Allergen‐free extracts from birch, ragweed, and hazel pollen activate human and guinea‐pig submucous and spinal sensory neurons

Background Non-allergenic, low molecular weight components of pollen grains are suspected to trigger changes in gut functions, sometimes leading to inflammatory conditions. Based on extensive neuroimmune communication in the gut wall, we investigated the effects of aqueous pollen extracts (APE) on enteric and spinal sensory neurons. Methods Using Ca2+ and fast potentiometric imaging, we recorded the responses of guinea-pig and human submucous and guinea-pig dorsal root ganglion (DRG) neurons to microejection of low (<3 kDa) and high (≥3 kDa) molecular weight APEs of birch, ragweed, and hazel. Histamine was determined pharmacologically and by mass spectrometry (LC–MS/MS). Key Results Birch APE<3kDa evoked strong [Ca+2]i signals in the vast majority of guinea-pig DRG neurons, and in guinea-pig and human enteric neurons. The effect of birch APE≥3kDa was much weaker. Fast neuroimaging in human enteric neurons revealed an instantaneous spike discharge after microejection of birch, ragweed, and hazel APE<3kDa [median (interquartile range) at 7.0 Hz (6.2/9.8), 5.7 Hz (4.4/7.1), and 8.4 Hz (4.3/12.5), respectively]. The percentage of responding neurons per ganglion were similar [birch 40.0% (33.3/100.0), ragweed 50.8% (34.4/85.6), and hazel 83.3% (57.1/100.0)]. A mixture of histamine receptor (H1–H3) blockers significantly reduced nerve activation evoked by birch and ragweed APEs<3kDa, but was ineffective on hazel. Histamine concentrations in ragweed, birch and hazel APE's < 3 kDa were 0.764, 0.047, and 0.013 μM, respectively. Conclusions Allergen-free APEs from birch, ragweed, and hazel evoked strong nerve activation. Altered nerve-immune signaling as a result of severe pollen exposure could be a pathophysiological feature of allergic and non-allergic gut inflammation

Calcium Imaging of Nerve-Mast Cell Signaling in the Human Intestine

Introduction: It is suggested that an altered microenvironment in the gut wall alters communication along a mast cell nerve axis. We aimed to record for the first time signaling between mast cells and neurons in intact human submucous preparations.Methods: We used the Ca2+ sensitive dye Fluo-4 AM to simultaneously image changes in intracellular calcium [Ca+2]i (%ΔF/F) in neurons and mast cells. Data are presented as median with interquartile ranges (25/75%).Results: We recorded nerve responses in 29 samples upon selective activation of 223 mast cells by IgE receptor cross linking with the antibody mAb22E7. Mast cells responded to mAb22E7 with a median [Ca+2]i increase of 20% (11/39) peaking 90 s (64/144) after the application. Only very few neurons responded and the median percentage of responding neuronal area was 0% (0/5.9). Mast cell activation remained in the presence of the fast sodium channel blocker tetrodotoxin. Specific neuronal activation by transmural electrical field stimulation (EFS) in 34 samples evoked instantaneously [Ca+2]i signals in submucous neurons. This was followed by a [Ca+2]i peak response of 8%ΔF/F (4/15) in 33% of 168 mast cells in the field of view. The mast cell response was abolished by the nerve blocker tetrododoxin, reduced by the Calcitonin Gene-Related Peptide receptor 1 antagonist BIBN-4096 and the Vasoactive Intestinal Peptide receptor antagonist PG97-269, but not by blockade of the neurokinin receptors 1–3.Conclusion: The findings revealed bidirectional signaling between mast cells and submucous neurons in human gut. In our macroscopically normal preparations a nerve to mast cell signaling was very prominent whereas a mast cell to nerve signaling was rather rare

Compound 48/80 (c48/80) evoked Ca⁺⁺ transients in primary cultured enteric neurons. A

<p>shows two images of a Fluo-4 AM stained ganglion before c48/80 application and at the time of the maximal response. <b>B</b> illustrates the Ca⁺⁺ signal of the neuron marked by an arrow in A. Two consecutive c48/80 applications (marked by the bars below the traces) evoked comparable responses. In this ganglion all 8 neurons responded to c48/80. <b>C</b> confirms that responses to c48/80 application were not caused by activation of mechanosensors because spritz application of Krebs solution using the same application parameters had no effect. <b>D</b> illustrates that c48/80 evoked [Ca⁺⁺]_i increase is concentration dependent.</p

Compound 48/80 (c48/80) evoked activation of visceral afferents <i>in vivo</i>. A

<p>Jejunal afferent discharge stimulated by c48/80 administered intraluminally. A second administration 5 min later evoked a similar afferent response but subsequent administrations are completely desensitized, despite similar mechanosensitivity during the luminal perfusions. <b>B</b> In sensitized animals intraluminal application of the antigen egg albumin (EA) evoked a response similar to that observed with c48/80 and which too rapidly desensitized so that subsequent antigen failed to evoke a response. <b>C</b> Following desensitization to c48/80, intraluminal antigen still evoked a response similar to that in naïve control animals. When the order of administration was reversed and the response to antigen was desensitized, c48/80 was still able to evoke a response <b>D</b> illustrates mean data from these cross-desensitization experiments. The left panel shows the afferent response to saline or antigen in sensitized animals that had received pretreatment with vehicle (saline, N = 11) or c48/80 (N = 5). The panel on the right shows similar data for the response to vehicle (saline) or c48/80 in naïve control animals (N = 9) or sensitized animals following desensitization to antigen (N = 6). The response to c48/80 was significantly augmented after desensitization to antigen (P<0.01).</p

Compound 48/80 (c48/80) evoked spike discharge in primary cultured enteric neurons. A

<p>Di-8-ANEPPS stained ganglion; the dye incorporates into the outer membrane revealing the outline of individual neuronal cell bodies. <b>B</b> illustrates action potential discharge in response to two consecutive c48/80 applications (marked by bars below the traces) from the neuron marked by white arrow in A. The traces show responses during three recording periods with non-recording periods of 6 s in between. In this ganglion 17 of 19 neurons responded to c48/80. <b>C</b> PGP9.5-positive cultured enteric neurons. Lack of c-kit immunoreactivity demonstrated lack of mast cells in the culture. <b>D</b> demonstrates that c48/80 application still evokes spikes in cultures treated with a combination of the H₁ and H₂ blockers pyrilamine (1 µM) and ranitidine (10 µM), respectively. <b>E</b> shows green PGP9.5-positive neurons in a whole mount preparation of the guinea pig submucous plexus (left panel) and red c-kit-positive round and smooth mast cells (some marked by white triangles in center panel), which are morphologically distinct from the spindle shaped interstitial cells of Cajal. The right panel shows the merged image of the two stainings.</p

Neuronal activation by mucosal biopsy supernatants from irritable bowel syndrome patients is linked to visceral sensitivity

Based on the discomfort/pain threshold during rectal distension, irritable bowel syndrome (IBS) patients may be subtyped as normo- or hypersensitive. We previously showed that mucosal biopsy supernatants from IBS patients activated enteric and visceral afferent neurons. We tested the hypothesis that visceral sensitivity is linked to the degree of neuronal activation. Normo- and hypersensitive IBS patients were distinguished by their discomfort/pain threshold to rectal balloon distension with a barostat. Using potentiometric and Ca(2+) dye imaging, we recorded the response of guinea-pig enteric submucous and mouse dorsal root ganglion (DRG) neurons, respectively, to mucosal biopsy supernatants from normosensitive (n = 12 tested in enteric neurons, n = 9 tested in DRG) and hypersensitive IBS patients (n = 9, tested in both types of neurons). In addition, we analysed the association between neuronal activation and individual discomfort/pain pressure thresholds. The IBS supernatants evoked Ca(2+) transients in DRG neurons and spike discharge in submucous neurons. Submucous and DRG neurons showed significantly stronger responses to supernatants from hypersensitive IBS patients as reflected by higher spike frequency or stronger [Ca(2+)]i transients in a larger proportion of neurons. The neuroindex as a product of spike frequency or [Ca(2+)]i transients and proportion of responding neurons correlated significantly with the individual discomfort/pain thresholds of the IBS patients. Supernatants from hypersensitive IBS patients caused stronger activation of enteric and DRG neurons. The level of activation correlated with the individual discomfort/pain threshold pressure values. These findings support our hypothesis that visceral sensitivity is linked to activation of peripheral neurons by biopsy supernatant