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

Distension evoked mucosal secretion in human and porcine colon in vitro

It was suggested that intestinal mucosal secretion is enhanced during muscle relaxation and contraction. Mechanisms of mechanically induced secretion have been studied in rodent species. We used voltage clamp Ussing technique to investigate, in human and porcine colonic tissue, secretion evoked by serosal (Pser) or mucosal (Pmuc) pressure application (2–60 mmHg) to induce distension into the mucosal or serosal compartment, respectively. In both species, Pser or Pmuc caused secretion due to Cl- and, in human colon, also HCO3- fluxes. In the human colon, responses were larger in proximal than distal regions. In porcine colon, Pmuc evoked larger responses than Pser whereas the opposite was the case in human colon. In both species, piroxicam revealed a strong prostaglandin (PG) dependent component. Pser and Pmuc induced secretion was tetrodotoxin (TTX) sensitive in porcine colon. In human colon, a TTX sensitive component was only revealed after piroxicam. However, synaptic blockade by ω-conotoxin GVIA reduced the response to mechanical stimuli. Secretion was induced by tensile rather than compressive forces as preventing distension by a filter inhibited the secretion. In conclusion, in both species, distension induced secretion was predominantly mediated by PGs and a rather small nerve dependent response involving mechanosensitive somata and synapses

Distension of human colonic tissue by serosal pressure application.

Pressure application (20 mmHg) from the serosal side led to a visible distension of human colonic mucosa/submucosa preparations into the mucosal compartment of the Ussing chamber. (MP4)</p

A porcine intestinal tract (with the stomach and small intestines removed) from a mature hybrid pig after removal from the carcass.

Porcine tissue samples for Ussing chamber experiments were taken from the transverse colon approximately 10 cm distal from the last spiral gyri centrifugales of the spiral colon. (TIF)</p

Secretory response to tensile forces.

Prevention of tissue distension during Pser by a mucosal placed filter significantly reduced the secretory response in human ((a); Wilcoxon test) and porcine (b) colonic tissue (unpaired t test); p values above brackets. Data shown are the medians with the 25th and 75th quartiles as a box plot and the minima and maxima as a whisker plot. N numbers are given in parenthesis.</p

Involvement of prostaglandins in mediating the distension induced secretory response.

Addition of piroxicam prior to Pser reduced secretory response in human (a) and porcine tissue (b). Simultaneous incubation with TTX further reduced the response in human tissue (human: Friedman test with Dunn’s multiple comparison test; porcine: Kruskal-Wallis test with Dunn’s multiple comparison test; p values above brackets). Data shown are the medians with the 25th and 75th quartiles as a box plot and the minima and maxima as a whisker plot. N numbers are given in parenthesis.</p

Ionic basis of the distension induced secretory response.

Distension induced secretory responses in human (a) and porcine (b) colonic tissue evoked by Pser was significantly reduced by depletion of chloride (Cl-) and Cl- and bicarbonate (HCO3-), respectively (human: Wilcoxon test; porcine: Paired t test; p values above brackets). Data shown are the medians with the 25th and 75th quartiles as a box plot and the minima and maxima as a whisker plot. N numbers are given in parenthesis.</p

TTX sensitivity of the distension induced secretory response.

Incubation of tissues with TTX prior to Pser (and Pmuc in porcine tissue) led to a reduced secretion only in the human ascending colon (a), and to a significant reduction in the secretory response in the porcine colon (b). Paired t test and Wilcoxon test; p values above brackets. Data shown are the medians with the 25th and 75th quartiles as a box plot and the minima and maxima as a whisker plot. N numbers are given in parenthesis.</p

Secretory response to different pressure values.

In human (a) and porcine (b) colonic tissue preparations, secretory responses to Pser significantly increased with increasing stimulus strength. Human: Intestinal regions pooled; Kruskal-Wallis test (p th and 75th quartiles as a box plot and the minima and maxima as a whisker plot. N numbers are given in parenthesis.</p

Location of human colonic specimens and diseases underlying the surgery.

Location of human colonic specimens and diseases underlying the surgery.</p