaviors and decision making processes (Craig, 2002; Berntson et al., 2003). The human insular cortex and related brain networks (including the anterior cingulate cortex, orbitofrontal cortex and amygdala), have emerged as the most plausible brain regions to support this integration (Craig, 2009).

#### **MICROBIAL LIFE AND SOCIALITY**

#### **SYMBIOSES ACROSS ANIMAL TAXA**

Symbioses have played a substantial role in the development of animal life. In their interaction with the geosphere, they shaped the ancient biosphere in which multicellularity and animal life emerged (Hickman, 2005). Multicellular organisms may have evolved as new conflict-mediation mechanisms (i.e., genetic codes) restricted lower-level individual fitness, increasing that of new individuals at higher levels of biological organization (Michod, 2003). From such humble beginnings and over extended periods of time, a diverse array of cooperative and organized groups of individuals (i.e., societies), have evolved. Since transmission vectors may originate across both the physical and the social environment, microbial communities found in an organism are dependent on its geoecology, physiology, and genotype, but also on the intensity of its social relationships (Archie and Theis, 2011). The following paragraphs provide an overview of selected research describing symbioses between microorganisms and different animal taxa.

Microbial endosymbionts have played an important role in the evolutionary and developmental modification of tissues and organs of several marine or *aquatic invertebrates*, for example in the construction of mineralized exoskeletons (Hickman, 2005). Hickman (2005) points out the example of sponges. This group acquires large symbiotic and diverse bacterial communities through vertical transmission, which perform functions like nutrient acquisition, stabilization of the sponge's skeleton, processing of metabolic waste, and production of important secondary metabolites. The phyla with the largest biomass (e.g., arthropods) are

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also those with more symbioses reported (McFall-Ngai, 2005). McFall-Ngai (2005) suggests that a crucial difference between vertebrates and invertebrates is based on the relationship between the immune system and its association with microbial life. On the one hand, invertebrates rely on an "innate immune system" consisting of a germline-encoded receptor system associated to cells like macrophages or epithelia. On the other hand, in addition to the innate immune system, vertebrates possess a "combinatorial" immune response (using T-cells and a major histocompatibility complex) which may have evolved as a more "permissive" form of association with long-term endosymbiont microbial consortia, but also as an improved capacity for distinguishing "friend from foe," leading to the identification of *specific* pathogens (McFall-Ngai, 2005). However, some recent studies suggest that these differences may be less clear-cut than previously thought. For instance, a study in *Daphnia magna* (an aquatic crustacean), found that, compared to those experimentally challenged with a different strain, individuals exposed to the specific strain of the pathogen to which their mothers had been exposed had better fitness, suggesting that some invertebrates may have some kind of specific adaptive immunity (Little et al., 2003). One of the best examples of a beneficial symbiotic-relationship in an aquatic invertebrate is the case of the mutualism between the squid *Euprymna scolopes* and the luminous, symbiotic bacteria *Vibrio fischeri*. This symbiosis is maintained by means of cyclic transmission, where the bacterial symbionts must be acquired from the environment each generation (McFall-Ngai, 1998). *V. fischeri* is first acquired directly from the environment, but then, the light organ of *E. scolopes* undergoes specific metamorphic changes that maintain the symbiosis (McFall-Ngai, 1994). Such relationship helps *E. scolopes* generating bioluminescence, camouflaging it from both prey and predators by eliminating the projection of its shadow (Ruby and McFall-Ngai, 1992). Finally, a study by Verschuere et al. (2000) suggests that *Artemia spp*. is protected from the pathogenic effects of *Vibrio proteolyticus* by specific bacterial strains.

Perhaps the best-studied examples of symbioses and transmission of microbiota is found among social *insects*. The phenomenon received close attention as a model for the study of the origins of sociality, permeating both scientific and popular accounts of the human society during the nineteenth and twentieth centuries (Sleigh, 2002). The niche of the hymenoptera, including a subterranean way of life, large biomass density at the nest, and frequent direct individual contact, can all make them particularly vulnerable to pathogens via a fast-spread of disease among conspecifics (Kaltenpoth and Engl, 2013). Therefore, based on selection at the individual and colony-level, social insects have developed different forms of prophylactic and active responses against some parasiterelated costs of social-living, such as: environmental parasite uptake, parasite intrusion (i.e., into a colony), parasite establishment and spread within the colony, and transmission between colonies (Cremer et al., 2007). These responses are referred to as "collective" or "social immunity" by Cremer et al. (2007). Among them is also the possibility for the social-transmission of beneficial microbiota. Recently, Koch and Schmid-Hempel (2011) suggested that both honey and bumble bees present bacterial communities not found among solitary species which, importantly, protect them against a virulent and naturally occurring parasite (*Crithidia bombi*). In an experimental setting, they demonstrate that in order to observe the protective effect of microbiota, individuals had to be exposed to feces from nest mates after pupal eclosion, providing strong evidence for an important benefit of the transmission of microbiota between individuals. On the other hand, Evans and Lopez (2004) suggest that nonpathogenic bacteria may have a positive effect on honey bee immunity, helping them to survive pathogen-infection across different life-stages. In turn, McFrederick et al. (2012) suggested that beneficial *Lactobacillus* found in bees were acquired by both vertical transmission or by contact with flowers, and that the *Lactobacillus* strains associated to Sweet bees could suppress mold-growth and other spoilage organisms at the nest. Other species of eusocial insects feed each other by regurgitation of liquid secretions originating in the crop or alimentary tract (Wilson, 2000), a phenomenon named *trophallaxis* by Wheeler (1918). In termites, trophallaxis allows for the social transmission of protozooans, which they lose after periodic molting but are crucial for the digestion of cellulose (Wilson, 2000).

Experimental studies focused on the transmission of microbiota have been practiced in a few non-eusocial insects. For example, germ-free desert locusts (*Schistocerca gregaria*) were associated with up to three species of locust gut bacteria and then fed with a pathogen (*Serratia marcescens*) by Dillon et al. (2005). Results of this study showed a negative relationship between the density of *Serratia marcescens* and the number of gut bacterial species present, as well as a negative relationship between bacterial community-diversity and the proportion of locusts harboring *Serratia*. A more recent study (Wang and Aksoy, 2012) investigated the role of *Wigglesworthia glossinidia* as an endosymbiont associated to the nutrition, fecundity, and development of the immune system in Tsetse flies (Diptera: Glossinidae). The study describes how the peptidoglycan recognition protein (PGRP-LB) is found only in adult flies (as an important component of the milk that nourishes developing progeny), and how the experimental reduction of PGRP-LB decreases female fecundity by damaging the transmission of *Wigglesworthia* through induction of an antimicrobial peptide (Attacin). The conclusion of Wang and Aksoy (2012) is that the transmission of PGRP-LB has a major role in the fitness of Tsetse flies by means of protecting such symbiosis.

There are fewer but equally interesting studies on this subject in fishes, amphibians, and reptiles. Coldwater *fish* appear to acquire their microbiota from the environment after hatching (Hansen and Olafsen, 1999), and there is some indication that different types of probiotic bacteria may have beneficial effects as biological control agents in aquaculture, including immune system improvements (Gómez and Balcázar, 2008) or enhancement of water quality (Verschuere et al., 2000). In the freshwater zebrafish (*Danio rerio*), an experimentally induced lack of microbiota arrests the development of the species' gut at specific points of differentiation, an effect than can, nevertheless, be reversed by the introduction of bacteria (Bates et al., 2006). In the case of *amphibians*, Walke et al. (2011) found that innate immune defenses with a beneficial effect on the inhibition of the fungal pathogen *Batrachochytrium dendrobatidis* can be vertically transmitted. Their work found that both antimicrobial skin peptides

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and mutualistic microbiota found in the adult Panamanian "glassfrogs" of the species *Hyalinobatrachium colymbiphyllum*, could be transmitted to embryos, with a possible role of different types of physical contact as a means for this transmission, including female-eggs contact during deposition, and/or male urination on the egg-clutch. Another study by Troyer (1984b) focused on the horizontal acquisition of microbiota in *reptiles*. She investigated how green iguana (*Iguana iguana*) hatchlings employ a significant amount of time in acquiring microbiota before fully exploiting the food resources in their habitat. They do this in a three-step process. First, before having a fully functional digestive activity, they consume soil from within the nest chamber, by which they increase their hindgut microbial populations. Then, up to a week after hatching, they leave the nest and begin eating both plants and soil. Finally, between 2 and 3 weeks after hatching they leave the area around the nest, associate with other conspecifics and eat the feces of older individuals, gaining access to more complex microbial communities.

In comparison, Kohl (2012) presents a thorough review of the many aspects by which microbial communities influence nutrition, development, immunity, and processing of toxins in many species of *birds*. One interesting aspect observed by Kohl is that the symbiotic relationships between birds and microbiota can be, on some occasions, extraordinarily similar to those found in the relationship between mammals and their endosymbionts, while on other instances, they are just slightly distinguishable, by a few, nevertheless remarkable, aspects. A particularly interesting study (Kyle and Kyle, 1993) described by Lombardo (2008) observed that food-provisioning by itself was insufficient for enhancing the survival of orphan chimney swifts nestlings. To achieve this objective, food needed to be coated with the saliva of adults. While all nestlings younger than 6 days receiving food that was not covered by an adult's saliva died, a high proportion of those that received the saliva-covered food survived.

There is also a vast literature describing the influence of microbiota on a variety of *mammals* (a portion of it reviewed by Lombardo, 2008). Therefore, as a preface for the following subsection, next we focus solely on aspects of the relationship between kinship, lactation and microbiota in human and non-human primates. Several studies have examined the bacterial microbiota of breast-fed and bottle-fed human infants using both conventional plating and molecular techniques. These studies have shown that the large gut microbiota of breast-fed infants is generally dominated by bifidobacteria and lactic acid bacteria, both considered beneficial (Penders et al., 2006). In contrast, the gut microbiota of formula-fed infants is more diverse, but less stable, often containing more *Bacteroides*, *Clostridium* and *Enterobacteriaceae*. Early start of feeding formula milk changes the composition of the intestinal-microbiota, promoting colonization by obligate anaerobes such as the *Clostridium coccoides* group, the *Clostridium leptum* subgroup, *Prevotella* and *Atopobium* cluster during the 3 months after birth (Tsuji et al., 2012). Human milk is a complex bio-fluid containing mainly lactose, lipids, and protein. However, it is not widely recognized that, after lactose and lipids, oligosaccharides are the third largest solid component of human milk. The majority of this type of sugars is not digestible by human infants, instead, their main function may be related to their interaction or

support of intestinal microbiota. Oligosaccharides in human milk encourage the growth of beneficial bifidobacteria in the colon, while they also bind competitively to cell adhesion receptors. This binding may prevent pathogen-binding to intestinal epithelial cells and thus pathogenesis. Analysis of the oligosaccharides in human milk resulted in 200 different molecules ranging in size from disaccharides up to approximately 22 residues (Ninonuevo et al., 2006). In this sense, through lactation, mothers provide food for both their infants and the bacteria helping assimilating milk's nutrients, allowing for the continued inoculation and establishment of infant's microbiota (Hinde and German, 2012). The importance of this "triangled" relationship is highlighted by the seminal work of Bailey and Coe (1999), who observed that due to strong emotional stress, the disruption of the mother-infant bond (i.e., by separation from the mother) in rhesus monkeys (*Macaca mulatta*) altered the composition of infants' gut microbiota, increasing their vulnerability to disease. The study of Bailey and Coe (1999) represents an important developmental piece of evidence of a health-related *cost* (i.e., increased vulnerability to disease) observed when the composition of microbiota is altered due to the interruption of microbial-transmission (i.e., by a disruption of the mother-infant bond).

Turnbaugh et al. (2009) found no significant correlations between separation of family members and the degree of similarity between their gut microbiota, as well as no significant differences in the composition of gut microbiota between monozygotic and dizygotic twins, suggesting a likely genetic factor underlying such commonalities. On the other hand,Yatsunenko et al. (2012), characterized bacterial species in fecal samples from 531 individuals from different nationalities. Groups included healthy children and adults from the Venezuelan Amazonas, rural areas of Malawi and US metropolitan areas, and included mono and dizygotic twins. An effect of kinship on gut microbiota was found across countries, focusing the discussion on how differences in social structures may influence the extent of vertical transmission of the microbiota and the flow of microbes among members of a group of people or family. Importantly, their results showed that the phylogeny of fecal microbiota of monozygotic twins was no more similar than the microbiota of dizygotic twins in all age groups tested. Likewise, there were no significant differences in the degree of similarity between the fecal microbiota of mothers and their teenage offspring, nor between teens and their biological fathers. Moreover, the microbiota of co-habiting couples was more similar to each other than to members of other households (Yatsunenko et al., 2012). These results, consistent across the populations studied, suggest that, as in other species, endosymbionts may have an important role in kin-recognition in humans.

#### **THE ASSOCIATION BETWEEN MICROBIAL TRANSMISSION AND SOCIAL BONDING IN PRIMATES**

In the case of primates, a behaviorally and cognitively complex social life has evolved across a range of group sizes and social structures as a prerequisite for individual's development, survival, and reproduction (Mitani et al., 2012). Either in the form of predator deterrence (e.g., Zuberbühler et al., 1997), cooperative breeding (e.g., Burkart and van Shaik, 2010) or group hunting (e.g., Watts and Mitani, 2002), primates use group-level cooperative strategies

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that in general enhance individual fitness. However, sociality also involves important costs, such as within-group competition for resources (van Schaik and van Noordwijk, 1986) or reduction of reproductive output due to socially induced stress (Dunbar, 1980; Altmann et al., 1988). Thus, a balance between the costs and benefits of social-life is by no means an easy task for primates. By increasing the frequency of contact and proximity between individuals, species living at higher densities, in larger groups, or with promiscuous mating are thought to be the most vulnerable to infection (Altizer et al., 2003). Nonetheless, primates exhibit cohesive and sometimes large groups with strong and long-lasting social bonds (Mitani, 2009) of a kind that, in other animal orders, are almost exclusively found among pair-bonded species (Shultz and Dunbar, 2007). These two observations are somewhat incompatible. However, this apparent paradox could be solved by recognizing the role of partner-choice mechanisms in the structuring of primate societies (Hinde, 1983a). Research suggests that endosymbionts are at the base of different mechanisms for individual recognition and partner selection (Archie and Theis, 2011) underlying social-bonding and the formation of cliques within larger social structures. When partner choice is exerted, not all subjects interact with all other group members, and thus, social bonds (or its absence) may act as a social barrier to pathogen transmission (Loehle, 1995). Hence, if endosymbionts' transmission is of any benefit to sociality, transmission should be facilitated as social bonds between individuals are stronger, and made more difficult as species use more frequent or more complex partner-selection mechanisms. As partner-choice limits the size of each individual's social network (Kudo and Dunbar, 2001), the increasing risk of pathogen transmission associated to large groups may set an upper limit to total group size (Freeland, 1976, 1979; Coté and Poulin, 1995; Bonds et al., 2005), leaving the beneficial link between endosymbionts and complex sociality (*sensu* Lombardo, 2008) intact in the form of bonded relationships. For example, the large social groups of the Hamadryas baboon (*Papio hamadryas*: Schreier and Swedell, 2009), classified as a multi-layered, fission-fusion society (Mitani et al., 2012), is a good example of the way social contact is less intense the larger a social-unit is, providing "borders" to microbial-transmission (**Figure 2**). Considering the role of social structure as a barrier against parasite transmission may explain why larger but more subdivided groups, tend to slow the spread of infectious diseases (Griffin and Nunn, 2011; Nunn, 2012), and connect the evolution of the MGB axis to primate sociality. This suggests that the *quality* of social relationships between subjects, but not necessarily the size of their social groups, should be associated to more frequent or direct mechanisms underlying endosymbionts' transmission between individuals.

#### *Indirect mechanisms*

In indirect mechanisms of microbial transmission, inoculation is somehow mediated (e.g., by either a free-living pathogen, inanimate environmental features or by another infected host species; Cortez and Weitz, 2013). *Coprophagy* and *urine ingestion* are good examples of indirect mechanisms: often considered abnormal behaviors appearing due to stress (e.g., Baker and Easley, 1996; Nash et al., 1999), they promote beneficial

mutualisms required for digestion of plant materials (as in colobus, howler monkeys and gorillas: Milton, 1981; Lambert, 1998; Graczyk and Cranfield, 2003). Even if they exacerbate exposure to parasites, risks can be circumvented by selectively ingesting excretes of relatives while avoiding those of parasitized conspecifics (Lombardo, 2008); two strategies made possible by the widespread individual-recognition capacities of primates (Langergraber, 2012).