TLR2 and TLR9 modulate enteric nervous system inflammatory responses to lipopolysaccharide

Joan F Burgueño
Albert Barba
Elena Eyre
Carolina Romero
Michel Neunlist
Ester Fernández
TLR2 and TLR9 modulate enteric nervous system inflammatory responses to lipopolysaccharide
10.1186/s12974-016-0653-0R E S E A R C H Open Access
Background: Accumulating evidence suggest that the enteric nervous system (ENS) plays important roles in gastrointestinal inflammatory responses, which could be in part mediated by Toll-like receptor (TLR) activation. The aim of this study was to characterise the expression and functionality of TLR2/4/9 in the ENS. Methods: TLR2/4/9 expression was assessed in the plexuses of adult rats and embryonic ENS cultures by immunofluorescence and quantitative PCR. Following stimulation with TLR2/4/9 ligands or their combinations, activation of NF-kB, production of TNF-α, IL-6 and MCP-1 and chemoattraction of RAW264.7 macrophages were evaluated by means of Western blot, ELISA, immunofluorescence and migration assays in transwell inserts.Results: TLR2/4/9 staining colocalised with enteric neuronal markers, whereas their presence in enteroglial processes was low to inexistent. Stimulation of ENS cultures with selective ligands induced NF-kB activation and release of cytokines and chemokines by neurons and resident immunocytes. TLR2 neutralisation before lipopolysaccharide (LPS) challenge reduced production of inflammatory mediators, whereas combination of TLR2/4 ligands promoted macrophage migration. Combined stimulation of cultures with LPS and the CpG oligonucleotide 1826 (TLR4/9 ligands) caused a synergic increase in chemoattraction and cytokine production.Conclusions:Our results suggest that the ENS, and particularly enteric neurons, can integrate a variety of microbial signals and respond in a relatively selective fashion, depending on the particular TLRs stimulated. These findings additionally suggest that the ENS is capable of initiating a defensive response against pathogens and expanding inflammation.

Background

The enteric nervous system (ENS) is the largest component of the peripheral nervous system and is constituted by two cell types, neurons and enteric glial cells (EGC). It is organized in two major ganglionated plexuses, namely the Meissner's submucosal (SMP) and the Auerbach's myenteric (LMMP) plexuses, which control gastrointestinal motility, secretion and blood flow, participate in maintenance of the epithelial barrier and modulate various processes of the local immune response [1,2]. Although a number of studies have demonstrated that the ENS undergoes structural and phenotypic plastic changes during inflammatory responses [3], growing evidence suggests that it is not only a bystander, but an active player during inflammation. Indeed, both enteric neurons and EGCs have been shown to contribute to the resulting inflammatory phenotype following chemically induced colitis [4,5], probably through the release of several immune mediators [6][7][8][9]. Moreover, perineural inflammation is dense in biopsies from inflammatory bowel disease patients and is correlated to major histocompatibility complex class II expression in EGCs [10], further illustrating the neuroimmune interactions existing in the intestine.

Toll-like receptors (TLR) are transmembrane receptors that recognise different highly conserved microorganismassociated molecular patterns (MAMP), as well as other molecules such as damage-associated molecular patterns [11,12]. Upon MAMP binding, the cytoplasmic domain recruits different adapter proteins to trigger a variety of signalling pathways that ultimately activate transcription factors such as nuclear factor-kB (NF-kB), activating protein-1 and interferon regulatory factors, which in turn promote the production of pro-inflammatory cytokines [13,14]. TLRs are expressed in most human tissues including the gastrointestinal tract [15], where they have been proposed to mediate the cross-talk between host cells and commensal microflora due to the key role they play in the innate immune response and in shaping adaptive immunity [16,17].

In recent years, expression of some TLRs in the ENS has been described [18,19], and their roles in intestinal motility, apoptosis and normal development have progressively been unravelled [19,20]. In addition, their immune functions have been also addressed by some authors [7,21], but there are still several questions to be answered, such as whether differential recognition of MAMPs by the ENS translates into microbial-selective responses, or whether TLRmediated signalling is involved in expansion of the inflammatory response. In this context, we aimed to characterise expression and functionality of TLR2/4/9 in the ENS, focusing on their responses in terms of cytokine, chemokine and chemoattraction induction after single or combined ligand challenges. Our results show that neurons are the main TLR-expressing cells in enteric plexuses and integrate TLR signals promoting diverse responses depending on the stimulus received, bringing some new evidence regarding their roles in defence against pathogens and inflammation enhancement.

Methods

Reagents and antibodies

Animals

For ex vivo experiments, 10-week-old male Sprague-Dawley rats were purchased from Charles River (Les Oncins, France) and housed in specific pathogen-free conditions, under a controlled temperature (20 ± 2°C) and photoperiod (12 h/12 h light-dark cycle), with free access to food and water. Animals were euthanized by CO 2 inhalation and distal ileum was removed and placed in ice-cold oxygenated Krebs solution for subsequent manipulation. Colons were flushed with Krebs solution, opened along the mesenteric border, pinned flat in a dissection dish and fixed in Lana's fixative (4 % paraformaldehyde, 14 % picric acid in 0.4 M phosphate buffer).

For in vitro experiments, pregnant Sprague-Dawley rats purchased from Charles River were killed by CO 2 inhalation followed by cardiac puncture exsanguination. Pregnant uteri were removed and kept in ice-cold phosphate-buffered saline (PBS) for further dissection.

All animal procedures performed were approved by the Ethical Committee of the Universitat Autònoma de Barcelona (code 2669).

Cell cultures

Isolation and culture of rat embryonic ENS was performed as described elsewhere [22]. Briefly, intestines of rat embryos (E16) were removed and finely diced in PBS. Tissue fragments were digested with trypsin and DNase I, and cells obtained were counted and seeded at a density of 2.4 × 10 5 cells/cm 2 on 24-or 48-well plates, previously coated with a 0.5 % gelatin solution in sterile PBS. Stimulation was performed for 24 h after 15-day culture in serum-free medium (DMEM-F12 (1:1)) containing 1 % of N-2 supplement.

The murine macrophage cell line RAW 264.7 (ATCC® TIB-71) was purchased from the American Type Culture Collection and cultured in DMEM supplemented with 10 % heat-inactivated foetal calf serum.

Stimulation experiments were performed for 24 h with either 100 ng/mL Pam2CSK4, 100 ng/mL LPS, 1 μM ODN 1826, 1 μM cODN 1826 or combinations of these ligands. For NF-kB inhibition and TLR2 neutralisation experiments, cultures were pre-treated for 1 h with 15 μM Bay 11-7082 or 10 μg/mL anti-TLR2 antibody before MAMP stimulation.

In costimulation experiments, comparison between expected additive effects and measured effects of TLR ligand combinations was calculated according to the model of functional interaction, represented by the following equation as described in [23]: E(ODN + LPS) expected = E(ODN)measured + E(LPS)measured -E(ODN)measured * E(LPS)measured.

Immunofluorescence

Fixed colon was dissected under a stereo microscope to obtain whole-mount preparations of the SMP and the LMMP. Adult tissues, as well as ENS cultures grown on cover-slips, were blocked for 1 h in PBS containing 4 % horse serum, 0.1 % Triton X-100 and 0.01 % NaN 3 . Samples were incubated overnight at 4°C with combinations of TLR2, TLR4 or TLR9 with Hu C/D, β-tubulin III (neuronal markers), GFAP, S100β (glial markers), IL-6, MCP-1 or IBA-1 antibodies. Secondary antibodies to rabbit, mouse or goat IgG and chicken IgY were used to detect bound primary antibodies. All samples were mounted in Vectashield aqueous anti-fading mounting medium (Vector Laboratories) and analysed under a Zeiss LSM 700 confocal laser microscope (Carl Zeiss, Madrid, Spain). To avoid overlapping, control preparations were previously single stained for one marker, and their fluorescence was evaluated with the three different lasers used for final image acquisition. Band-pass filters were set up to avoid cross-talk between channels, and sequential acquisition was performed during experiments to excite fluorochromes one at a time.

Real-time RT-PCR analysis

Total RNA from ENS culture was extracted using the RNeasy Mini Kit (QIAGEN, Las Matas, Spain), quantified by optical densitometry and assessed for integrity by on-chip gel electrophoresis with the Experion™ System (Bio-Rad Laboratories, el Prat de Llobregat, Spain). Then, 100 ng of RNA were retro-transcribed by using the Transcriptor First-strand cDNA Synthesis Kit (Roche Applied Science, Mannheim, Germany) for reversetranscriptase polymerase chain reaction (RT-PCR). Primer sequences listed in Table 1 were designed to span introns using the Universal ProbeLibrary Assay design Center (https://lifescience.roche.com/webapp/wcs/stores/servlet/ CategoryDisplay?tab=Assay+Design+Center&identifier=-Universal+Probe+Library&langId=-1), and checked for specificity through BLAST search. PCR amplifications were performed using the LC480 SYBR Green I Mastermix (Roche Applied Science) according to manufacturer's protocol, and run on a LightCycler 480 II instrument (Roche Applied Science). Absence of coamplification products was assured by generating a final melting curve for each reaction and by loading PCR products on a denaturing 2 % agarose gel, stained with SYBR safe (Life Technologies) and visualized under UV transillumination. Specificity of the primers was also determined by sequencing these amplification products. Messenger RNA (mRNA) level of expression of the genes of interest was corrected to that of the S6 housekeeping gene and calculated by the ΔΔCt method.

In order to compare mRNA expression levels of the receptors in basal conditions, absolute mRNA levels were estimated by determining the difference between the cycle threshold (Ct) of the target receptor and the Ct of the housekeeping gene, as described elsewhere [21,24]. According to their ΔCt to the S6 gene, genes were classified as high-expressed (ΔCt less than 5 cycles), intermediate-expressed (ΔCt from 5 to 15 cycles), low or Bound antibodies were detected with HRP-conjugated anti-rabbit or anti-mouse antibodies, and visualized by enhanced chemiluminescent detection (ECL advance, GE Healthcare). Membranes were stripped for 15 min in Reblot buffer (Millipore), followed by extensive washing in TBST before reblocking with 5 % non-fat dry milk in TBST and reprobing for β-actin determination. Bands were imaged in a LAS-3000 Imager (Fujifilm, Tokyo, Japan) and quantified with Multigauge 3.0 software (Fujifilm). To allow comparison between different membranes, the density of the bands was referred to that of untreated controls and normalized to the amount of β-actin in the same sample.

TNF-α, IL-6 and MCP-1 ELISA

Culture supernatants were centrifuged, aliquoted and frozen, and further assayed with the corresponding BD OptEIA™ ELISA Sets (BD), following manufacturer's recommended assay procedures. Final cytokine or chemokine values were related to the total protein amount of the sample, which was determined by using the BCA protein assay kit (Pierce, Rockford, USA).

Migration assays

Twenty-four hours after stimulation of ENS primary culture with MAMPs, conditioned supernatants were centrifuged, placed into 24-well plates and left to equilibrate for an hour with the transwell insert. Then, 10 5 RAW 264.7 macrophages were seeded in the upper chamber of the 8-μm-pore transwell inserts, and allowed to migrate for 4 h at 37°C and 5 % CO 2 . After fixation in 4 % paraformaldehyde, cells on the upper surface of the transwell membrane were removed by rubbing with a sterile cotton swab, and cells on the lower surface were stained with DAPI. The average number of migrating cells was determined by counting eight fields per membrane at ×100 under a Carl Zeiss Axioskop 40 FL epifluorescence microscope equipped with a Zeiss AxioCam MRm camera (Carl Zeiss, Germany). Each experiment was performed in duplicate.

Statistical analysis

Results are presented as mean values ± S.E.M. of at least three independent experiments. All data were compared using one-way or two-way ANOVA, followed by Tukey's post hoc test (unless otherwise stated). Where stated, randomised block design analysis was performed to minimise the variability due to differences between individual culture responses. Data analysis and plot were performed with GraphPad Prism 5.0 software (GraphPad Software Inc., La Jolla, USA). Randomised block design analyses were performed with Minitab 15 Statistical Software (Minitab Inc., Pennsylvania, USA). A P value <0.05 was considered to be significant.

Results

Enteric neurons express TLR2/4/9 in adult rat colon

Expression of TLR2/4/9 in whole-mount preparations from adult rat colon was assessed by immunofluorescence. Prominent immunoreactivity for the three receptors was found in neurons from both SMP and LMMP ( Fig. 1), as demonstrated by colocalisation with the neuronal markers Hu C/D or β-tubulin III. However, distribution patterns were different for each receptor. TLR2 staining was found in all neurons of the SMP and the LMMP (Fig. 1, upper panels), whereas TLR4 was only observed in discrete neuronal somas and fibres ( Fig. 1, middle panels, and Additional file 1). TLR9 reactivity was found in neurons and interganglionic bundles, fully colocalising with the β-tubulin III marker ( Fig. 1, lower panels). Since expression of TLR2 and 4 has previously been described in EGCs [18,19,21], we additionally performed colocalisation studies of the three receptors with the enteroglial marker glial fibrillary acidic protein (GFAP). Our results demonstrate minor localisation of the TLRs studied in EGCs in the SMP (Additional file 2, left panels). In the same vein, TLR4 and 9 showed minor colocalisation with GFAP + cells in the LMMP (Additional file 2, right middle and lower panels). Conversely, TLR2 immunoreactivity was observed in some enteroglial processes in the LMMP (Additional file 3), indicating that this receptor is expressed in both neurons and EGCs.

TLR2/4/9 show similar expression patterns in rat embryonic ENS culture

To determine whether rat embryonic ENS cultures might be a good in vitro model to study the immune functions of TLRs, we characterised their expression in such set-up. The three receptors were found in ENS cultures, displaying similar expression levels (Fig. 2a, b).

Distribution of such receptors was circumscribed to neuronal structures, either somas or axons. TLR2 was found in all neurons (Fig. 2c, upper panels), whereas TLR4 was seen in most but not all somas, as well as in discrete fibres (Fig. 2c, middle panels). TLR9 positive staining colocalised with β-tubulin III + structures, including neuronal somas (arrows) and nerve bundles (Fig. 2c, lower panels).

Microbial motifs induce TLR-mediated activation of the NF-kB signalling pathway

Recognition of MAMPs through TLRs leads to activation of different signalling cascades, including the NF-kB pathway. Phosphorylation of the inhibitor of kB (IkB)-α protein is necessary to target it for ubiquitination and release NF-kB subunits, which are then able to translocate to the nucleus [14]. Therefore, p-IkBα was used as an indicator of TLR-induced activation of ENS cultures.

The three TLRs described were functional, as the ligands Pam2CSK4 (TLR2/6), LPS (TLR4) and ODN 1826 (TLR9) caused a time-dependent increase in phosphorylation of this protein (Fig. 3a, b). Furthermore, the cODN 1826, which does not bear the CG motifs, did not induce NF-kB activation, demonstrating the selectivity of the TLR9 ligand. Pre-incubation of ENS cultures with 15 μM Bay 11-7082 abrogated LPS-induced activation, confirming the specific involvement of the NF-kB pathway (Fig. 3a).

Activation kinetics observed for all MAMPs were similar, lasting from 1 to 48 h and peaking at 4-8 h (Fig. 3b). However, activation intensity was ligand-specific, LPS being the most potent inducer (LPS 0.1 = 386.6 ± 61.8, Pam2CSK4 100 = 328.3 ± 29.14 and ODN 1826 1 = 110 ± 15.98 AUxhour; Additional file 4A-E).

Neuronal activation was determined through immunofluorescent localisation of the NF-kB p65 subunit. Unstimulated ENS culture showed cytoplasmic staining for p65 in neurons and other undetermined cell types (Fig. 3c, upper panels). However, 1 h after LPS stimulation, p65 reactivity was preferentially seen in nuclear localisation, colocalising with DAPI. Interestingly, some of the TLR4-activated cells were HuC/D + neurons (Fig. 3c, lower panels), indicating that TLRs expressed in these cells signal through the NF-kB pathway.

LPS stimulation induces a pro-inflammatory microenvironment in ENS culture through the NF-kB signalling cascade Tumour necrosis factor (TNF)-α and interleukin (IL)-6 are secreted in rat embryonic ENS culture in response to LPS [7]. In addition, IL-8 is also released in vitro by LPS-stimulated jejunum biopsies [25]. Therefore, we LPS addition to embryonic ENS culture elicited a marked increase in all inflammatory mediators analysed 24 h after stimulation (Fig. 4a-c). In contrast, the TLR2/6 ligand Pam2CSK4 induced a slight, though not significant, increase in TNF-α and MCP-1, whereas ODN 1826 was not able to induce secretion of such molecules (Fig. 4a-c). These results suggest that Gram-negative signalling induces pro-inflammatory responses in embryonic ENS culture, while Gram-positive structures and bacterial DNA do not, despite their ability to activate the NF-kB pathway.

Inhibition of the NF-kB pathway by Bay 11-7082 significantly decreased the production of all studied mediators, but did not completely abrogate it (Fig. 4a-c). This observation points out that other signalling pathways, such as the mitogen-activated protein kinases (MAPK) cascade [7], might also be involved in responses to these MAMPs.

TNF-α expression after LPS challenge has been described in embryonic ENS neurons [7]. Since TLR4 staining in ENS cultures was found circumscribed to neurons, we sought to determine whether TLR4 + cells were responsible for the production of IL-6 and MCP-1 24 h after LPS challenge. Colocalisation studies revealed that TLR4-expressing neurons produced IL-6 ( Fig. 4d, upper panels) and MCP-1 (Fig. 4d, lower panels). However, macrophages did also show immunoreactivity to IL-6 ( Fig. 4d, upper panels, white arrows, and Additional file 5) and MCP-1 antibodies (Additional file 6, white arrows), pointing out that resident immunocytes are also involved in responses to MAMPs in these cultures. Nevertheless, neurons were the only IL-6-producing cells that stained positive for TLR4 (Additional file 7), suggesting they act as primary sensors of LPS in these cultures.

TLR2 is up-regulated upon stimulation with TLR2/4/9 ligands We next aimed to evaluate whether stimulation with one particular MAMP might have cross-regulatory effects on the expression of other TLRs, as has been previously described [26,27]. Quantitative PCR assays of ENS culture showed consistent TLR2 up-regulation after an 8-h exposure to the tested MAMPs (TLR2 Pam2CSK4 = 18.78 ± 3.02, TLR2 LPS = 40.78 ± 7.84 and TLR2 ODN 1826 = 8.29 ± 2 vs. TLR2 Ctrl = 1.23 ± 0.33 folds; Fig. 5a-c). Moreover, a slight

Neutralisation of TLR2 modulates TLR4-mediated production of inflammatory mediators

Consistent overexpression of TLR2 after MAMP challenge prompted us to study the involvement of this receptor in LPS-elicited pro-inflammatory responses. Pre-incubation with a TLR2-neutralising antibody significantly decreased the release of TNF-α, IL-6 and MCP-1 (Fig. 6a-c). These results point out that TLR2 shapes TLR4-driven responses to its ligand LPS, increasing their final effect.

TLR4/9 synergise amplifying the pro-inflammatory responses elicited by LPS

Given that the described alterations in TLR expression might influence subsequent responses to LPS, we studied the production cytokines and chemokines after combined stimulation with the selected MAMPs.

Since TLR2 is strongly up-regulated after LPS exposure, we expected that costimulation with Pam2CSK4 would increase cytokine and chemokine production. However, addition of this ligand to LPS-challenged ENS culture did not induce a higher mediator release (Fig. 7a-c). Nevertheless, the inhibitory effects of Bay 11-7082 in production of IL-6 and MCP-1 were reverted by this combined treatment (Fig. 7b, c), suggesting that Pam2CSK4 + LPS incubation might involve activation of signalling pathways other than NF-kB.

TLR2/4 and TLR4/9 interact enhancing chemoattraction of a macrophage cell line

The release of MCP-1 regulates migration and infiltration of different cell types, especially monocytes/macrophages [28]. In addition, other chemokines might be secreted in ENS culture upon stimulation with MAMPs [29]. Therefore, we finally aimed to study whether the supernatants of challenged ENS primary cultures could induce the recruitment of immune cells.

MAMP-enriched DMEM culture medium did not induce RAW 264.7 cell migration to the lower compartment of the transwell inserts (data not shown). Similarly, conditioned medium from non-stimulated ENS culture did not increase migration when compared to DMEM culture medium (data not shown). However, conditioned medium obtained from LPS-stimulated ENS culture showed a tendency to increase migration to the lower chamber (59 % increase, P = 0.08; Fig. 8). This chemotactic effect was further enhanced to a 107 and 112 % increase by media conditioned by a combination of Pam2CSK4 or ODN 1826 with LPS, respectively (P < 0.001 for both, Fig. 8). Taken together, these data suggest that MAMPs themselves are not chemoattractant, but LPS alone or in combination with other TLR ligands elicits secretion of chemotactic substances by enteric neurons and resident immunocytes, promoting migration of RAW 264.7 macrophages.

Discussion

Since their initial characterisation in human in the late 90s [13], TLRs have been widely described in several tissues and cells, including central and peripheral nervous systems [15,30]. In recent years, the study of their role in the ENS has gained attention; however, there are still only a few works addressing their participation in the immune response to microbes. In this study, we report the expression and functionality of TLR2/4/9 in embryonic ENS primary cultures, emphasising in their interactions in terms of cytokine and chemokine production. Our results indicate that enteric neurons respond to MAMPs through secretion of inflammatory mediators, integrating their signals to trigger a tailored response to each challenge. Indeed, activation of TLR2 or TLR9 upon LPS stimulation enhanced macrophage chemoattraction,  The mean production of TNF-α, IL-6 or MCP-1 elicited by ODN 1826 + LPS was considered as 100 % effect. The expected additive effect of the combination of two ligands was calculated according to the model of functional interaction, represented by the equation E (ODN+LPS)expected = E (ODN)measured + E (LPS)measured -E (ODN)measured * E (LPS)measured . Statistical comparisons were performed by means of randomised block design analysis to minimise the random effects due to intrinsic culture responsiveness while potentiating different pro-inflammatory responses. Taken together, these findings suggest that TLRs confer the ENS the ability to discriminate microbial signals and expand inflammation by promoting particular proinflammatory microenvironments and interacting with resident immunocytes to chemoattract immune cells. These facts further support the idea that the ENS is an immunologically active tissue. Expression of TLRs within the ENS has been previously assessed by other groups in full-thickness tissue sections of the small intestine [18,25]. In this regard, colocalisation studies in SMP and LMMP whole-mounts from adult rat colon offer an improved perspective to discern immunoreactive cell populations. Our findings in these tissues confirm previous data about TLR2 expression, which had been described in both HuC/D + and GFAP + cells [19]. Conversely, we could only observe TLR4 staining in some subsets of enteric neurons [20,25], even though expression in EGCs has been already reported [18,21]. In this regard, our results agree with parallel studies in central nervous system cultures pointing out that neurons have more prominent TLR4 expression than astrocytes [29]. Finally, the TLR9 expression patterns we report here match with previous work that localise this receptor in neuronal somas and β-tubulin III + microtubules from dorsal root ganglia cultures [31] and various brain sections [32]. Interestingly, TLR distribution in embryonic ENS primary cultures was very similar to that of adult tissues, which might indicate that few modifications affect their localisation during development. Indeed, minor TLR2 and TLR4 expression variations have been observed in the mouse developing brain, whereas TLR9 increases in adulthood [32].

Functional TLRs activate the NF-kB pathway in several cell types, including neurons [33] and EGCs [21]. This transcription factor has been shown to convey signals from lipopeptides, LPS and class B CpG ODNs, leading to production of cytokines and chemokines [13,14,34].

Our results indicate that NF-kB is also involved in ENSmediated recognition of MAMPs, but additionally suggest that other signalling cascades may participate in cytokine and chemokine production. Indeed, inhibition with Bay 11-7082 resulted in significant decrease in TNF-α, IL-6 and MCP-1 release, but not complete abrogation. Therefore, it is likely that other pathways, such as the MAPK and the 5-adenosine monophosphate-activated protein kinase, are responsible for the observed mediator production after NF-kB blockade, as they have been previously shown to play important roles in production of TNF-α in embryonic ENS culture [7] and central nervous system neurons [29].

Release of cytokines and other immunomodulatory molecules is a common feature following TLR recognition of MAMPs [13]. However, a number of adaptions have developed in the gastrointestinal tract to prevent TLRdriven immune responses to commensal microbes [35]. Some of these adaptions might also occur in the ENS, as we observed no significant pro-inflammatory or chemoattractive responses in embryonic ENS culture upon Pam2CSK4 or ODN 1826 stimulation, despite the fact that they induced NF-kB activation. Similar observations have been reported in human EGCs, in which translocation into the nucleus of the NF-kB p50 subunit takes place after exposure to enteropathogenic bacteria, but not to probiotic strains [21]. These findings might underlie selective mechanisms to signal microbe presence while minimising responses to those non-pathogenic.

We have reported an active participation of resident macrophages in IL-6 and MCP-1 production, which poses an important limitation to the use of these embryonic ENS cultures for immunologic research purposes. Indeed, the neuronal or macrophage origin of the released cytokines could not be quantified, making it difficult to define the actual contribution of each cell type to the inflammatory microenvironment and chemoattractive effects. We propose a major role for enteric neurons because (1) expression of TLR2/4/9 was preferentially seen in these cells; (2) TNF-α expression, which has been localised by our collaborators in enteric neurons following LPS exposure [7], was altered upon TLR2/4 and TLR4/9 interactions; (3) MCP-1 reactivity following LPS stimulation accumulated majorly in neurons and (4) TLR2 up-regulation upon LPS challenge occurred in neurons [7]. Even though the involvement of resident immunocytes may be important, embryonic macrophages display important phenotypic differences with adult macrophages, such as no major histocompatibility complex (MHC) class II expression or poor cytokine production [36,37]. Similarly, embryonic EGCs did not release IL-6 upon MAMP challenge, which is in contrast with previous observations in adult cells [8,21].

Consistent up-regulation of TLR2 following MAMP or cytokine stimulation has been described in astrocytes, neurons and other cell types [7,24,38,39]. Induction of TLR2 mRNA is dependent on reactive oxygen species [39] and ultimately on NF-kB, which has different binding sites on the TLR2 promoter region [40]. Overexpression of this receptor is necessary to reach maximal NF-kB activation after MAMP recognition [38], possibly through signalling the formation of lipoproteins bearing lipid oxidation end-products [41,42]. Our experiments using a TLR2-blocking antibody agree with these explanations, showing that in our setup, TLR2 induction is crucial to obtain maximal TNF-α, IL-6 and MCP-1 production following LPS stimulation. Furthermore, increased chemoattraction after combined challenge of TLR2/4 suggests that these receptors may interact in ways other than those we determined to enhance the release of cytokines or chemokines. These additional interactions might implicate activation of additional signalling pathways, since we report reduced responsiveness to NF-kB inhibitors in ENS-mediated release of IL-6 and MCP-1 after Pam2CSK4 + LPS stimulation.

In addition to TLR2/4 cross-responsiveness phenomena [43,44], other TLR interactions after combined MAMP challenge have been documented [23,45,46]. Here, we demonstrate strong synergic responses in TNF-α release after costimulation with TLR4/9 ligands. Furthermore, IL-6 production and chemoattraction of macrophages by supernatants from ODN 1826 + LPS-treated embryonic cultures were also significantly enhanced, suggesting that additional chemokines might be induced after combined TLR4/9 challenge. Similar interactions between both receptors have been characterised in microglia, dendritic cells and bone marrow-derived macrophages [23,46,47]. The molecular mechanisms which may account for such effects are TLR9 up-regulation following LPS stimulation, as we and others have described [46], as well as enhanced signalling and duration through alternative pathways, such as the MAPK [47]. From a functional point of view, the ability to integrate different stimuli from the same microorganism contributes to discrimination of commensals and pathogens, inducing robust proinflammatory responses against the latter [48]. Recognition of Gram-negative motifs from both membrane (LPS) and nucleus (CpG DNA) is hence integrated in a synergic way, triggering strong pro-inflammatory responses and promoting chemoattraction of immune cells. In contrast, detection of Gram-positive and Gram-negative molecular patterns (Pam2CSK4 and LPS) lead to similar chemotactic responses but to a not so harsh pro-inflammatory environment, which might result into a milder priming of the subsequent effector response.

Different studies have addressed the involvement of central and peripheral nervous system neurons in defence against pathogens. These studies demonstrate that LPS stimulation activates NF-kB and MAPK pathways in these cells to induce production of cytokines like TNF-α and IL-6, and chemokines such as RANTES and KC [29,33]. Participation of enteric neurons in inflammation has been also demonstrated. Transgenic mice displaying increased and reduced numbers of neurons develop, respectively, more and less severe experimental colitis than wild-type littermates, suggesting that neurons play pro-inflammatory roles [4]. Furthermore, different signalling pathways are activated in these cells upon IL-1β or LPS challenge [7,49], leading to production of IL-8 and TNF-α [6,7]. Our findings show that enteric neurons express TLR2/4/9,signal through the NF-kB pathway and release IL-6 and MCP-1 in response to LPS challenge. These observations add further evidence to the pro-inflammatory roles of neurons, and set them as active players in neuroimmune interactions in the gastrointestinal tract, participating in microbial recognition and priming subsequent immune responses.

Conclusions

Our data suggest that the ENS is capable of recognising and discriminating between different microbial challenges, triggering robust or moderate inflammatory responses depending on the combination of TLR stimulated, as well as interacting with resident immunocytes to induce chemoattraction of macrophages. Given the fact that neurons are the main TLR-expressing cells in the enteric plexuses and are activated upon MAMP challenge to produce TNF-α, IL-6 and MCP-1, they arise as important players in enhancement of neuroimmune interactions.

Additional files

Additional file 1: TLR2/4/9 antibodies specifically stain ENS structures in WT mouse tissues. Tissue from WT, TLR2 −/− and TLR4 −/− mice was stained with TLR2/4 antibodies to test their staining specificity. In micrographs from WT mice, positive neurons were distinguished by strong immunoreactivity (white arrows) when compared to negative ones (open arrows). Knockout mice tissues displayed background immunoreactivity in neurons and unspecific secondary antibody precipitates. Pictures in this Add. File were taken in a Leica TCS SP5 microscope. Scale bars: 25 μm. The unconjugated TLR9 antibody (clone 26C593.2) raised in mouse could not be used to detect this receptor in mouse whole mounts, due to secondary antibody cross-reactivity with neurons, muscle fibres and supportive cells in the submucosa (TLR9 MOM). Conversely, the biotinylated form of this antibody displayed strong staining in the LMMP (black arrows) that was not observed in TLR9 −/− preparations (Biot TLR9). Antigen retrieval was achieved by boiling sections in 10 mM sodium citrate pH 6, followed by quenching of peroxidases and avidin/biotin blocking. After primary antibody incubation overnight at 4°C, sections were washed, incubated with ABC-HRP kit and developed with DAB (both from Vector Laboratories). Haematoxylin was used to counterstain tissues. Scale bars: 20 μm. (TIF 12107 kb) Additional file 2: TLR2/4/9 display minor colocalisation with GFAP in adult rat plexuses. TLR colocalisation with the EGC marker GFAP in whole-mount preparations of the SMP and the LMMP. White arrows point to enteroglial processes colocalising with TLR expression. Scale bars: 25 μm. (TIF 2537 kb) Additional file 3: TLR2 colocalises with glial structures in adult LMMP. Different insets from LMMP micrographs in Fig. 1 and Additional file 2 were generated to demonstrate colocalisation between TLR2 and GFAP-expressing EGCs Abbreviations DAPI, 4',6-diamidino-2-phenylindole; EGC, enteric glial cell; ENS, enteric nervous system; GFAP, glial fibrillary acidic protein; HRP, horseradish peroxidase; IBA-1, ionized calcium-binding adapter molecule; IkB, inhibitor of kB; IL-6, interleukin-6; LMMP, longitudinal muscle myenteric plexus; LPS, lipopolysaccharide; MAMP, microorganism-associated molecular pattern; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein-1; NF-kB, nuclear factor-kB; ODN, CpG oligonucleotide; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; SMP, submucosal plexus; TLR, toll-like receptor; TNF-α, tumour necrosis factor-α

Fig. 1
1TLR2/4/9 are expressed in enteric neurons of adult rat colon tissue. TLR localisation in whole-mount preparations of the SMP and the LMMP. B-tubulin III was used instead of HuC/D as neuronal marker for colocalisation with TLR9 due to antibody detection incompatibilities. Scale bars: 25 μm assessed the production of TNF-α, IL-6 and the monocyte chemoattractant protein (MCP)-1 upon TLR2/4/9 ligand challenge, as well as the involvement of the NF-kB pathway in these responses.

Fig. 2
2Embryonic ENS culture neurons express TLR2/4/9. a Agarose gel showing specific products of real-time PCR for the assayed genes in ENS culture (PC); water was used as a no-template control (H 2 O) and rat colon cDNA as positive control (+C). b TLR relative expression in ENS culture in basal conditions (n = 8). c Localisation of TLR in ENS culture ganglia. B-tubulin III was used instead of HuC/D as neuronal marker for colocalisation with TLR9 due to antibody detection incompatibilities. White arrows point to neuronal somas. Scale bars: 25 μm

Fig. 3
3MAMP stimulation induces activation of the NF-kB pathway in embryonic ENS culture. a Rat embryonic ENS culture was incubated for 8 h with the indicated MAMPs or the IkB phosphorylation inhibitor Bay 11-7082, and cell protein extraction and Western blot were performed to determine phosphorylated IkB (P-IkBα). B-actin was used as a loading control. b Time-course densitometric quantification of P-IkBα bands corrected to β-actin and related to basal activation levels; representative bands are shown in Additional file 4. Statistical analysis was performed independently for each ligand, using one-way ANOVA followed by Dunnett's test (n = 4 for each ligand and time point; *P < 0.05, **P < 0.01 and ***P < 0.001). c Representative micrographs showing NF-kB p65 subunit localisation in basal conditions (Ctrl) or 1 h after LPS challenge (LPS). White arrows point to neuronal nuclei displaying p65 positive staining. Scale bars: 25 μm increase in TLR4 (TLR4 LPS = 1.78 ± 0.28 vs. TLR4 Ctrl = 1.06 ± 0.13 folds;Fig. 5b) and TLR9 expression (TLR9 LPS = 2.13 ± 0.53 vs. TLR9 Ctrl = 1.1 ± 0.17 folds;Fig. 5b) was observed after LPS challenge. Finally, ODN 1826 stimulation did also up-regulate TLR9 expression (TLR9 ODN 1826 = 2.28 ± 0.43 vs. TLR9 Ctrl = 1.1 ± 0.17 folds;Fig. 5c).

Fig. 4
4ENS culture releases cytokines and chemokines in response to LPS. Rat embryonic ENS culture was stimulated for 24 h and culture supernatants were collected and centrifuged prior to measuring cytokine and chemokine secretion. a TNF-α (n = 4-8; LPS vs. control and Bay 11-7082 + LPS, ***P < 0.001; Bay 11-7082 + LPS vs. control, ***P < 0.001). b IL-6 (n = 4-8; LPS vs. control and Bay 11-7082 + LPS, ***P < 0.001). c MCP-1 (n = 4-8; LPS vs. control, ***P < 0.001; LPS vs. Bay 11-7082 + LPS, **P < 0.01). d Distribution studies show IL-6 and MCP-1 colocalisation with TLR4 + neurons. White arrows show TLR4 -IL-6-producing cells. Scale bars: 25 μm

Fig. 5
5TLR activation in ENS culture is associated to TLR2 up-regulation.

Fig. 7
7TLR4/9 combined stimulation elicits synergic pro-inflammatory responses in ENS culture. Embryonic ENS cultures were incubated with the specified combinations of MAMPs for 24 h. Randomised block design analysis followed by Tukey's post hoc test was applied to minimise statistical differences due to intrinsic culture responsiveness. a TNF-α (n = 4-9; ODN 1826 + LPS vs. LPS, ***P < 0.001; ODN 1826 + LPS vs. Bay 11-7082 + ODN 1826 + LPS, **P < 0.01). b IL-6 (n = 4-9; ODN 1826 + LPS vs. LPS, ***P < 0.001; ODN 1826 + LPS vs. Bay 11-7082 + ODN 1826 + LPS, *P < 0.05). c MCP-1 (n = 4-9; LPS vs. Bay 11-7082 + LPS, ***P < 0.001)

Fig. 8
8Stimulated ENS culture conditioned media induce chemoattraction of RAW 264.7 macrophages. RAW 264.7 macrophages were seeded in the upper chamber of 8-μm-pore transwell inserts and left to migrate for 4 h towards lower chambers filled with stimulated ENS culture conditioned media. Representative micrographs of the evaluated fields in the lower chamber of a transwell insert mounted upon unstimulated (Ctrl), LPS-stimulated (LPS) or ODN 1826 + LPS-stimulated (ODN 1826 + LPS) ENS culture medium. Scale bars: 200 μm. Number of migrating cells per field (n = 4, with two replicates for experiment; LPS vs. unstimulated ENS culture supernatant, #P = 0.08; Pam2CSK4 + LPS and ODN 1826 + LPS vs. unstimulated ENS culture supernatant, ***P < 0.001). Randomised block design analysis was performed to minimise the variability in migration due to RAW 264.7 passage, followed by Tukey's post hoc test

(arrows). (TIF 2932 kb) Additional file 4: Time-course activation of NF-kB and dose-dependence. Time-course activation after stimulation of ENS cultures with (A) 100 ng/mL Pam2CSK4, (B) 100 ng/mL LPS, (C) 1 μM ODN 1826 or (D) 1 μM control ODN 1826. (E) Quantification of the overall NF-kB activation after treatment with different doses of the indicated MAMPs. Densitometric measurements for each experiment were represented as time-course curves by means of the GraphPad Prism 5.0, and the area under the curve was calculated for each replicate. The resulting values were used to compare the NF-kB inducing strength of each TLR ligand (n = 4; *P < 0.05, **P < 0.01 and ***P < 0.001, one-way ANOVA followed by Dunnett's post hoc test). Statistics were performed independently for each ligand. (TIF 13694 kb) Additional file 5: Resident immunocytes, but not EGCs, express IL-6. ENS cultures were costained for S100β (EGC marker), IBA-1 (macrophage marker) and IL-6 after 24 h stimulation with LPS. Scale bars: 25 μm. (TIF 1791 kb) Additional file 6: MCP-1 staining is more prominent in TLR4-expressing cells. ENS cultures were stimulated for 24 h with LPS and costained for MCP-1 and TLR4. Although cells displayed different staining intensities for TLR4, positive populations showed increased MCP-1 intensity (open arrows). MCP-1 was also observed in TLR4-negative cells, presumably macrophages, but the staining intensity was lower (white arrows). Scale bars: 25 μm. (TIF 857 kb) Additional file 7: Neurons are the only TLR4-expressing cells in ENS cultures. ENS cultures were stimulated for 24 h with LPS and costained for Hu C/D and TLR4. IL-6 antibody was also added to visualise macrophages (arrows). TLR4 was located exclusively in Hu C/D positive cells. Scale bars: 25 μm. (TIF 1806 kb)

Table 1
1List of primers used for real-time RT-PCR analysisGenes
Sense primer
Antisense primer
Reference

rS6
CCAAGCTTATTCAGCGTCTTGTTACTCC
CCCTCGAGTCCTTCATTCTCTTGGC
NM_017160

rTLR2
CAGATGGCCAGAGGACTCA
AATGGCCTTCCCTTGAGAG
ENSRNOT00000013025.3

rTLR4
GGATGATGCCTCTCTTGCAT
TGATCCATGCATTGGTAGGTAA
NM_019178.1

rTLR9
TCCGTGACAATCACCTCTCTT
GGTCCAGGTCTCGCAGATT
NM_198131.1

Table 2
2Comparison between expected additive effects and measured effects of TLR ligand interactions in embryonic ENS cultureCytokine
TLR ligand combination
Expected additive effect (%)
Measured effect (%)
N
P

TNF-α
Pam2CSK4 + LPS
53.9 ± 8.5
52.1 ± 7.8
5
0.744

ODN 1826 + LPS
51.9 ± 8.5
100 ± 20.4
5
0.02

IL-6
Pam2CSK4 + LPS
43.7 ± 5.7
45 ± 8.5
5
0.729

ODN 1826 + LPS
42.3 ± 5.9
100 ± 24.8
5
0.048

MCP-1
Pam2CSK4 + LPS
91.5 ± 3.6
85.2 ± 5.3
5
0.102

ODN 1826 + LPS
88.2 ± 4.3
100 ± 5.4
5
0.02

AcknowledgementsWork in EF's group is also supported by Generalitat de Catalunya Funding JFB and CR were supported by personal grants from the Spanish Ministry of Education & Science (AP2005-1160 and AP2006-02705, respectively). All material, reagents and services required to perform the experiments included in this manuscript were financed by the grants (BFU2006-15063-C03-01/BFI) and the Spanish Ministry of Health (PS09/01127) assigned to EF.Availability of data and materialsAll data supporting the conclusions of this manuscript are shown in the text, tables and figures included in the main body of the manuscript and in Additional files 1, 2, 3, 4, 5, 6 and 7.Authors' contributions JFB participated in the design of experiments, performed studies, analysed data and drafted the manuscript. AB carried out the immunoassays. EE and CR performed immunofluorescence studies. MN participated in conception of study and design of experiments. EF conceived and coordinated study, participated in data analysis and drafted the manuscript. All authors have read and approved the final version of the manuscript.Competing interestsThe authors declare that they have no competing interests.Consent for PublicationNot applicable.Ethics approval and consent to participateIn this research article, we include data obtained from primary cultures prepared from rat embryonic intestines, as well as tissues from adult rats.All these experiments were approved by the Ethical Committee of the • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal Submit your next manuscript to BioMed Central and we will help you at every step:
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Interactive Informed Consent: Randomized Comparison with Paper Consents
2013

M C Rowbotham
J Astin
K Greene
S R Cummings
Interactive Informed Consent: Randomized Comparison with Paper Consents

PLoS ONE
8358603201310.1371/journal.pone.0058603Received October 9, 2012; Accepted February 7, 2013;United States of America Editor: Tobias Preis, University of Warwick, United Kingdom Funding: The study was funded by departmental funds at California Pacific Medical Center Research Institute (CPMCRI) under the control of MCR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: MCR, JA, and KG have no conflict of interest with reference to Mytrus, the maker of the interactive iPad device. The study was funded by departmental funds at CPMCRI under the control of MCR. SRC is a founder and officer of Mytrus but was not involved in any contact with subjects or statistical analysis. JA was responsible for final data analysis. Declaration of competing interests for author SRC because of his relationship to Mytrus. SRC is Chief Scientific Officer and Board Chairman at Mytrus. The study was not funded by Mytrus. No other author has any relationship to Mytrus. No other author has a competing interest to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. *
Informed consent is the cornerstone of human research subject protection. Many subjects sign consent documents without understanding the study purpose, procedures, risks, benefits, and their rights. Proof of comprehension is not required and rarely obtained. Understanding might improve by using an interactive system with multiple options for hearing, viewing and reading about the study and the consent form at the subject's own pace with testing and immediate feedback. This prospective randomized study compared the IRB-approved paper ICF for an actual clinical research study with an interactive presentation of the same study and its associated consent form using an iPad device in two populations: clinical research professionals, and patients drawn from a variety of outpatient practice settings. Of the 90 participants, 69 completed the online test and survey questions the day after the session (maximum 36 hours post-session). Among research professionals (n = 14), there was a trend (p = .07) in the direction of iPad subjects testing better on the online test (mean correct = 77%) compared with paper subjects (mean correct = 57%). Among patients (n = 55), iPad subjects had significantly higher test scores than standard paper consent subjects (mean correct = 75% vs 58%, p , .001). For all subjects, the total time spent reviewing the paper consent was 13.2 minutes, significantly less than the average of 22.7 minutes total on the three components to be reviewed using the iPad (introductory video, consent form, interactive quiz). Overall satisfaction and overall enjoyment slightly favored the interactive iPad presentation. This study demonstrates that combining an introductory video, standard consent language, and an interactive quiz on a tablet-based system improves comprehension of research study procedures and risks.

Introduction

Informed consent is the cornerstone of human research subject protection

Research studies are growing in complexity and duration. Concerns persist about the adequacy of the consent process, in particular whether subjects are fully informed and actually comprehend the study. The consent form is a detailed -and generally long -description of the protocol, possible benefits and harms, study procedures, and patient rights. Most often, the first step is giving a paper consent form to the potential subject to review. Staff and the study investigator may assist the potential subject in reviewing the consent form and answering questions, but this may be insufficient and is generally not standardized. Many subjects then sign -and thereby provide consent -without understanding the study purpose, procedures, risks, benefits, and their rights. Documentation of comprehension is not required and rarely obtained before a subject is allowed to sign the form. The standard process of informed consent is risky because the subject may have complications that he or she did not realize were inherent in the research or procedure. Misunderstandings can become contentious.

Features of an ideal informed consent process

Understanding may be improved by providing potential subjects with several options to hear, view or read the consent form at their own pace. If done interactively, subjects can be given immediate feedback about their level of understanding of study procedures, risks, and so on, thereby theoretically increasing their overall comprehension. A test can then verify comprehension of key study elements before the subject is allowed to sign the ICF and enter the study.

The need for a comparative study

There has been little study of interactive methods to improve consent. A few studies have tested informed consent on computers -with colorful graphics and multimedia -and compared them with written documents before elective surgeries with mixed results about whether understanding and satisfaction were improved [1]. Furthermore, the consent forms evaluated were for surgery or single procedures, not complex multi-visit clinical research studies that present the greatest risk of an inadequate consent process. There have been no studies of whether an interactive process, including home-based learning, audiovisual options, and the requirement to complete an interactive quiz improves understanding of the study or procedure.

Interactive Consent System

An online interactive consent system (Mytrus, San Francisco) has been developed with several features that might improve potential subjects' understanding of the study, including video and audio summaries of the consent form with options to read the document online or in printed form. The consent process in this system includes an interactive quiz.

This prospective randomized study compared the IRB-approved paper ICF for an actual clinical research study with an interactive presentation of the same study and its associated ICF using an iPad device in two populations: clinical research professionals, and patients drawn from a variety of outpatient practice settings. Outcome measures included comprehension, delayed recall, and ratings of user acceptability of the standard paper ICF or the interactive consent process (as delivered via an iPad and including a comprehension quiz).

Materials and Methods

Overview

The prospective randomized study, approved by the Institutional Review Board (IRB) of California Pacific Medical Center, was conducted in two parts:

In Part 1, clinical research professionals (primarily IRB panel members) at California Pacific Medical Center Research Institute who agreed to participate were randomized by computer to learn about a chemotherapy neuropathy clinical research study by reviewing either the IRB-approved standard paper consent form or the same consent form via an iPad based interactive system. After orientation to the study by a research coordinator, the session took place in the subject's office and could last up to onehour. Each participant completed an online survey the next day (minimum 18 hours post-session) that consisted of a test of delayed recall, included questions about the user experience, and free text space for comments. Participants had to complete the online survey within 36 hours of the study session.

In Part 2, patients drawn from a variety of clinical practices at CPMC who agreed to participate were randomized by computer to learn about the same clinical research study by reviewing either the IRB-approved paper consent form or via the same iPad based interactive system used for Part 1. The session took place after they finished their medical appointment in either a separate room or a quiet area of the waiting room. To be eligible, participants had to be between age 18-80, literate in written and spoken English, have internet access available to complete the online survey the next day, be able to stay after their medical visit is completed for the one-hour study session. Participants could not have cancer or have undergone cancer chemotherapy. As in Part 1, each participant completed an online survey the next day that tested delayed recall and includes questions about the user experience. Participants had to complete the online survey within 36 hours of the study session.

Consent form used to assess the interactive system

The complete consent form from a previously approved longitudinal study in patients with cancer undergoing chemotherapy of the possible nerve damaging effects of chemotherapy ('chemotherapy neuropathy study') was used because it provided the needed level of complexity required for assessing how an interactive consent would perform when used for subjects enrolling in an actual trial. The purpose of the chemotherapy neuropathy study was to evaluate the development of peripheral neuropathy (a problem with the nerves that carry information between the brain and spinal cord and the rest of the body that can produce pain, loss of sensation, and an inability to control muscles) and neuropathic pain (chronic pain caused by nerve damage) in patients undergoing chemotherapy. The chemotherapy neuropathy study did not involve an intervention or invasive procedures. The three study visits over 6 months relied on questionnaires, simple bedside sensory testing, and quantitative sensory testing (QST) to look at changes in peripheral nerve function over time in patients receiving chemotherapy. The consent form included relatively standard information about compensation and outcomes of study-related injury or harm to subjects was included in the consent form as follows: ''Investigators have no special program to provide compensation if injury occurs during biomedical research. If you are injured or made ill as a result of participation in this study, treatment will be made available. Because insurance companies may not pay for research-related costs, they may not pay for an injury resulting from your participation in this study. Any costs not paid by your insurance company will be your responsibility.''

Session procedures

With IRB approval, an informational letter in lieu of signed informed consent was used in order to maintain participant anonymity. The informational letter contained all required consent elements including the study purpose (that the study is designed to test their comprehension and recall of a consent form that could be used in a clinical research study), the study design, what will be asked of the participant if he/she agrees to participate, risks of participation, the voluntary nature of participation, and compensation ($27 gift card). The study was completely anonymous; no personally identifying information was recorded on any study documents.

Interested subjects were then given the iPad and asked to enter the following information: their assigned subject username, year of birth, gender, and the registration number on their gift card for reimbursement purposes. The iPad then randomly assigned the subject to either the paper or interactive iPad group in a ratio of 1:1. The paper group returned the iPad and received a paper consent form for review; the iPad group proceeded with the electronic interactive version of the consent. The interactive version began with an informational video outlining the main features of the study, then presented a series of screens of the same consent form as in the paper participants (in the same font), followed by quiz questions at the end of the chemotherapy neuropathy consent form.

Participants were encouraged to fully review the consent form before signaling to the research coordinator when they felt they had completed their review to a sufficient degree that they would actually sign (or are sure they would refuse to sign) the consent form to participate in the cancer chemotherapy study. The iPad recorded the amount of time spent on the device and the number of correct/incorrect questions. The study coordinator recorded the number of minutes spent reviewing the consent for the paper group.

Participants randomized to the interactive iPad consent were encouraged to complete the quiz that followed the consent form. The quiz consisted of 7 multiple-choice questions covering the following areas: (1) the main reason for the study; (2) the role of the study doctor; (3) what happens 'if you agree to participate'; (4) risks of quantitative thermal sensory testing; (5) reimbursement for participation; (6) what happens 'if you were accidentally injured'; (7) total duration of the study. Each multiple-choice question had 4 possible answers. If the participant marked the wrong answer on the iPad, the device would signal the answer was incorrect and return to the screen on the consent relevant to that particular question before allowing the subject to answer again. Participants had three chances to answer correctly before the device moved to the next question.

At the end of the session, the study coordinator provided an information sheet with the participant's subject ID, the website URL for accessing the followup online questionnaire, time window for online questionnaire completion, and contact information for the study coordinator in case the participant has questions or problems accessing the online survey. Participants were also given their gift card and notified that the gift card had no value until the online questionnaire was completed and that lost or stolen gift cards could not be replaced due to the anonymous nature of the study. The exact time window for completion of the online questionnaire was reviewed. Participants were told that they could not complete the online survey until the next day and if they did not complete the survey within a 36 hour window they would no longer be able to access the website and would not be compensated. In actuality, the gift cards were already activated to ensure that all participants would be able to use them as soon as they completed the online survey. Once the subject has completed the online questionnaire, their participation in the study was complete.

The web survey began with questions about age, gender, years of education, and race/ethnicity. Twelve multiple-choice comprehension and recall questions were then asked, summarized as follows: 1) main reason for the study 2)

if you think you have had an injury as a result of the study, whom should you call? 3) if you have questions about your rights as a participant in this study, who should you call? 4) if you decide to participate, then which of the following will happen? 5) which statement about quantitative thermal sensory testing is true? 6) risks of quantitative thermal sensory testing 7)

if you were accidentally burned during the study and needed treatment, which is true? 8) if you agree to participate, which one of the following will you receive? 9) total duration of study 10) which one of these statements is true? (you will have QST; study doctor may prescribe treatments for painful neuropathy that develops during the study; if you stop chemotherapy, you will not need to have further visits for this study; results of your QST will be sent to your doctor) 11) during the quantitative thermal sensory testing procedure, you will be asked to: (continue until probe feels painfully cold; push button whenever you want probe to return to neutral temp; rate the heat sensation 0-100; all of above) 12) which of the following statements is true? (if you agree to participate you must complete all 3 exams; you may stop for any reason at all; if you stop before final you will not receive payment for participating; none of the above)

Five of the questions (1,4,6,8,9) were duplicates of the questions appearing in the iPad quiz. Participants were then asked about their willingness to participate in the cancer chemotherapy study if they actually had cancer and were an eligible subject; user experience questions; and a free text box for likes/dislikes/ problems they perceived with the system they were assigned to (paper or interactive).

Methods of Data Analysis

The target sample size was set at a low number (N = 15) for Part 1 because the aim of Part 1 was to pilot-test the two presentations of the consent document (paper and electronic/ tablet-based) for differences in comprehension and collect data on the functionality of and user experience with the interactive iPad consent. The sample size for Part 2 was determined based on an estimated 20% difference in comprehension scores between those randomized to the standard written consent and subjects receiving the interactive iPad consent. With 27 subjects per group (N = 54), there would be 95% power to detect this level of difference at the p , .05 level. Participants who failed to complete the follow-up online survey provided no evaluable data and were not included in the statistical analysis. Estimating a 25-30% failure rate in completing the follow-up online survey, a target sample size of 75 participants for Part 2 was set. Depending on the outcome measure, chi-squared, unpaired t-test, and Wilcoxon signed ranks tests were used to determine if significance was present at the p , .05 level.

Results

Participation

In Part 1, 15 subjects agreed to participate and 14 completed the followup online survey. In Part 2, 75 subjects agreed to participate and 55 completed the online survey. Of Part 2 participants, 66% were female. The mean age was 50 (18-35: 18%; 36-55: 35%; 56-80: 57%). Overall, the Part 2 group was well educated: 43% had a college degree and another 30% had an advanced degree (only 4% had no education following high school diploma). Consistent with San Francisco's population, the largest racial/ethnic groups were Caucasian (76%) and Asian (17%).

Online survey results (Table 1) Table 1 provides a breakdown of results by question for each group.

Part 1: Among the 14 research professionals, comparing iPad vs. Paper groups, there was a definite trend (p = .07, chi-squared test) in the direction of iPad subjects scoring better (mean correct = 77%) compared with paper (mean correct = 57%). For the questions that were duplicates of the quiz presented on the iPad, the differences were greater. Participants randomized to the iPad were correct 90.2% of the time on the five duplicated questions, and correct only 68.1% of the time for the seven non-duplicated questions. In contrast, participants randomized to review the paper consent scored similarly for duplicated and non-duplicated questions (53.2% and 59.4%). Interestingly, although the research professionals scored very well on questions related to compensation, they scored poorly on technical questions related to quantitative sensory testing (the main procedure being used to detect nerve damage) at 38% correct in the iPad group and zero% correct in the paper group.

Part 2: Among the 55 patients, comparing iPad vs. Paper groups, there was a significant difference (p , .001, chi-squared test) in total score on the online next day survey with iPad subjects scoring better (mean correct = 75%) compared with standard paper consent (mean correct = 58%). Participants randomized to the iPad did much better than paper on the five duplicated questions (85.4% vs 63.4%). However, the iPad group also outperformed the paper group to nearly the same extent on the seven non-duplicated questions (67.6% correct vs 54.1% correct).

Time spent reviewing the consent form

Considering subjects in both parts of the study, the total time spent reviewing the paper consent was 13.2 minutes (range 4.0-21.5). Subjects randomized to the iPad spent an average of 22.7 minutes total (range 12.4-50.4) on the three components to be reviewed, significantly more than the paper subjects (p,.001, unpaired t-test). In the iPad subjects, a mean of 445 seconds (7.4 minutes) was spent viewing the video (three subjects watched it twice), 685 seconds (11.4 minutes) were spent reviewing the consent document, and 235 seconds (3.92 minutes) were spent on the quiz (range 2-6 minutes).

There was no significant correlation between time spent reviewing the consent form and comprehension as measured by % correct responses in the online survey (whether looking at groups together or separately).

User experience and user feedback questions

The research professionals in Part 1 who were randomized to the iPad reported slightly greater overall satisfaction on the 10 point scale (7.5 vs. 7.0), and iPad users reported slightly more enjoyment/interest (2.3 vs 1.7 on the 4 pt. scale). Only two participants provided feedback related to the consent form, writing "The consent form is overloaded with information. The way definitions of medical terms are incorporated is very awkward." Another wrote ''I was randomized to the iPad however in reality would always prefer a hard copy of any ICD so that I have it for reference and if I was a patient I could take it away with me and share it with family members etc.''

In Part 2, 62% of iPad subjects said they would participate in the study compared with 69% of paper-based subjects (p = NS). Stated reasons for non-participation in order of frequency were: ''worry about time'' (25%); ''lack of interest'' (21%); ''privacy concerns'' (17%); ''concern about QST pain'' (12%); and, ''skin biopsy'' (4%). In terms of user experience, the differences were also not statistically significant. There was a trend in the direction of greater overall satisfaction on the 10 pt. scale for iPad users (7.7 vs. 6.5). Differences between reported enjoyment and understandability of the consent were not significant.

User feedback for Part 2 was noteworthy for concerns about the amount and way information is presented in the consent form (''overloaded with information'', ''babble of words'', ''boring and made me not want to read the details''). A few subjects randomized to the paper version wished they had had the opportunity to see the iPad version (''Illustrations and interactive programs would be much smarter and a better way to explain what is really going to occur. A form is just a form and shouldn't be.''). Multiple users asked for a bullet point format with access to background information if more was desired. User feedback in Part 2 was also noteworthy for concerns about who would pay for treatment of any injury related to the study. Some subjects felt very strongly that there should not be any possibility of a research subject being financially responsible for care in the event of an injury. A few subjects were concerned about the possible response of their health insurer (''Would they have a negative response to my participating with the study?'').

When all participants were combined, the differences in overall satisfaction rating favoring the iPad approached significance (p = .09, Wilcoxon signed-ranks test). The iPad users reported slightly greater overall enjoyment (3.2 vs. 2.9; p,0.05, Wilcoxon signedranks test), but self-reported differences in comprehension of the consent were not significant.

Discussion

A major issue in human subjects research is the quality of the consenting process. IRBs and external monitors are able to audit whether all the paper consent forms are signed, but unless additional steps are taken to ensure comprehension the quality of the interaction between research personnel and potential subjects is unknown. Riecken and Ravich's 1982 study examined informed consent in a group of research patients in Veterans Administration hospitals [2]. Even though signed consent forms were on file, 28% did not know they were research subjects. Readability analysis of the consent forms indicated a college education was necessary for comprehension, and also found subjects were more likely unaware of their participation when someone other than the investigator took exclusive responsibility for explaining the study. Three studies have examined how long research subjects actually spend 'reading' consent documents before signing. Baren and colleagues conducted a study in the emergency department of the informed consent process for an intimate partner violence survey [3]. A research assistant first gave a scripted verbal description of study. Of those agreeing to participate (1,312 or 82%), only about half (53%) read the brief (2 pages, 11 paragraphs) consent form at all. Of those that did read the consent form, the majority spent less than one minute reviewing it before signing. The study by McNutt and colleagues provides a similar example [4]. After providing information verbally to two samples of women, research assistants observed that over half of the women read their consent forms in thirty seconds or less before signing. Lee and colleagues gave 73 subjects a comprehension quiz right after the consent form was signed [5].

Of six elements of the form, about half the subjects were not aware of two or more study-related risks. Actual time spent reading the consent form was a mean of 2 minutes, less than the estimated 4-5 minutes needed based on standard reading rates for high school students and a 1300 word consent.

In their systematic review, Flory and Emanuel raised the distinction between rote memorization of the content of the ICF and true understanding of the research study [6]. Of twelve trials of multimedia interventions (computer or video in place of or in addition to the written ICF), only three significantly increased understanding. They concluded that for most of the trials of enhanced consent forms that showed a significant effect, the simulation of the consent process was unrealistic in that there was no active discussion, only a reading of the form. In such a setting, the form becomes the participant's only source of information and this exaggerates the impact of changes to the form. In research, the informed consent process is already formalized through federal regulations that require a written consent form, and video-based and computer-based interventions may not add much to this relatively thorough disclosure process. In contrast, the five trials with testing and/or feedback all improved apparent understanding. Flory and Emanuel also pointed out the strong links between higher educational level, higher reading level, and higher understanding scores. The 2009 study by Tait and colleagues showed that baseline knowledge and younger age, as well as the use of an interactive video in the setting of non-research cardiac procedures were associated with 'complete' understanding of the ICF [1].

The recent commentary by Schenkel and Meisel elaborated three practical issues to be considered in improving comprehension of ICFs [7]. First, more is not always better. More information in consent forms may produce the opposite effect of the subject spending even less time reviewing the form before signing. Second, timing matters -in both clinical practice and research the informed consent process may take place immediately before a procedure, after the patient or research subject is already psychologically committed to proceed and the optimum time for weighing risks and benefits has passed. Third, technology can help. Strategies that do not involve physicians (because they are often in a rush), such as an interactive consent aided by a nurse or other educated health care professional, are needed. However, the physician-investigator must play an important role as the study by Riecken and Ravich showed the subjects were ''more likely unaware'' when someone other than investigator took exclusive responsibility for explaining the study.

Creative, non-technological interventions continue to advance. Antoniou and co-workers attempted to ensure subjects would stay engaged by constructing a three level patient information sheet for an online survey study [8]. The first level was brief but provided enough information for most to make a decision; the second level corresponded to a 'standard' patient information sheet. Of the 552 participants, 77% went to first level (23% never accessed the information sheet); only 18% went to the second or third level. Fink and colleagues asked patients to recount what they had been told in the informed consent to confirm understanding the key risks of the (non-research) surgical procedure they were to undergo [9]. In this large study of 575 patients, there was a small but significant effect on comprehension compared to subjects who were consented without using ''repeat-backs'' (68.2% vs 71.4% correct on the test). Low cost, non-technological solutions to improving consent form comprehension are spreading, such as including a comprehension quiz or having both the research coordinator and the potential subject initial each page before proceeding. Some research projects utilize an online video or slide deck describing the study, in some cases including the actual consent form.

In the present study, freedom to stop participating at any time was the only question where participants exceeded an 80% correct threshold. Part 1 and Part 2 subjects assigned to the paper consent scored below 50% on multiple questions (four questions with Part 1 and three questions with Part 2). Interestingly, research professionals did not do particularly well with the more technical questions regarding quantitative thermal sensory testing. Overall, the research professionals performed almost exactly the same as the participants drawn from clinic waiting rooms with both the iPad and paper versions.

How should the results of the current study be interpreted and applied? The central aim of this randomized study was to compare an interactive, tablet-based presentation of a clinical research consent form with a paper version of the same consent on comprehension of study procedures and risks. The interactive system was tested in a simulated situation where research professionals and patients were asked to pretend they had cancer and were considering entering a research study. They knew that comprehension was going to be tested after a time delay. This likely added to the length of time spent reviewing the consent form compared to the earlier studies of Baren, McNutt, and Lee [3][4][5]. Among both research professionals and patients, next-day comprehension was better in subjects randomized to the interactive iPad consent form. The iPad group received study information in multiple ways; via a brief video before viewing the consent form, and followed by a 7-item quiz where incorrect responses resulted in seeing relevant text from the consent form before trying to answer again. For those online survey questions that had been a part of the quiz presented at the end of the iPad version of the consent form, the probability of a correct answer was higher in both Part 1 and Part 2 participants. However, simple repetition accounts for only part of the improvement as participants randomized to the iPad also performed better on questions that hadn't been part of the quiz. The iPad participants spent more time with the device, but the amount of time spent reviewing the actual consent document was actually shorter (13 minutes for paper, 11.4 minutes on the iPad). The introductory video about the study was relatively brief at just under six minutes in length, and a few subjects chose to view it twice. The quiz can be time consuming if a participant makes multiple incorrect choices on the same question. If the interactive consent were to be used in an actual study, participants making too many errors would not be allowed to proceed further before reviewing the areas of misunderstanding with the investigator or appropriate staff member. The errors could guide the discussion toward specific areas of the study.

Although the interactive consent system used adds cost to conducting a study, features such as automatic archiving of consent documentation are useful independent of the enhanced consenting process. An interactive consent process on a tabletsuch as an iPad -would allow the patient to go through the consent process in the office and even take the tablet home. For long term studies or therapies requiring multiple visits, the iPad is applicable to research and clinical practice as the portal for completing symptom ratings, reporting outcomes and adverse events, and completing questionnaires. In summary, this study demonstrates that combining an introductory video, standard consent language, and an interactive quiz on a tablet-based system improves comprehension of procedures and risks.

Table 1 .
1Post-review online survey test question results.group1
group 1
group 2
group 2

PLOS ONE | www.plosone.org
March 2013 | Volume 8 | Issue 3 | e58603
AcknowledgmentsThe authors thank Leigh Pruneau and Angie Stone of the CPMC Institutional Review Board for their assistance.Author Contributions
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