T-cell derived acetylcholine aids host defenses during enteric bacterial infection with Citrobacter rodentium

The regulation of mucosal immune function is critical to host protection from enteric pathogens but is incompletely understood. The nervous system and the neurotransmitter acetylcholine play an integral part in host defense against enteric bacterial pathogens. Here we report that acetylcholine producing-T-cells, as a non-neuronal source of ACh, were recruited to the colon during infection with the mouse pathogen Citrobacter rodentium. These ChAT+ T-cells did not exclusively belong to one Th subset and were able to produce IFNγ, IL-17A and IL-22. To interrogate the possible protective effect of acetylcholine released from these cells during enteric infection, T-cells were rendered deficient in their ability to produce acetylcholine through a conditional gene knockout approach. Significantly increased C. rodentium burden was observed in the colon from conditional KO (cKO) compared to WT mice at 10 days post-infection. This increased bacterial burden in cKO mice was associated with increased expression of the cytokines IL-1β, IL-6, and TNFα, but without significant changes in T-cell and ILC associated IL-17A, IL-22, and IFNγ, or epithelial expression of antimicrobial peptides, compared to WT mice. Despite the increased expression of pro-inflammatory cytokines during C. rodentium infection, inducible nitric oxide synthase (Nos2) expression was significantly reduced in intestinal epithelial cells of ChAT T-cell cKO mice 10 days post-infection. Additionally, a cholinergic agonist enhanced IFNγ-induced Nos2 expression in intestinal epithelial cell in vitro. These findings demonstrated that acetylcholine, produced by specialized T-cells that are recruited during C. rodentium infection, are a key mediator in host-microbe interactions and mucosal defenses.


Introduction
The recently revealed degree of integration between the nervous and immune systems are remarkable [1]. While it is well accepted that neurotransmitters can act on immune cells to alter cell activation and consequently host immune response, recent evidence demonstrates that select immune cell populations not only respond but can also produce neurotransmitters. Among these immune cells are the CD4 + T-cells that express choline acetyltransferase (ChAT), the enzyme required for acetylcholine (ACh) biosynthesis [2][3][4]. These T-cells are crucial intermediaries between the nervous and immune system, functioning to relay neuronal signals and prevent aberrant immune cell activation. Neural inhibition of inflammation can inhibit innate immune cell function in preclinical models of inflammatory bowel disease [5], rheumatoid arthritis [6], ischemia reperfusion injury [7,8], and post-operative ileitis [9]. Immune regulation in this pathway requires norepinephrine (NE) released from neurons to activate β2 adrenergic receptors (β2AR) on ChAT + T-cells causing the release of ACh [2].
Mucosal immunity is crucial to restricting access of commensal and pathogenic bacteria to the host. Host defenses are comprised of overlapping mechanisms that bind, flush away, exclude, or kill pathogenic enteric bacteria [10]. These roles are in part fulfilled by differentiated intestinal epithelial cells (IECs) that not only act as a physical barrier, but also produce and release mucus [11], bactericidal antimicrobial peptides [12,13], and free radicals such as nitric oxide (NO) that are bactericidal or bacteriostatic [14,15]. Loss of these protective mechanisms can result in aberrant immune responses to otherwise innocuous commensal bacteria, increased mucosal inflammation, or susceptibility to infection. In addition, mucosal homeostasis and host-resistance to pathogens is dependent on composition of the intestinal microbiota, with bacterial species that can reduce, or enhance susceptibility to pathogens including Citrobacter rodentium [16][17][18]. Physiological processes that govern these mechanisms of host defense and host-bacterial interactions are therefore paramount to the health of the host.
In the gastrointestinal tract, ACh enhances mucosal protection by controlling IEC functions ranging from release of mucus and antimicrobial peptides to increasing ion and fluid secretion [12,19,20]. Together, these mechanisms of mucosal defense maintain homeostatic interactions between the host and commensal microbiota, while limiting access of pathogens such as C. rodentium. Although the source of ACh regulating these functions of IEC has long been attributed to ChAT + secretomotor neurons within the gastrointestinal tract, we and others have previously described ChAT + T-cells that serve as essential non-neuronal sources of ACh [2][3][4]. This unique source of ACh appears to participate in mucosal immunity and host commensal interactions. As evidence of this, conditional ablation of ChAT in T-cells was found to reduce production of antimicrobial peptides in the small intestine of naïve mice, and induce changes in the jejunal but not colonic microbiota composition [13]. Despite these key observations, the role of ACh released from specialized T-cells during enteric infection is unknown. With these issues in mind, we have used ChAT-GFP reporter mice, and conditional ablation of ChAT in T-cells to assess the role of T-cell derived ACh in host mucosal immune function during C. rodentium infection.
Using this approach, we have identified that ChAT + T-cells are recruited to the colon during C. rodentium infection, and that conditional ablation of ChAT in T-cells significantly increases C. rodentium burden in the colon. This increased susceptibility to infection is due to decreased expression nitric oxide synthase isoform 2 in IEC, with ACh acting to enhance IFNγ-induced gene transcription.

Mice
Mice used in this study are on a C57BL/6 background and were originally purchased from Jackson laboratories (Bar Harbor, ME), including CXCR5-/-, ChAT-GFP (B6.Cg-Tg(RP23-268L19-EGFP)2Mik/J)), ChAT f/f and LCK.Cre to establish a breeding colony. ChAT T-cell conditional knockout (cKO) mice were produced by breeding ChAT f/f and LCK.Cre mice to generate LCK.Cre -ChAT f/f (WT) and LCK.Cre + ChAT f/f (cKO) mice. This breeding scheme permitted use of littermate cKO and WT controls. At 6-8 weeks of age, mice were gavaged with either LB, or Citrobacter rodentium (10 8 CFU (colony-forming unit), strain DBS100, generously provided by Dr. Andreas Baumler). In a subset of experiments, colitis was induced by administration of dextran sodium sulfate (DSS, 3% v/v) in the drinking water for 5 days followed by normal water for 3 days as previously published [21].

Ethics statement
All procedures were approved by the Institutional Animal Care and Use Committee at UC Davis under protocol number 20170 in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals. Mice were euthanized by CO 2 asphyxiation followed by cervical dislocation according to American Veterinary Medical Association guidelines for collection of tissues.

Quantification of colonic C. rodentium
A 1 cm segment of colon was cut and placed into a pre-weighed microcentrifuge tube to determine tissue weight. Samples were homogenized in sterile PBS using a 5 mm sterile stainlesssteel bead (Qiagen, Germantown MD) in a bead beater (Qiagen). Sample homogenates were then diluted in sterile PBS, and (100 μL) plated onto MacConkey agar plates, and colonies counted after 16 h of incubation at 37˚C.

Quantitative PCR
Analysis of gene expression was performed by quantitative real-time PCR (qRT-PCR) as previously described [3]. Briefly, a 1 cm long segment of colon was homogenized in Trizol (Invitrogen, Carlsbad, CA) using a 5 mm stainless steel bead in a bead beater (Qiagen). RNA was extracted as directed by manufacturer's instructions, with isolated RNA dissolved in ultrapure H 2 O (Invitrogen). Synthesis of cDNA was performed using an iSCRIPT reverse transcriptase kit (Bio-Rad, Hercules, CA), and Real time qPCR was performed for the following targets using the indicated primer pairs from Primerbank [24]: CIIta forward 5'-TGCGTGTGATGGATGTCCAG-3', reverse 5'-CCAAAGGGGATAGTG GGTGTC-3', Irf1 5'-ATGCCAATCACTCGAATGCG-3', reverse 5'-TTGTATCGGCCTG TGTGAATG-3'. Amplification and data acquisition were conducted using a QuantStudio6 (ThermoFisher Scientific). Data were analyzed using the delta delta CT method normalizing gene expression to Actb in each sample followed by normalization to experimental control sample.

Analysis of short-chain fatty acids (SCFA) in fecal pellets by LC-MS
Fresh fecal samples from mice were collected and stored at -80˚C until analysis. Pellets were extracted with nano-pure water (10 mg/mL) and gently agitated overnight at 4˚C. The homogenized samples were centrifuged at 21,000 g for 5 min. Supernatants (100 μl) were transferred and centrifuged at 21,000 g again for 20 min. For each sample, 20 μl of the supernatant was mixed with 20 μl of 100 mM N-(3-Dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (1-EDC HCl) (Sigma-Aldrich, cat. # E7750) in 5% pyridine (Sigma-Aldrich cat. # 270407) and 40 μL of 200 mM 2-Nitrophenylhydrazine (2-NPH) (Sigma-Aldrich, cat. # N21588) in 80% acetonitrile (ACN) (Sigma-Aldrich) with 50 mM HCl. The mixture was incubated at 40˚C for 30 min. After reacting, 400 ml of 10% ACN was added to the solution. Then 1 μl of the solution was injected into an Agilent 6490 triple quadruple mass spectrometer for analysis. Chromatographic separations were carried out on an Agilent C18 stationary phase (2.1 x 50 mm, 1.8 um) column. Mobile phases were 100% ACN (B) and water with 10% ACN (A). The analytical gradient was as follows: time = 0 min, 10% B; time = 4.5 min, 10% B; time = 10 min, 35% B; time = 10.1 min, 85% B; time 11.6 min, 90% B; time 12 min, 90% B. Flow rate was 0.3 ml/min and injection volume was 1 μL. Samples were held at 4˚C in the autosampler, and the column was operated at 40˚C. The MS was operated in selected reaction monitoring (SRM) mode, where a parent ion is selected by the first quadrupole, fragmented in the collision cell, then a fragment ion selected for by the third quadrupole. Product ions, collision energies, and cone voltages were optimized for each analyte by direct injection of individual synthetic standards. The MS was operated in positive ionization modes with the capillary voltage set to 1.8 kV. Source temperature was 200˚C and sheath gas temperature 200˚C. Gas flow was 11 L/min, sheath gas flow was 7 L/min, and collision gas flow was 0.2 mL/min. Nebulizer pressure (nitrogen) was set to 25 psi. Argon was used as the collision gas. A calibration curve was generated using authentic standards for each compound.

Histology
Colonic tissue specimens were fixed in 10% normal buffered formalin for 24 h prior to gradual dehydration in ethanol, embedded in paraffin and 6 μm thick cross sections were cut onto glass slides. Slides with tissue sections were de-paraffinized and rehydrated according to standard protocols, stained with hematoxylin and eosin to allow for evaluation of histopathology. Crypt lengths were measured using bright field microscopy on these sections with FIJI (Fiji Is Just ImageJ) [25], measuring at least 20 crypts per animal.

Confocal microscopy
Slides with colonic tissue section were also used for confocal analysis with antibodies raised against specific proteins of interest according to standard protocols. In brief, after slides were de-paraffinized and rehydrated, antigen retrieval was performed in citrate buffer (10 mM, pH 6.0, 30 min., 95˚C). After blocking in 5% BSA and normal donkey serum, samples were incubated in primary antibody overnight (16 h 4˚C). Primary and secondary antibodies used are detailed in Table 1. Slides were washed extensively (3 x 5 mins) in TBS-tween20 and incubated in appropriately labeled secondary antibodies (Invitrogen) for 1 h at room temperature, washed, counterstained with DAPI in TBS-tritonX100 0.1% v/v, washed and mounted in Prolong gold (Invitrogen). Staining using anti-mouse CDH1 (E-cadherin) was revealed using a mouse on mouse kit according to manufacturer's instructions (Vector laboratories, Burlingame, CA). Slides were imaged on a Leica SP8 STED 3X confocal microscope with a 63X 1.4 NA objective. Areas larger than the field of view of the objective were acquired using a tiling approach, whereby adjacent images were acquired with a 10% overlap.

Confocal image analysis
Analysis of standard confocal data sets was performed by opening Leica image format files in Imaris Stitcher (v9.0, Bitplane Scientific) to fuse overlapping fields of view together. These reconstructed areas were then analyzed using Imaris software. Expression of NOS2 in IEC was determined by creating a mask based on regions of CDH1 staining (i.e. IEC) that contained DAPI + cells. This defined region was then interrogated for the number of IEC present, and the intensity of NOS2 staining. Counting of DAPI + Ki67 + IEC, or T-cells (CD3+ DAPI+) cells were performed in a similar manner in 3-5 fused fields of view from each animal counted.

Ussing chambers
Following excision of the intestine, segments of colon were cut along the mesenteric border to allow for mounting in the Ussing chamber (Physiologic Instruments, San Diego, CA). Tissues were maintained in oxygenated Kreb's buffer consisting (in mM) of: 115 NaCl, 1.25 CaCl 2 , 1.2 MgCl 2 , 2.0 KH 2 PO 4 and 25 NaHCO 3 at pH 7.35 ± 0.02 and maintained at 37˚C. Additionally, glucose (10 mM) was added to the serosal buffer as a source of energy, which was balanced osmotically by mannitol (10 mM) in the mucosal buffer. Agar-salt bridges were used to monitor potential difference (PD) across the tissue, and to inject the required short-circuit current (Isc) to maintain the PD at zero by an automated voltage clamp. Data from the voltage clamp

Statistical analysis
Data were analyzed using one-way analysis of variance (ANOVA) in Prism (Graphpad, San Diego CA), with a P value of less than 0.05 denoted as significant.

C. rodentium infection induces the recruitment of ChAT + T-cells
Only sparse numbers of ChAT + T-cells have been observed in the intestine of naïve mice [13], however the potential role of ChAT + T-cells in the mucosal immune response during enteric bacterial infection has not been established. To assess if ChAT + T-cells are recruited during infection, ChAT-GFP + mice were infected with C. rodentium and the number of CD3 + ChAT-GFP + T-cells determined by confocal microscopy on days 6, 10, 21, and 30 post-infection (p.i.). Mice infected with C. rodentium had a significant increase in the number of CD3 + ChAT-GFP + T-cells in the colon beginning 10 days p.i. which persisted until 30 days p.i. (Fig 1A & 1B). In order to characterize these recruited cells, flow cytometry was conducted on isolated lamina propria lymphocytes (LPL). These colonic lamina propria ChAT-GFP + T-cells (Single, Live, CD3 + , CD4 + ) 10 days post-C. rodentium infection produced IFNγ, IL-17A, and IL-22 (Fig 2A). Quantification revealed that ChAT-GFP + T-cells express more IFNγ, IL-17A, and IL-22 by MFI (mean fluorescence intensity) compared to ChAT-GFP -T-cells (Fig 2B).
Despite this, it is important to note that the frequency of ChAT-GFP + T-cells actively producing IFNγ and IL-17A were significantly less compared to ChAT-GFP -T-cells. ChAT-GFP + IL-22 + T-cell population appears to be persistent in the naïve colon and does not increase significantly during infection. These data demonstrate that the ChAT-GFP + T-cells are not unique to Th1/Th17/Th22 T-cells subsets, and can be polarized to these three different phenotypes (Fig 2).
In contrast to the recruitment induced by C. rodentium infection, induction of colonic inflammation by the chemical irritant DSS failed to increase the number of ChAT-GFP + Tcells compared to naïve control, despite evidence of overt inflammation (S1A Fig). Together, these results suggest that ChAT + T-cells are a specific component of the host response to C. rodentium infection and their recruitment is driven by specific signals and rather than a simple consequence of intestinal inflammation.

Ablation of T-cell derived acetylcholine increases colonic C. rodentium
The functional role of T-cell-derived ACh during C. rodentium infection was determined using a T-cell conditional knockout (cKO) approach. Accordingly, infected ChAT T-cell cKO mice had increased CFU/g of C. rodentium in colonic tissue at day 10 p.i. as compared to infected WT mice (Fig 3A). To determine if the increased bacterial burden of C. rodentium resulted in altered localization of the pathogen in the colon, confocal microscopy analysis using antibodies directed against C. rodentium was performed (Fig 3B). Compared to WT, we observed increased C. rodentium in the colonic lumen, adjacent to IEC (CDH1 + DAPI + ), and ChAT+ T-cells mediate protection against Citrobacter rodentium the presence of microcolonies within the colonic crypts in ChAT T-cell cKO mice. Despite the increased bacterial burden in ChAT T-cell cKO mice, no significant increase in the number of proliferating (DAPI + CDH1 + Ki67 + ) IEC cells (Fig 3D &3E), histopathological damage, or crypt hyperplasia was observed compared to infected WT mice (Fig 3C). Together these data indicate that T-cell derived ACh is a critical component of host defense during C. rodentium infection but does not influence epithelial barrier integrity or effect crypt hyperplasia. To assess what factors could contribute to recruitment of these CD3 + ChAT + T-cells during C. rodentium infection, we performed qRT-PCR for chemokines that are cognate ligands for previously identified receptors expressed by this population of T-cells [4,26].

C. rodentium-induced inflammation is enhanced with loss of T-cell derived ACh
To determine the immunological consequences of conditional ablation of ChAT in T-cells during C. rodentium infection, qRT-PCR was conducted on colon from LB control and infected WT and ChAT T-cell cKO mice for proinflammatory gene expression. At day 10 p.i., expression of Il-1β, Il-6, and Tnfα were significantly increased in C. rodentium infected mice compared to LB control mice (Fig 4). Expression of these cytokines was significantly enhanced 10 days p.i. in the ChAT T-cell cKO mice compared to WT infected animals. In contrast, expression of Ifnγ, Il-17a, Il-22were increased 10 days p.i. to a similar extent in WT and ChAT T-cell cKO mice (Fig 4). These data indicate that ablation of ChAT in T-cells can significantly alter the host immune response to C. rodentium, but in a manner that does not alter local Th1, Th17, or Th22 responses.

Conditional ablation of ChAT in T-cells does not affect antimicrobial peptide production
As ChAT T-cell conditional knockout mice were previously observed to have reduced expression of antimicrobial peptides [13], we questioned if this could result in an increased C. rodentium burden. Using qRT-PCR we observed no significant differences in antimicrobial peptide expression in the small intestine or colon (S4 Fig) in naïve WT and ChAT T-cell cKO mice. As expected, colonic expression of RegIIIγ was significantly increased after C. rodentium infection in both WT and ChAT T-cell cKO mice, however there was no difference between the two genotypes in the terminal ileum or colon (S4A & S4B Fig). As the commensal microbiota actively produces bioactive metabolites, we assessed if production of short-chain fatty acids ChAT+ T-cells mediate protection against Citrobacter rodentium (SCFA) was different in WT compared to ChAT T-cell cKO mice. Mass spectrometry revealed significant changes in specific SCFA during C. rodentium infection. Significantly reduced lactic acid was observed in infected WT but not in ChAT T-cell cKO mice. Butyric acid was significantly enhanced in both WT and ChAT T-cell cKO infected mice compared to uninfected WT or cKO control mice. While significantly increased production of pyruvic acid was detected in the feces from uninfected ChAT T-cell cKO mice, infection reduced the concentration of this metabolite to levels observed in uninfected or C. rodentium infected control mice. (S5 Fig). Together these findings indicate that the increased C. rodentium burden in ChAT Tcell cKO mice was not due to an inability to produce antimicrobial peptides or alterations in SCFA produced by the microbiota.

T-cell derived ACh regulates host Nos2 expression during infection
The increased expression of certain pro-inflammatory cytokines coupled with increased colonic C. rodentium burden in ChAT T-cell cKO mice lead us to ascertain if innate effector responses were intact. First, we considered if lack of T-cell derived ACh could increase differentiation of alternatively activated macrophages, disrupting the ability to mount and effect innate responses to C. rodentium. As indicated in Fig 5, no significant differences were noted in arginase1 (Arg1), mannose receptor C-type 1 (Mrc-1), chitinase-like 3 (Chi3l3), or resistinlike molecule α (Retnla) expression by qRT-PCR in colonic tissues between WT and ChAT Tcell cKO mice. Expression of Nos2 ("iNOS") however was significantly abrogated 10 days p.i. in ChAT T-cell cKO mice compared to infected WT. These data indicate that lack of T-cell derived ACh does not increase alternatively activated/M2 macrophage polarization, but significantly impacts the expression of Nos2. As numerous cell types can express NOS2, we assessed the localization and quantity of NOS2 protein by confocal microscopy on colonic tissue from infected WT and ChAT T-cell cKO mice and uninfected controls. As indicated in Fig 6, IEC (CDH1 + DAPI + ) were the predominant cell type that were immunoreactive of NOS2 during C. rodentium infection. In keeping with the qRT-PCR data, C. rodentium induced NOS2 expression was significantly reduced in ChAT T-cell cKO compared to WT mice (Fig 6A). Quantification of NOS2 expression in IEC further demonstrate reduced NOS2 expression in C. rodentium infected ChAT T-cell cKO mice (Fig 6B). This reduced ability to increase NOS2 expression in IEC during 10 days p.i. was further validated by flow cytometry conducted on IEC (Single, live, CD45 -, EpCAM + ) from naïve and infected WT and ChAT T-cell cKO mice (Fig 6C & 6D). These data demonstrate that T-cell deficiency in ChAT significantly impairs C. rodentium induced increases of NOS2 expression in IEC.

Acetylcholine enhances expression of NOS2 in IEC
As ACh has been previously demonstrated to induce NOS2 expression in lung epithelial cells [27], we sought to determine if ACh could induce similar effects in IEC. The mouse colonic epithelial cell line CMT-93 was treated with IFNγ ± carbachol (ACh mimetic), with Nos2, irf1, and CIIta expression assessed by qRT-PCR. As expected, stimulation with IFNγ (1 ng/mL, 3 h, time and dose determined empirically) induced expression of Ciita, Irf1, and Nos2. Co-treatment with carbachol further significantly increased expression of Nos2 compared to IFNγ alone, but did not enhance Ciita or Irf1 expression (Fig 7). Treatment with carbachol alone failed to significantly increase expression of any of the target genes. These results suggest that cholinergic signaling in IEC can synergistically enhance select IFNγ induced genes including Nos2.

Discussion
The finding that the nervous system is an active participant during inflammation has been an unexpected and intriguing finding. At the interface between these two systems is a unique type of T-cells that can produce and release ACh in response to sympathetic neurotransmitters [2,28]. Although the predominant focus on these ChAT + T-cells has been on their ability to reduce the severity of disease in a number of clinically relevant immunopathologies [5][6][7][8][9], ChAT + T-cells can also help to establish host-commensal interactions. While a prior study demonstrated increased diversity of the small intestinal microbiota in ChAT T-cell cKO mice, due to reduced antimicrobial peptide expression [13], the role of these cells during enteric bacterial infection was not known.
Using a combination of ChAT-GFP reporter and ChAT T-cell cKO mice, our studies are the first to demonstrate recruitment of ChAT + T-cells and a functional role for these cells during C. rodentium infection. These recruited ChAT-GFP + T-cells do not appear to be restricted to a unique Th subset, with ChAT-GFP + T-cells found to produce IFNγ, IL-17A, or IL-22 in agreement with prior studies [3].
As we and others have previously demonstrated that ChAT + T-cells express the chemokine receptors CXCR5 [4] and CCR8 [29]; we sought to characterize the production of cognate ligands to these receptors during C. rodentium infection, Cxcl13 and Ccl8 respectively. Our analysis indicates that Cxcl13 but not Ccl8 expression is induced beginning 10 days p.i. until day 30 p.i., a period during infection that closely mirrors when the number of ChAT + T-cells increased in the colon. This temporal pattern of chemokine expression is corroborated by other studies demonstrating increased Ccl8 during C. rodentium infection [30]. CXCL13 is well established as critical to organization of secondary lymphatic organs [31], tertiary lymphoid tissues and recruitment of IL-22 producing ILC3 [32] and can be induced by vagal nerve stimulation [33]. Despite this, our studies using CXCR5 KO mice indicate that this signaling axis is either not critical or functionally redundant with respect to the host response to C. rodentium 10 days post-infection. The importance of ACh derived from T-cells to host mucosal immune response during enteric bacterial infection was determined using a ChAT T-cell conditional knockout. Highlighting the host protective role of ACh, we observed an increased bacterial burden following enteric C. rodentium infection in ChAT T-cell cKO compared to WT mice. This increased bacterial burden in ChAT T-cell cKO mice was associated with significantly increased expression of the pro-inflammatory cytokines Il-1β, Il-6, Tnfα, with equivalent expression of Ifnγ, Il-17a, or Il-22 compared to infected WT mice. Loss of T-cell derived Ach however did not impinge on IL-22 production, typically produced by ILC or T-cells in response to C. rodentium infection [34]. Together these findings, supported by the literature, suggest that ChAT + T-cells are important in eliciting host-protective responses.
Mucosal immunity is comprised of a multitude of overlapping mechanisms that serve to protect the host from pathogens including the production and secretion of antimicrobial peptides. T-cell derived ACh has been implicated in regulation of host-microbial interactions at the mucosal surface by controlling antimicrobial peptide production. Conditional ablation of ChAT in CD4 + cells using CD4.Cre ChAT f/f mouse line resulted in reduced lysozyme, defensin A, and ang4 expression in the small intestine, consequently increasing the diversity of commensal microbiota in the jejunum but not the cecum, or colon [13]. In contrast to these findings we noted no significant reductions in antimicrobial peptide expression in ChAT T-cell cKO compared to WT mice. As expected [35], expression of RegIIIy was significantly enhanced in WT and ChAT T-cell cKO mice during C. rodentium infection irrespective of genotype. These data suggest that the increased bacterial burden in ChAT T-cell cKO mice was not due to a deficit in antimicrobial peptide expression.
Host production of free radicals including NO are critical factors in protection against several bacterial pathogens [36][37][38]. NO also functions as a short-lived cell signaling molecule and is produced by three distinct isoforms of nitric oxide synthase that are each uniquely regulated in a tissue-or context-dependent manner [37]. In contrast to the constitutively expressed NOS found in endothelium or neurons, bacterial products or inflammation can induce NOS2 expression in a variety of cell types [37,39]. Infection with C. rodentium increases NOS2 expression, functioning to limit bacterial burden and disease [14,40]. In agreement with this literature, our data demonstrate that IEC in the colon are the predominant cell type expressing NOS2 during C. rodentium infection in WT mice. Conditional ablation of ChAT in T-cells, however, resulted in significantly reduced Nos2 expression compared to WT mice. Confocal microscopy on colonic tissue sections and flow cytometry experiments confirmed NOS2 expression was significantly increased in colonic IEC of WT mice, but not in ChAT T-cell cKO mice during infection. Together these data demonstrate that lack of T-cell derived ACh significantly reduced the induction of NOS2 in IEC during C. rodentium infection. Although NOS2 expression is characteristically elicited by IFNγ-induced activation of STAT1-dependent gene transcription [41], expression of this cytokine was not affected by ChAT T-cell deficiency. Additionally, we observed that acetylcholine mimetics significantly enhance IFNγ-induced Nos2 expression in IEC in vitro, in agreement with previously reported experiments in lung epithelial cells [27].
There are striking similarities in the aberrant host response to C. rodentium infection in ChAT T-cell cKO and the previously described Nos2 -/mice [14]. For example, both mouse lines exhibit increased bacterial burden at day 10 p.i. without resulting in increased mortality or enhanced colonic histopathology [14]. Although Nos2 deficiency in mice, or inhibition of NO production increases Th17 differentiation [42], no significant increase in Il-17a expression was observed in the colonic tissue from C. rodentium infected ChAT T-cell cKO mice compared to WT mice. This is likely due to the short half-life of NO in biological fluids [43], and the expression of NOS2 in colonic IEC far from differentiating T-cells in draining lymph nodes.
Our data further substantiate the unique role of ACh producing ChAT + T-cells in modulating immune function. These unique T-cells appear to function as a critical component of the mucosal immune system, limiting the number and detrimental effects of enteric bacterial pathogens. How these specialized T-cells that are recruited to the colon, become activated, and release ACh during C. rodentium infection warrants future study. Given the requirement for NE signaling through the β2AR receptor on ChAT + T-cells in septic shock [2], activation by the sympathetic innervation is a strong possibility. Supporting this contention, Salmonella typhimurium induces activation of the sympathetic innervation within the small intestine, and the release of NE adjacent to muscularis macrophages [44]. While it is tempting to speculate that infection induced activation of a neuronal circuit is host protective, it is important to note that host NE induces bacterial expression of virulence genes by enteric pathogens such as C. rodentium [45] and enterohemorrhagic Escherichia coli [46]. Future studies will only further illuminate the integrated nature of the nervous system and immune system with ChAT + Tcells as a critical node mediated host protection during enteric bacterial infection.