Vagus Nerve through α7 nAChR Modulates Lung Infection and Inflammation: Models, Cells, and Signals

Cholinergic anti-inflammatory pathway (CAP) bridges immune and nervous systems and plays pleiotropic roles in modulating inflammation in animal models by targeting different immune, proinflammatory, epithelial, endothelial, stem, and progenitor cells and signaling pathways. Acute lung injury (ALI) is a devastating inflammatory disease. It is pathogenically heterogeneous and involves many cells and signaling pathways. Here, we emphasized the research regarding the modulatory effects of CAP on animal models, cell population, and signaling pathways that involved in the pathogenesis of ALI. By comparing the differential effects of CAP on systemic and pulmonary inflammation, we postulated that a pulmonary parasympathetic inflammatory reflex is formed to sense and respond to pathogens in the lung. Work targeting the formation and function of pulmonary parasympathetic inflammatory reflex would extend our understanding of how vagus nerve senses, recognizes, and fights with pathogens and inflammatory responses.


Introduction
From 2000, Tracey and colleagues have been working on the mechanisms by which electric stimulation of vagus nerve suppresses activation of NF-B and production of proinflammatory cytokines in 7 nicotinic acetylcholine receptor-( 7 nAChR-) expressing macrophages and lessens severity of sepsis in animal models [1,2]. These findings led to establishment of a novel theory of cholinergic anti-inflammatory pathway (CAP) [1]. Most experiments regarding the modulatory effects of CAP on inflammation were tested in the models of sepsis, a syndrome of systemic proinflammatory responses. The experimental results support that spleen is the functional hub of CAP. In 2011, Tracey and colleagues found that innervation of vagus nerve in the spleen, CHAT-expressing T lymphocytes, and 7 nAChR-expressing macrophages forms a neural circuit to finely tune the proinflammatory responses [3]. In this review, we summarized the progress regarding the modulatory effects of CAP on inflammation and pointed out the future research directions towards brain center of CAP, activation of 2 adrenergic receptor, synthesis of acetylcholine in the T lymphocytes, and others.
ALI is a devastating inflammatory disease [4]. It is pathogenically heterogeneous and involves many cells and signaling pathways. From 2007, the modulatory effects of CAP have been tested in a variety of animal models with ALI [5]. Activation of CAP also affects many types of cells and signaling pathways involved in ALI. In this review, we compared the deferential pulmonary inflammatory responses during sepsis (systemic) and ALI (local). Considering different features of modulatory effects of CAP on pulmonary inflammatory responses, we put forward a new working model, pulmonary parasympathetic inflammatory reflex, to extrapolate how vagus nerve through 7 nAChR modulates acute lung infection, inflammation, and injury. The pulmonary parasympathetic inflammatory reflex works locally and may not require spleen. In accordance with this assumption, vagus nerve coupling with 7 nAChR-expressing resident macrophages also modulates intestinal inflammation independent of spleen [6]. Thus, future studying local modulatory effects of CAP on inflammation may be an emerging avenue to explore how vagus nerve senses, recognizes, and responds to pathogens.

Inflammatory Reflex and CAP.
Studies suggest that afferent and efferent vagus nerves, 7 nAChR-expressing inflammatory cells, and central vagal nucleus in the brain form an inflammatory reflex that could finely tune inflammation and immunity [2,7]. Strictly speaking, CAP is the efferent arm of vagal inflammatory reflex and spleen may be the antiinflammatory hub in this neural circuit [8][9][10]. Activation of this pathway provides the host with a fast, discrete, and localized means of modulating the inflammatory and immune responses in variety of animal models [11][12][13].

The Role of Spleen in Inflammatory
Reflex. As Figure 1 shows, in the vagal inflammatory reflex, the sensory neurons may sense the changes of pathogen associated molecular patterns (PAMPs) or damage associated molecule patterns (DAMPs) in the peripheral afferent vagal nerve endings and then feedback to nucleus tractus solitarii (NTS) in the brain stem. After the information is processed in the NTS, the efferent vagus nerve transmits integrated information by action potentials to the celiac ganglion and then delivers in the spleen. Anatomically, the splenic vagus nerve endings are closely in contact with a group of 2 adrenergic receptor-( 2 AR-) expressing T memory lymphocytes (CD4 + CD44 high CD62L low ) and release norepinephrine (NE), a sympathetic neurotransmitter. NE activates 2 AR in the T lymphocytes, initiates transcription of choline acetyltransferase (ChAT), and synthesizes acetylcholine (ACh). ACh could activate splenic 7 nAChR-expressing macrophages, inhibit NF-B activity, promote STAT3 phosphorylation [14], and therefore dampen proinflammatory cytokine production (especially TNF-and HMGB1) [2,3,12,15,16].
We have to point out that Figure 1 is a hypothetical model that shows a direct connection of the efferent vagus nerve to the spleen via the celiac ganglion [3]. However, a recent finding has demonstrated there was no neural connection from the vagus to splenic sympathetic by neuroanatomical tract tracing and neurophysiological measurements [17]. Moreover, one study has showed that sympathetic nerves rather than vagi contribute to anti-inflammatory effects revealed by a LPS-challenged splanchnic-nerve or vagus nerve-cut rat model to compare changes of splanchnic sympathetic nerve activity and peripheral blood TNF- [18,19]. But this study has its limitations, for example, short experimentation (1-2 h), small sample size, and unknown mechanisms. More recently, Torres-Rosas et al. have found that electroacupuncture controls systemic inflammation in BioMed Research International 3 sepsis via the sciatic and vagus nerves and catecholamines from the adrenal glands [20]. Therefore, the controversy regarding anti-inflammatory roles of vagus and sympathetic nerves should be sorted out in the future study.

Information-Integrating Center of Inflammatory Reflex.
It should be mentioned that vagus nerve originates from medullar oblongata, which consists of four nuclei: dorsal nucleus, nucleus ambiguous, NTS, and spinal nucleus of trigeminal nerve [21,22]. About 80% afferent sensory fibers are contained in the vagus nerve and responsible for transmission of the information to the NTS [21]. For example, animals were intravenously or intraperitoneally challenged with LPS or IL-1 could induce c-fos expression in the nodose ganglia and the NTS [23,24]. NTS also plays a very important role in projecting information to the nuclei (including the locus ceruleus and dorsal raphe nuclei) of the brain [21]. It is unknown whether and how PAMPs or DAMPS can be recognized by the afferent vagus nerve endings in the lung and how NTS processes the information that is collected from the lung during infection and inflammation.

Transcription of ChAT in 2 AR-Expressing T Lymphocytes.
Upon the vagus nerve stimulation, transcription of ChAT gene in the splenic 2 AR-expressing T lymphocytes might be regulated by cAMP, which is a major second messenger following activation of 2 AR [25,26]. However, study has also shown that stimulation of efferent vagus nerve induces plasma norepinephrine via the 7 nAChR in a mouse model [27]. This finding raises a possibility that transcription of ChAT and biosynthesis of ACh in the splenic lymphocytes are positively regulated by 2 AR or 7 nAChR. Experimental data have demonstrated that 2 AR-expressing CD3 lymphocytes and 7 nAChR-expressing CD11b/c monocyte or macrophages are present in the spleen. The defects in response to 2 AR or 7 nAChR stimulation or quantity in the spleen lead to the dysfunction of inflammation resolution and postoperative cognition decline [28]. In addition, the 2 agonist is well recognized for its anti-inflammatory property for ALI [29,30]. Whether this protective effect of 2 agonist on ALI [31] is via activation of splenic 2 AR-expressing T lymphocytes or CAP requires to be investigated.

Synthesis of Acetylcholine in Nonneuronal Cells.
It needs to emphasize the important role of high-affinity choline transporter (CHT1) or choline transporter like proteins (CTLs) in the process of ACh synthesis in the nonneuronal cells (e.g., lymphocytes and lung cells) [32][33][34]. ACh is synthesized from choline and acetyl-CoA by the enzyme choline acetyltransferase (ChAT), and this event may be limited by choline availability [35]. In neurons, loss of CHTmediated presynaptic choline uptake might result in neonatal lethality [36]. ChAT contains nuclear localization signals and is also localized in the nuclei of neural and nonneuronal cells [37]. Enzymatic activity and nuclear translocation of ChAT are required for its transcriptional enhancement of CHT gene [37]. Pulmonary nonneuronal cholinergic system (including ChAT-, CTLs-, VAChT-, and OCT-mRNA) is downregulated in acute allergic airway inflammation [35], suggesting that synthesis of ACh is regulated locally during inflammation.

Acute Lung Inflammation and Injury and Modulatory Effects of CAP
3.1. Acute Lung Inflammation and Injury. Adult respiratory distress syndrome (ARDS), characterized by ALI, has a mortality of 40% even if the patients receive advanced intensive care [4]. Pneumonia, severe sepsis, and acid aspiration are the most serious causes of ARDS [4,38,39]. Gram-negative sepsis derived ALI is characterized by neutrophil alveolitis and increased permeability of the lung microvascular endothelial and alveolar epithelial barriers [40][41][42]. Aspiration of gastric contents is reported to be associated with a 26-36% incidence of ARDS [43,44]. Aspirated hydrochloric acid may evoke direct damage to the alveolar-capillary membrane and promote adhesion, activation, and sequestration of neutrophils.

Direct and Indirect Animal Models of Acute Lung
Inflammation and Injury. Alveolar epithelial cells are the main target cells in the epithelial respiratory compartment exposed to noxious substances such as E. coli or acid [45]. Injury to the alveolar epithelial barrier is a major determinant of severity of clinical ALI [46,47]. Our experiments have demonstrated that, at the same dosage, intratracheal challenge of E. coli could induce much severe lung inflammation than intraperitoneal challenge of E. coli. As Figure 2 shows, mice were divided into three groups: control group received PBS; E. coli pneumonia group received an intratracheal challenge of E. coli (10 7 cfu); E. coli peritonitis ALI group was given an intraperitoneal challenge of E. coli (10 7 cfu). All mice were also given I 125 -albumin intratracheally or intravenously to measure lung wet-to-dry weight ratio and lung epithelial and endothelial permeability as previously reported [5,48]. At 4 h after challenge, three parameters were markedly higher in the E. coli pneumonia compared to E. coli peritonitis ALI.

Category of ALI.
ALI experimental models can be categorized into direct and indirect lung injury based on the route of insults. Acid-induced ALI, LPS-induced ALI, E. coli pneumonia, and other experimental ALI models were considered direct models because the injurious agents (such as HCl, bleomycin, endotoxin, E. coli, and influenza virus) were instilled into the air spaces with initial direct contact with pulmonary epithelium [5,[49][50][51]. Ventilatorinduced ALI caused by overstretch of lung epithelial cells is also considered as direct lung injury [52][53][54]. Thioureainduced lung vascular injury [55], oleic acid-induced ALI [56,57], peritonitis-induced ALI (including cecal ligation and puncture (CLP)) [16], and transfusion-related ALI (TRALI) (by intravenous MHC I monoclonal antibody) [58] were considered as indirect models because the injurious agents initially interacted with the lung endothelium after intravenous challenge [55].  on proinflammatory cytokines also alter when the challenge route of pathogens is different. Numerous studies have demonstrated that TNF-is a proinflammatory cytokine and is well regulated by CAP. The spleen is identified as the source of 90% of the serum TNF during endotoxemia and in particular the marginal zone-and red pulp-macrophages of the spleen [10,59]. Compartmentalization of TNFin the blood or alveolus is dependent on route of LPS challenge. For example, intravenous endotoxin significantly increases TNF-production in the spleen by a factor of 30 as compared with six-and twofold increases in the lung and liver, respectively. Vagus nerve stimulation significantly reduces TNF levels in the spleen (94%) and liver (40%) but not in the lung (20%). However, in a lung injury model by an intratracheal challenge, compartmentalization of TNFin alveolus is preserved before alveolar-capillary injury [60]. Once compartmentalization of alveolar TNF-is lost, injured lung may contribute to a systemic inflammatory response and subsequent multiorgan failure [60]. Similarly, intratracheal LPS induced a significant increase in MIP-2 in BAL fluid, whereas MIP-2 in the plasma was not detectable. In contrast, intravenous LPS caused a marked increase in plasma MIP-2, whereas only a small elevation of MIP-2 concentration in BAL fluid was observed [61]. In a LPS-induced ALI (intratracheal), administration of 7 nAChR agonists could inhibit NF-B activity in the BAL proinflammatory cells and reduce both TNF-and MIP-2 levels in the BAL [5]. Vagotomy and deficiency of 7 nAChR worsen lung inflammation [5,50]. (Table 1). By analyzing Table 1, we can conclude that activation of CAP might affect the development of lung infection, inflammation, or injury in a PRR-(pattern recognition receptors-) dependent manner. For example, nicotine administration worsens Gram-positive bacterial pneumonia (TLR2) [50] and influenza viral pneumonia (TLR3, TLR7, or RIG-I-MAVS) [62][63][64] but improves Gram-negative bacterial pneumonia or LPS-induced ALI (TLR4) [65]. It has to be noted that activation of 7 nAChR universally suppresses TLR2, TLR3, TLR4, or TLR9 agonist (rather than live pathogens) induced TNF-production in monocytes [66]. These findings suggest that vagus nerve through 7 nAChR responds to PAMPs or DAMPs differently. Therefore, vagus nerve may play pleiotropic roles in modulating lung infection and inflammation.

Opposite Effects of CAP on Lung Infection and Inflammation.
The discovery that splenectomy inactivates CAP strongly supports that spleen determines the function of CAP [9]. In the splenectomized animals, nicotine therapy worsens animals with lethal polymicrobial sepsis [9]. This finding suggests that once CAP is dysfunctional, activation of 7 nAChR would paradoxically compromise immunity and worsen lung infection. Traumatic brain injury or stroke might cause functional impairment of CAP and activation of 7 nAChR worsened Gram-negative bacterial pneumonia [59,67].  (ii) Vagal denervation increased MIP-2 production and airway neutrophil accumulation and increased mortality.
(iii) 7 nAChR deficient mice developed severe lung injury and had higher mortality compared with wildtype mice.
Protective [50] Gram-negative Stroke, then IT PA Direct: lung epithelial cells (i) Exacerbation of P. aeruginosa-induced lung injury and mortality by prior stroke is reduced by loss of 7 nAChR receptors.
(ii) Genetic deletion of the 7 nAChR attenuates the effect of stroke on bacterial clearance in P. aeruginosa pneumonia.
(iii) Pretreatment with PNU-282987, a pharmacologic activator of the 7 nAChR significantly increased lung injury caused by P. aeruginosa pneumonia, significantly decreased the release of KC, a major neutrophil chemokine, and significantly decreased intracellular bacterial killing by a mouse alveolar macrophage cell line and primary mouse neutrophils.

Streptococcus pneumoniae
IT Direct: lung epithelial cells (i) Nicotine treatment was associated with a transiently enhanced growth of S. pneumoniae in both lungs and blood.
(ii) Mice treated with nicotine showed enhanced lung inflammation at 24 h after infection.
(iii) Both lung and plasma concentrations of the proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma were higher in nicotine-treated animals at this time point.
(ii) Nicotine administration increased lung PMN infiltration and mortality.
VNS: protective Nicotine: worse [103] 6 BioMed Research International and KC compared to sham), and resulted in decreased pO 2 (compared to sham-operated animals).
(ii) VNS did not affect any of the studied parameters in both SP and MV animals.
(iii) MV with moderate tidal volumes potentiates the pulmonary inflammatory response elicited by systemic LPS administration.
(iv) No beneficial effects of vagus nerve stimulation performed following LPS administration were found.
VNS is protective in LPS challenge but not in LPS + MV model [104] Oleic-acid induced ALI (dogs) [49,57] Oleic acid IV Indirect: lung endothelial cells (i) In the dogs with normal lungs, bilateral vagotomy per se did not cause lung injury during 3 h of observation.
(ii) In oleic acid-induced ALI, vagotomy significantly deteriorated pulmonary edema by increasing pulmonary intravascular pressures.
(iii) Inhibition of vagal or sympathetic innervation will aggravate pulmonary edema in the dog.

Pulmonary Parasympathetic
Inflammatory Reflex 4.1. Vagus Nerve Helps Sensing, Recognizing, and Responding to Pathogens. We have to mention that classical CAP theory was mostly tested in the experimental models of sepsis (intravenous LPS) or CLP peritonitis animal models in which spleen is required for dampening inflammation; however, these models only present mild lung inflammation which is manifested by less impressive intra-alveolar inflammation and hyaline membrane formation [68]. Different from the regulatory effects of the classical CAP on sepsis, vagus nerve might modulate lung infection and inflammation using new machinery: pulmonary parasympathetic inflammatory reflex [69], and the spleen may not be involved in this regulatory mechanism. As illustrated in Figure 3, the pulmonary parasympathetic inflammatory reflex may consist of three components: the afferent arc residing in the distal airway or alveolus; the NTS information-integrating center in the brain stem; and the efferent arc innervating the distal lung epithelial cells. Vagus nerve endings are reported to innervate the distal airway of the lung, possibly in the alveoli [70,71] (though it is entirely unclear how efferent fibers traveling in the vagus nerve might exert influence upon the alveolar region), where varieties of sensors or PRR in the vagal afferent arc are located. Via this apparatus, mechanical, chemical, biological, and other stimuli in the alveoli can be sensed. Sensory neurons express TLR3, 4, 7, and 9, which can recognize different pathogens [72][73][74]. Lung neuroendocrine cells also are complex airway sensors, which are predominantly innervated by vagal afferent fibers derived from the nodose ganglion [75]. The information is transmitted via the afferent arm to NTS, a processing center, which is capable of differentiating types of infection, inflammation, or challenges. After processing, the active potentials are remitted from NTS to the alveoli via the vagal efferent arc. The vagal nerve endings could synthesize and release ACh, which in turn activates 7 nAChR in the proinflammatory cells (e.g., macrophages and neutrophils) or epithelial cells to regulate the production of proinflammatory cytokines via NF-B or other signaling pathways.

Recruitment of 7 nAChR-Expressing Cells and Nonneural
ACh in the Lung. During lung infection and inflammation, alveolar macrophages produce MIP-2, a key chemokine, which could attract neutrophil migrating into the alveoli [76]. These infiltrated neutrophils also express 7 nAChR. Apart from release of ACh from the vagal nerve endings in the distal airway, lung epithelial cells, immune cells, and neuroendocrine cells also produce nonneuronal ACh [32,35,77]. 7 nAChR in bronchial epithelial cells can be upregulated by the stimulation of nicotinic agonists [78,79]. This positive feedback between acetylcholine and 7 nAChR might facilitates maintenance of concentration of acetylcholine in the alveoli.

The Role of Local CAP Is
Emerging. In addition, the theory of classical CAP has also been challenged by recent researches. In a rat model, one group reported that vagal efferent neurons in the rat neither synapse with splenic sympathetic neurons nor drive their ongoing activity using vagal terminals anterograde and Fast Blue labeling technology and electrophysiological stimulation [17]. A recent study has shown that gastrointestinal CAP plays a protective role in a mouse of postoperative ileus [6]. In this study, denervation of spleen and depletion of T lymphocytes could not deactivate the protective property of vagus nerve stimulation. Anterograde labeling revealed that vagal efferents closely make contacts between cholinergic myenteric neurons and resident 7 nAChR-expressing macrophages. Therefore, the protective effects are attributable to local vagal nerve innervation and resident macrophages independent of spleen [6].  (Table 2).

Modulatory Effects of Activation of 7 nAChR on Inflammatory Cells May Be Dynamic.
On average, 20-25% of total cells are 7 nAChR-expressing cells in the bone marrow (BM), blood, spleen, lymph nodes, and Peyer's patches [82]. Lung 7 nAChR + Gr1 or 7 nAChR + CD11b + granulocytes (neutrophils and monocytes) were increased to 40% after being infected with E. coli [50], suggesting that more granulocytes migrate into the lung. In addition, vagus nerve stimulation significantly attenuates CD11b + cells in the spleen during sepsis [83]. These findings support that 7 nAChRexpressing proinflammatory cells can dynamically migrate among lung, spleen, and other organs during different stages of inflammation [84]. This dynamic movement of 7 nAChRexpressing cells might facilitate them being activated by acetylcholine released from the vagus nerve.

Signaling Pathways Involved in Acute Lung Inflammation.
It was reported that activation of p38 MAPK, AKT1, and NF-B in neutrophils contributes to ALI [85]. Lack of AKT1 could worsen acid, LPS, or bacteria induced acute lung infection and inflammation [86][87][88]. LPS activates the STAT kinases, Src, and JAK. LPS treatment could activate STAT3 in the resident lung cells and recruited inflammatory cells [89]. In a rat model of intrapulmonary deposition of IgG immune complexes, STAT3 activation was dramatically suppressed by depletion of neutrophils or lung macrophages, resulting in reduced gene expression of IL-6 and IL-10 in whole lung tissues [90].

Modulatory Effects of Activation of 7 nAChR on Sig-
naling Pathways. Activation of 7 nAChR in macrophages, monocytes, and other immune cells may downregulate production of proinflammatory cytokines and attenuate the inflammatory responses by several possible mechanisms: NF-B activation, JAK-STAT3 pathway, and PI3K-AKT1 pathway. Activation of 7 nAChR by its agonists in monocytes and macrophages could reduce nuclear translocation of NF-B and the transcription of proinflammatory cytokines [1,2,91,92]. In the sepsis and lung injury mouse models, administration of 7 nAChR agonist also suppresses activation of NF-B [5,16]; however, one study showed that vagus nerve electrical stimulus could attenuate the proinflammatory cytokine responses in vivo but did not decrease the NF-B activation [93]. Whether activation of 7 nAChR affects TLR4 signaling pathway, for example, MyD88, TRIF, IRAK, TARF, and other adaptor proteins in the proinflammatory cells, needs further study. The modulatory effects of activation of 7 nAChR on signaling pathways are summarized in Table 3. (ii) Cholinergic agonists inhibit TNF-production and HMGB1 release from macrophages. (iii) Vagus nerve stimulation does not inhibit TNF production in 7-subunit-deficient mice.
(v) Nicotinic treatment prevents lethal endotoxemia.  (ii) LPS-stimulated TNF-production could not be inhibited by an 7 nAChR agonist LCR rat MNCs, whereas inhibition by the 2 adrenergic agonist, salmeterol, was significantly less (−35%) than that obtained in HCR rats.
Rats with the metabolic syndrome have ineffective CAP [28]   (iv) Nicotine had equal efficiencies to 2 ng/mL IFN-in DCs-mediated T cell proliferation.
Beneficial effects for HBV immunotherapy [107] T lymphocytes; B lymphocytes Mouse Sepsis infection 7 nAChR genetic depletion; bone marrow transplantation (i) CD4 + T cell population that is stimulated by norepinephrine to release ACh.
(ii) ChAT + B cells release ACh after stimulation with sulfated cholecystokinin.
(iii) 2-Adrenoreceptors of regulatory lymphocytes are essential for vagal neuromodulation of the innate immune system. (iv) Cholinergic lymphocytes reestablish splenic protection and the potential of cholinergic agonists to rescue immunocompromised animals from established sepsis.
(v) Increased DSS-induced inflammation was associated with reduced CD4 + CD25 + Foxp3 + regulatory T cell numbers in recipients.
Adoptive transfer of CD4 + T cells from vagotomized animals (but not CD4 + T cells from sham-operated controls) to naive DSS-treated recipients resulted in increased inflammatory scores.
T and B lymphocytes synthesize ACh, regulating neutrophil recruitment and innate immunity [3,77,[108][109][110] Epithelial cells (iv) nAchR activation by topical agonist application or deletion of the nAChR antagonist catestatin reduced antimicrobial peptide (AMP) activity in skin extracts and increased susceptibility to methicillin-resistant Staphylococcus aureus and group A Streptococcus infections.
(v) 7 nAChR is in fundamental cellular processes relevant to lung development, injury and repair, and carcinogenesis In the lung epithelial cells, involvement of 7 nAChR in controlling bacteria growth, cell growth, and repair [5,78,[111][112][113]  (ii) ACh and nAChR agonists inhibit TNF-induced adhesion molecule expression by HuMVECs.
(iii) ACh and nAChR agonists reduce TNF-induced chemokine production by endothelial cells.
(iv) Changes in molecular (upregulation, affinity, and conformational states) and cellular (distribution, association with membranes) properties of the 7AChR related to angiogenesis (wound-repair cell migration) and atherogenesis (alterations in cholesterol content) were studied in living endothelial cells.
(v) The nAChRs on endothelial cells modulate key angiogenic processes, including endothelial cell survival, proliferation, and migration.
(iii) At nontoxic concentrations, nicotine increased spontaneous migration of hMSCs, whereas chemotaxis of hMSCs toward C3a and bFGF in vitro and migration of intravenously infusion hMSCs into bone marrow and spleen in vivo were inhibited.
(iv) The antagonist for the alpha 7 homopolymer, bungarotoxin, blocked the inhibitory effect of nicotine on chemotactic factor-induced migration of hMSCs.
Regulation of MSC migration [117,118]  (iv) Nicotine treatment increased the number of EPCs in the bone marrow and spleen and increased their incorporation into the vasculature of ischemic tissue. Administration of nicotine increased markers of EPC mobilization.
Mobilization of EPCs facilitates angiogenesis [119,120] Fibroblast Human Mouse Arthritis patients and models Immunofluorescence; depletion of 7 nAChR (i) Fibroblasts from synovial tissue of arthritis patients expressed 7 nAChR.
(ii) In 7 nAChR knockout mice, a significant increase in the incidence and severity of arthritis and increased synovial inflammation and joint destruction were seen.

Activation of 7
nAChR is protective in arthritis [121,122] HMGB1   (ii) The anti-inflammatory effect of nicotine required the ability of phosphorylated STAT3 to bind and transactivate its DNA response elements.
(iii) In a mouse model of intestinal manipulation, stimulation of the vagus nerve ameliorated surgery-induced inflammation and postoperative ileus by activating STAT3 in intestinal macrophages.
(iv) GTS-21 inhibits proinflammatory cytokine release independent of the Toll-like receptor stimulated via a transcriptional mechanism involving JAK2 activation. The inhibitor -BTX could reverse these effects.
(vi) Inhibition of STAT3 protein expression enhanced cytokine production and abrogated 7 nAChR signaling.
(vii) 7 nAChR controls TNF production in macrophages through a mechanism that requires STAT3 protein expression but not its tyrosine phosphorylation.   (iii) Consistent with allosteric modulation of alpha 7 nAChRs, PNU-120596 enhanced both the agonist potency and efficacy in activating ERK.

Spatial and Temporal Effects of 7 nAChR Activation on p-STAT3.
Anti-inflammatory effect of nicotine in murine macrophages acts through the recruitment of JAK2 to the 7 nAChR and subsequent phosphorylation of JAK2, thereby initiating the anti-inflammatory STAT3 cascade [14]. JAK2 inhibitor AG490 inhibited the anti-inflammatory effect of GTS-21 in the human PBMCs (peripheral blood mononuclear cells) [94], suggesting that p-STAT3 mediates inhibitory role of activation of 7 nAChR. However, in an endothelial cell line, GTS-21 significantly reduced STAT3 activation by phosphorylation and DNA binding [95]. In the splenocytes or myocardium tissue, cardiac troponin I (TnI) induced STAT3 activation and IL-6 is inhibited by nicotine [96]. In macrophage cell line, both 7 nAChR activation and inhibition of JAK2 blunt STAT3 phosphorylation. Inhibition of STAT3 phosphorylation mimicked the 7 nAChR signaling, inhibiting NF-B and cytokine production in macrophages. These findings suggest the proinflammatory role of p-STAT3. In addition, unphosphorylated STAT3 might compete with NF-B. Inhibition of STAT3 protein expression enhanced cytokine production and abrogated 7 nAChR signaling [97].

Modulatory Effects of 7 nAChR Activation Might Involve
CREB and c-fos. It has been assumed that interaction between 7 nAChR and adenylate cyclase 6 increases intracellular cAMP, a secondary messenger, which in turn promotes phosphorylation of CREB. P-CREB translocates into the nucleus and initiates transcription of c-fos, an early response gene. Activation of c-fos could inhibit NF-B activity and production of proinflammatory cytokines [11,59]. So far, there is no scientific evidence to prove this hypothesis, but some previous findings indicate that it might be testable. For example, in epithelial cells, 7 nAChR physically binds adenylate cyclase 6 [98]. In response to LPS stimulation, Fos −/− macrophages and mice showed significantly enhanced production of TNF-, IL-6, and IL-12 p40 but reduced production of the anti-inflammatory cytokine IL-10 compared with wildtype controls. Activation of c-fos inhibits NF-B activity [99].

Concluding Remarks
How nervous system, especially vagus nerve, modulates inflammation and immunity has been a puzzle for many years. In past decade, a large body of evidence has shown that the classical CAP could systemically modulate proinflammatory responses via spleen. More recently, the regulatory role of local CAP is emerging and challenging. The immediate questions we have to answer are the following. How vagus nerve senses the PAMPs or DAMPs in the airspaces of the lung? What sensors and receptors are used by sensory nerve endings during this process? To where and how does vagus nerve send the pathogenic signals? How are pathogenic signals being integrated or transformed in the brain center? What are targeting cell population of vagus nerve in the deferent stages of infection and inflammation? How signaling pathways are finely tuned by vagus nerve spatially and temporally during infection and inflammation? In summary, the overall task of this review is to extend our understanding of how nervous and immune systems work collaboratively during infection and inflammation.