High prevalence and functional effects of serum antineuronal antibodies in patients with gastrointestinal disorders

Antineuronal antibodies can be associated with both gastrointestinal (GI) and brain disorders. For example, antibodies against the potassium channel subunit dipeptidyl‐peptidase‐like protein‐6 (DPPX) bind to neurons in the central nervous system (CNS) and myenteric plexus and cause encephalitis, commonly preceded by severe unspecific GI symptoms. We therefore investigated the prevalence of antineuronal antibodies indicative of treatable autoimmune CNS etiologies in GI patients.


| INTRODUCTION
In recent years, autoantibodies targeting neuronal epitopes have received increasing attention in patients with neurological syndromes, as the antibodies can be directly pathogenic leading to neuronal dysfunction and various neuropsychiatric abnormalities. 1 Similar etiologies were observed in gastrointestinal (GI) disorders, for example the new clinical entity called "autoimmune gastrointestinal dysmotility", a type of autoimmune dysautonomia associated with various antineuronal antibodies targeting among others voltage-gated calcium channels, voltage-gated potassium channels, acetylcholine receptors, and intracellular Hu (ANNA-1) proteins. 2 Traditionally, autoantibodies such as anti-Hu antibodies were described in paraneoplastic disorders, characteristically in patients with small cell lung carcinoma and paraneoplastic GI motor dysfunction. 3,4 However, tumor-negative syndromes are increasingly considered, for example, with antibodies against ganglionic nicotinic acetylcholine receptors which cause a well-defined syndrome called autoimmune autonomic ganglionopathy (AAG). 5 Symptoms include orthostatic hypotension, reduced pupil constriction, and GI dysmotility, reflecting failure of the sympathetic, parasympathetic, and enteric nervous system (ENS). 6 Autoantibodies are suspected to play a role also in more common GI syndromes such as irritable bowel syndrome (IBS). They might cause neuropathic degeneration of enteric neurons in a subgroup of patients, 7,8 although other studies have not found increased frequencies of neuronal antibodies in functional GI disorders compared to healthy controls. 9 Additionally, a rat model of postinfectious IBS has demonstrated that antibodies against cytolethal distending toxin B cross-react with the cytoskeletal protein vinculin in the ENS. 10 Clearly, further research is needed to better understand the link between functional GI disorders and autoantibodies. The same holds true for other common GI diseases, such as inflammatory bowel disease (IBD), in which antineuronal autoantibodies 11 could either represent an epiphenomenon of neuronal degeneration or a contributing factor.
Only recently, a new fascinating overlap has been discovered between autoantibodies and the fields of Neurology and Gastroenterology. Antibodies directed against dipeptidyl-peptidaselike protein-6 (DPPX), a regulatory subunit of Kv4.2 potassium channels, bind to hippocampus, cerebellum, striatum, and the myenteric plexus. 12, 13 We could show that anti-DPPX antibodies are directly pathogenic ex vivo in both, brain and bowels, by regulating the activity of enteric neurons and membrane expression of DPPX and Kv4.2 in hippocampal neurons. 14 The majority of DPPX antibody-positive patients complained of GI symptoms, including constipation, abdominal pain, nausea, vomiting, early satiety, and seemingly unexplained diarrhea with weight loss that often antedated immunotherapy-responsive neurological symptoms. 12,[14][15][16][17][18][19] It is tempting to speculate whether earlier detection of DPPX antibodies on a gastroenterology ward could have led to immunotherapy and prevented the severe neuropsychiatric abnormalities in these patients. Toward this goal, we analyzed the associations of antineuronal antibodies with common GI diseases in a routine clinical setting. For this, we prospectively collected serum samples of patients with diverse GI symptoms. Sera were examined for the frequencies of anti-DPPX, anti-Hu, and a large panel of further antineuronal antibodies using cell-based assays, immunofluorescence staining on rat brain and intestine sections, and functional assays on guinea pig colon.

| Study population
We tested serum samples of 107 adult patients from the Charité-Universitätsmedizin Berlin, Department of Gastroenterology. This study was approved by the Charité University Hospital Institutional Review Board (EA1/080/15), and written informed consent was given by all subjects. Patients were selected for any kind of GI complaints (e.g., diarrhea, constipation, often more than one) being severe enough to require in-patient treatment, comprising unexplained causes or being the major symptoms of Crohn's disease (CD), ulcerative colitis (UC), IBS, or other diagnoses. Patients with an infectious cause of symptoms were excluded. Clinical documentation for each patient included major diagnoses of GI diseases, medication, coexisting neoplasia, autoimmune diseases, and accompanying neurologic or psychiatric symptoms. Serum samples of 44 healthy blood donors, not complaining of any GI symptoms, were used as a control group. Serum samples were coded and stored at −80°C.

| Immunohistochemistry
The serum of each patient or control was tested for the presence of IgG and IgA antibodies using both, rat intestinal and rat brain tissue. For brain sections, adult Wistar rats were sacrificed, the brain removed, frozen in 2-methylbutane, and stored at −20°C until 20μm sections of the cerebellum and hippocampus were made with a cryostat. For intestinal sections, rats were perfused with 4%

Key Points
• Antineuronal autoantibodies can cause both, neurological and gastrointestinal symptoms. We here determined the prevalence and functional effects of antibody-positive human sera.
• Autoantibodies against central and enteric nervous system antigens were frequent in patients with diverse gastrointestinal diseases. Antibody-positive sera led to increased intestinal secretion in functional assays.
• Antineuronal antibodies potentially contribute to the clinical phenotype and might help to identify gastrointestinal patients who could benefit from immunotherapy.
Future work should relate functional effects to antibody target epitopes and disease course.

| 3 of 11
LÜTT eT aL. paraformaldehyde (PFA), the intestine removed, washed, placed in 4% PFA overnight, followed by 30% sucrose. After freezing in 2-methylbutane, 15μm cryostat sections of different parts of the small intestine and colon were used for immunohistochemistry.
Brain sections were fixed with 4% PFA for 10 minutes. Brain and intestinal sections were washed in phosphate-buffered saline (PBS) and preincubated for 1 hour at room temperature with blocking solution which either contained 5% normal goat serum, 2% bovine serum albumin, and 0.1% Triton X-100 (for IgA detection protocol) or 4% horse serum and 0.5% Triton X-100 (for IgG). Sections were incubated with serum overnight at 4°C at screening dilutions of 1:200 on brain and 1:500 on intestinal tissue. Sections were washed with PBS and incubated with the secondary antibody for 2 hours at room temperature: fluorescein (FITC)-labeled goat anti-human IgA

| Evaluation
Fluorescence stainings were viewed using a CKX41 cell culture microscope from Olympus (Hamburg, HH, Germany); confocal and widefield photographs were taken with a LEICA DMI8 (Wetzlar, HE, Germany).
Evaluation of fluorescence intensity and anatomical distribution was performed by two experienced histologists using a semiquantitative fluorescence score. This scale defined "0" as fluorescence not exceeding the background of a representative negative control, "1" = weak fluorescence (occasionally seen background intensity in serum stainings), "2" = moderate fluorescence (clearly positive with reproducible anatomical distribution), and "3" = very high fluorescence (seen, e.g., in a positive control serum containing anti-Hu antibodies). Intensities of ≥2 were defined as antibody-positive.
In comparison with control stainings, the slides were evaluated by fluorescence microscopy, using 1:10 for brain tissue and 1:100 for intestinal plexus as cutoff for positivity.

| Organ bath
The motility of guinea pig muscle strip preparations of the distal colon was measured as described previously and were performed accord-

| Ussing chamber
The effects of serum samples on intestinal secretion were tested with the Ussing chamber voltage clamp technique as explained previously in detail. 21 Briefly, colonic mucosa/submucosa tissue specimens were mounted in Ussing chambers (Easy Mount chambers; Physiologic Instruments, San Diego, CA, USA) with an exposed tissue area of 0.5 cm 2 . Both sides of the tissue were bathed separately in 5 ml carbogenated Krebs solution at 37°C. We measured the electrogenic, active short circuit current I sc in μA/cm 2 across the epithelium with positive currents indicating a net ion current from the basolateral to the apical side. After an initial equilibration period of 30 minutes, the viability of the tissue was tested with EFS (Grass SD-9; Astro-Med Inc., West Warwick, RI, USA) via platinum electrodes that were placed on either side of the tissue (100 pulses with 20 V, 10 Hz, 1 ms pulse duration). Serum samples were added to the basolateral solution. As in the organ bath experiments, we analyzed changes ∆I sc relative to the baseline I sc . Statistical test procedures for paired or unpaired data are indicated in the results section (SigmaPlot 12.5; Systat Software Inc.). A P < .05 was considered as significant.

| Patient cohorts and clinical data
Of the 107 patients, 60 (56.1%) were female and 50.1 ± 18 years

| Frequency of autoantibodies in patient subgroups
Autoantibodies against ENS neurons, detected as fluorescence labeling of an intensity score ≥2, were found in 21 of 107 patients and in F I G U R E 1 Antineuronal autoantibody frequency in patients and controls. (A) Antineuronal autoantibodies against ENS were significantly more frequent in patients (19.6%) than in controls (4.5%). (B) Antineuronal antibodies varied between patient subgroups and were most frequent in IBS patients. (C) Anti-CNS antibodies occurred significantly more frequent in patients (20.6%) than in controls (4.5%). (D) Similar to anti-ENS antibodies, anti-CNS antibodies showed a differential distribution among patient subgroups with frequent presence of both IgA and IgG isotypes in immunostainings of brain but not intestine. CD, Crohn's disease (n = 42); HC, healthy controls (n = 44); IBS, irritable bowel syndrome (n = 13); UC, ulcerative colitis (n = 16); others (n = 36) Female patients (24 of 60; 40%) had ENS/CNS antibodies significantly more often than male (9 of 47, 19.1%) patients (P = .02; chi-squared test

| Patterns of intestinal plexus immunoreactivity
Immunohistochemistry of patient sera on intestinal sections resulted in a wide range of staining patterns. In four cases, single ganglion cells of the myenteric or submucosal plexus could easily be discriminated and showed cytoplasmic reactivity (Figure 2A). Sera titers were usually high ranging from 1:1000 to 1:20 000. Several further sera with titers between 1:500 and 1:80 000 bound rather diffusely to plexus neurons, staining somatodendritic and nuclear structures ( Figure 2B). Similar patterns, but with lower intensities and slightly different shapes, were found with further four patient sera. Only one patient showed an intense (1:20 000 titer) nuclear staining ( Figure S1A) that was not restricted to ENS cells but was detectable in almost every cell in the intestine and brain. The finding likely corresponds to the antinuclear antibodies (ANA) and extractable nuclear antigen antibodies present in this patient who had the diagnosis of mixed connective tissue disease.
Another pattern of ENS reactivity was the dendritic or axonal signal found in three sera with titers between 1:5000 and 1:20 000 ( Figure 2E-G). Double-labeling with MAP2 ( Figure 2F) or Tau ( Figure 2G) confirmed the dendritic or axonal staining, respectively.
Apart from antineuronal immunoreactivity in the ENS, several sera showed binding to further structures in the intestine (Table S1) probably mucins ( Figure S1C). The significance of this is unclear as the use of human tissue instead of rat colon is essential for the determination of goblet cell antibodies. 22 The lamina propria was another structure commonly stained with variable patterns by patient sera in different intensities and patterns (8.4% of patients vs 0% of controls) ( Figure S1D).

| Patterns of brain immunoreactivity
The predominant immunostaining pattern, seen in all 22 anti-CNS positive cases, was a cellular signal in different parts of the brain, with titers ranging from 1:200 to 1:80 000. The hippocampus frequently showed a strong signal (19 of 22 positive sera; Table S2), with variable predominance of a more intracellular ( Figure 3A, antibody-negative serum shown for control in Figure 3B) vs neuronal surface distribution ( Figure 3C). Similarly in the cerebellum, neurons of the cerebellar cortex showed strong immunoreactivity in 15 sera, for example, Purkinje neurons ( Figure 3D) or granule cells ( Figure 3E). Apart from the staining of neuronal cell bodies, immunoreactivity was also seen against somatodendritic or astrocytic structures in several brain areas ( Figure 3F-H). Two sera, one of a patient with unexplained GI symptoms, one of a CD patient, showed a GFAP-like pattern with a strong staining of the radial processes of Bergmann glia and the glia limitans ( Figure 3G and H). Interestingly, the reactive isotype in both sera was IgA with a titer of 1:1000.
Several sera contained antibodies that reacted with structures beyond neurons and glia cells. For example, further staining patterns included nuclear or nucleolar signals, large blood vessels, or capillaries. Immunoreactivity to blood vessels was also frequently found in control sera (18.7% patients, 20.5% controls) ( Figure S1E-G).

| Functional studies in organ bath and Ussing chamber
Patient sera with the highest anti-ENS antibody titers in the staining experiments were selected and compared to sera of age-and sex- were not significantly different between antibody-positive and antibody-negative patients ( Figure 4A and B). Responses to EFS consisted of an "on response" during the train of stimulation pulses and an "off response" starting immediately after cessation of the stimulation.
We could not find significant differences in the EFS responses before and after addition of the sera (Figure 4C-E).
In the Ussing Chamber experiments, we used the same sera and dilution (1:200) as in the organ bath experiments. Responses to all antibody-positive patient sera specifically targeting the ENS and to the one polyreactive serum showed a similar response, consisting of a slow and sustained increase in short circuit current (I sc ) starting approximately 10 minutes after addition of the serum to the serosal chamber. This increase in I sc was significantly larger than the responses to sera from antibody-negative patients ( Figure 4F and G). In fact, we observed in no case an increase in I sc after addition of the sera from antibody-negative patients (∆I sc = −0.65 ± 1.3 μA/cm 2 for n = 6 antibody-negative patient sera vs 7.2 ± 2.7 μA/cm 2 for n = 5 antibodypositive patient sera, P = .00013, t test).

| DISCUSSION
In this comprehensive analysis, we could show that the prevalence of antineuronal antibodies against the ENS and CNS is significantly higher in patients across a wider range of GI disorders compared to healthy controls. Immunofluorescence analyses revealed that autoantibodies reacted with diverse surface and intracellular structures in the intestine and brain, including not only neuronal or astrocytic elements but also epithelium and endothelium. Autoantibodies were more common in female than male patients and showed the highest prevalence in patients with IBS. However, differences were not statistically significant for most patient subgroups in our exploratory study.
Higher numbers of patients are needed for sufficient statistical power in future cohorts. Additionally, no pathognomonic patterns were observed that could clearly distinguish patient groups. The findings rather suggest a high variability of antibody titers, anatomical regions and epitopes.

| Pathogenic antibodies or immunological bystanders?
The most intriguing question is whether the autoantibodies found in the patient sera contribute to the variable clinical spectrum of GI symptoms or-in particular in the many patients with neuropsychiatric comorbidities-to CNS symptoms. Although the cross-sectional design of this pilot study does not provide a definite answer to this question, data suggest a clear tendency.
The assumption of antibody pathogenicity is supported by the common binding of patient sera to soma and processes of ENS neurons, while ANA were seen in few cases only. As ANA commonly associate with autoimmunity, their absence argues against autoimmune diseases as confounders in patients. Similarly, the autoantibody prevalence did not differ significantly in patients with and without coexisting autoimmune diseases in the present cohort. However, we cannot exclude that the autoantibodies are a rather general sign of However, given the limited amount of available patient material, we cannot exclude that significant effects would have been detected with higher serum concentrations, even though IgG-mediated cytotoxic effect in cell cultures has been shown at similar dilutions that we used here. 24 The lack of serum effects on smooth muscle/myenteric plexus structures suggests that the yet to be explored target of the serum antibodies must be specifically expressed in mucosa/submucosa preparations but not within the muscle layers.
Knowledge on the exact antibody target might be a prerequisite for prediction of pathogenic effects. Along these lines, previous stud- The pathogenic effects on GI dysfunction were less clear in studies on achalasia and gastro-esophageal reflux disease. Although antibodies bound to enteric neurons in the small intestine and esophagus, there was no specificity to subpopulations of enteric neurons or to disease groups suggesting that the antibodies were an epiphenomenon. 11 Similarly, a study on patients with achalasia demonstrated effects of sera on neurochemical coding in the myenteric plexus and on motility in fundic specimens despite the absence of antienteric antibodies. 35 Autoantibodies might potentially be a secondary phenomenon in some patients, for example as a response to neurodegeneration in enteric ganglionitis 36 or related to an independent autoimmune process. In this way, they could be a disease biomarker rather than driving the disease.

| Can the antibody search in GI patients accelerate diagnosis of neurological diseases?
An aim of this study was to experimentally approach potential overlapping autoimmunity in the brain and in the gut, as can be observed in anti-DPPX encephalitis. Given that none of the examined patient sera harbored antibodies against DPPX or one of the here tested brain/ gut epitopes, the data support the notion that these well-defined F I G U R E 4 Sera with high titers of anti-ENS IgG had no effects on smooth muscle activity but enhanced mucosal secretion in guinea pig colon preparations. (A) Addition of serum (1:200) containing high-titer ENS-reactive IgG or non-ENS-reactive IgG/IgA caused no changes in the spontaneous motility pattern of guinea pig colon. (B) Although some preparations showed small contractions or relaxations after addition of the sera, these changes were not statistically different between IgG/IgA-negative and IgG-positive sera (P > .1 for all comparisons, Wilcoxon signed rank test). (C, D) Responses of longitudinal muscle preparations to EFS before and after addition of non-ENS-reactive IgG/IgA serum (C) or ENSreactive IgG serum (D). (E) Comparison of responses to EFS before and after addition of patient sera showed no significant differences (P > .1 for all comparisons, paired t test or Wilcoxon signed rank test). (F) Anti-ENS IgG-positive (left) but not IgG/IgA-negative serum (right) caused an increase in short circuit currents (∆ Isc ) in Ussing chamber experiments. (G) The change in ∆ Isc was significantly larger for anti-ENS IgG-positive sera (n = 5) than for IgG/IgA-negative sera (n = 6). Scale bar in C also applies to D. All ENS-reactive or non-ENS-reactive serum samples are represented by the same symbols throughout B, E and G encephalitis-related antibodies are rare in a routine gastroenterology setting. Anti-DPPX encephalitis should still be a differential diagnosis and antibody testing should be performed when unexplained severe diarrhea, weight loss, and other intestinal symptoms precede or go along with neurological symptoms. 14 Future studies should include further antibodies targeting CNS but also enteric epitopes, for example, acetylcholine receptors.
Nonetheless, the results of our functional assays suggest that the here detected autoantibodies could potentially participate in bowel dysfunction, in particular in the regulation of secretion. Future studies will determine whether the presence of such antibodies can help to identify patients with GI disorders who might benefit from a more specific, antibody-directed therapy. Analyses will have to include the specificity of these antibodies, that is, the target epitopes, affinity, and causal relationship in functional assays.
Even more interesting, we found a high prevalence of brainbinding antibodies in patients with GI diseases, often with overlapping reactivity to ENS and CNS neurons. In one patient, antibodies were directed against the brain protein LGI1, and in two patients, IgA antibodies showed a GFAP-like pattern. Both LGI1 and GFAP antibodies were recently associated with different forms of encephalomyelitis 37,38 which could not be recognized in the clinical phenotype of the here described patients. Shared pathology is also suggested by the fact that many neuron-targeting autoantibodies were of the IgA isotype, which is normally thought to result from immunity in the GI tract and mucosal surfaces. IgA antibodies are indeed increasingly considered in CNS autoimmunity, such as in progressive cognitive impairment, 39,40 limbic encephalitis, 41 or antigliadin IgA antibodies in celiac disease cross-reacting with neuronal proteins. 42 It will be an interesting field of future research to determine the target epitopes of autoantibodies contained in patient sera. A promising strategy might be the generation of monoclonal recombinant human antibodies as recently shown by our group for braintargeting autoantibodies in patients with encephalitis. 43 Monoclonal antibodies will enable functional assays in exactly determined concentrations, which would be superior to human samples. In addition, further studies should include functional data of larger, clearly defined patient groups and age-matched controls and should analyze correlations of sera-derived autoantibodies with disease initiation and progression.
Taken together, antineuronal autoantibodies are significantly more prevalent in patients with GI dysfunction than in healthy controls. Our data stimulate the question whether serum-derived antibodies can participate in the clinical phenotype, even though the size of the patient cohort and the study design do not allow correlations between antibody prevalence, functional data, and patient subgroups. Nonetheless, routine testing for well-established antineuronal antibodies seems not warranted on a routine gastroenterology ward at present, with only minimal risk of overlooking treatable autoimmune diseases such as anti-DPPX encephalitis.
Further research should aim at the identification of the antibodies' target epitopes and prospective correlations with clinical diagnoses and symptoms.