Introduction

Inflammatory bowel disease (IBD) is characterised by inflammatory flares with excruciating pain, for which effective analgesics are lacking. Cocaine- and amphetamine-regulated transcript (CART) peptide regulates peripheral pain in mice.¹ However, its role in visceral nociception as well as the underlying molecular signalling mechanism in primary sensory neurons remains elusive. CART is highly expressed in TRPV1-positive colon-projecting sensory neurons.² In addition, the enzyme prohormone convertase 1/3, which is involved in processing CART into its bioactive fragment 55-102, is upregulated in colonic biopsies of IBD patients (internal data), suggesting a potential role for the peptide in colonic pain.

Aims & Methods

Here, we aimed to test if CART would affect firing of the colon-innervating lumbar splanchnic nerve (LSN) as model of visceral nociception and characterise its effect and signalling pathway in sensory neurons innervating the colon. Studies were performed using tissue from male and female C57BL/6J mice euthanised by cervical dislocation and exsanguination in accordance with Schedule 1 of the Animals (Scientific Procedures) Act 1986. Thoracolumbar dorsal root ganglia (DRG) neurons were dissociated and cultured overnight for Ca²⁺ imaging using Fluo-4 AM as previously described.³ Cells were perfused with extracellular solution (ECS) alone or supplemented with either rat CART 55-102, (20-300nM, ECS), thapsigargin (10mM, DMSO), xestospongin C (1mM, ECS), edelfosine (10mM, ECS), capsaicin (10mM, ethanol) or KCl solution (60mM, ddH₂O) for respective challenge or pre-treatment. All conditions were blinded and randomised. Colorectum with attached lumbar splanchnic nerve was carefully removed and mounted on a tissue bath superfused with Krebs buffer as previously described.³ Rat CART 55-102 was superfused at 5ml/min, resulting in a final concentration of 200nM. Statistical analysis was performed using Student´s t test or ANOVA and Dunnett´s multiple comparisons test. Data is displayed as mean ± SEM.

Results

CART elicited a concentration-dependent increase in intracellular Ca²⁺ in DRG neurons, which at the highest concentration tested (300nM) induced a significant increase in intracellular Ca²⁺ in 29.2 ± 2.98% of DRG neurons relative to 8.35 ± 1.25% of vehicle responders (p < 0.0001). 55.05% of CART-responsive neurons were putative nociceptors due to co-sensitivity to capsaicin. Further investigation revealed a statistically significant reduction in the proportion of CART-sensitive neurons compared to CART (300nM) alone following pre-treatment with edelfosine, a phospholipase C inhibitor (10.5 ± 3.88%, p = 0.006), thapsigargin, a SERCA pump inhibitor (7.25 ± 1.78%, p = 0.001), or xestospongin C, an inositol triphosphate receptor inhibitor (6.74± 1.87%, p = 0.006). Furthermore, tissue bath application of CART (200nM) elicited a marked transient increase in ongoing LSN discharge, which increased from a baseline of 12.8 ± 3.9 (spikes/s) to a peak firing of 19.7 ± 6.1 spikes/s (p = 0.0385).

Conclusion

CART stimulates intracellular Ca²⁺ release in capsaicin-sensitive neurons consistent with a role in pain processing. This response was mediated by the release of Ca²⁺ from intracellular stores such as the endoplasmic reticulum and attenuated by inhibitors of the G_q pathway, suggesting activation of a G_q-coupled GPCR. The pronociceptive activity of CART on nerve firing suggests a potential role in colonic pain. Further research is necessary to identify the role of CART in IBD.

References

1. Ohsawa M, Dun SL, Tseng LF, Chang J, Dun NJ. Decrease of hindpaw withdrawal latency by cocaine- and amphetamine-regulated transcript peptide to the mouse spinal cord. Eur J Pharmacol. Jul 7 2000;399(2-3):165-9. doi:10.1016/s0014-2999(00)00374-5
2. McGuire C, Boundouki G, Hockley JRF, et al. Ex vivo study of human visceral nociceptors. Gut. Jan 2018;67(1):86-96. doi:10.1136/gutjnl-2016-311629
3. Higham JP, Bhebhe CN, Gupta RA, et al. Transcriptomic profiling reveals a pronociceptive role for angiotensin II in inflammatory bowel disease. Pain. Jan 29 2024;doi:10.1097/j.pain.0000000000003159

Disclosure

The PhD research is partially funded by Metrion Biosciences.

Introduction

A wide range of relationships between the microbial community and various environmental or individual factors have been proposed. Monozygotic and dizygotic twins provide a unique model for assessing host-microbial interactions. The mycobiome has been increasingly implicated in the development of several diseases, but knowledge of the dynamics of the fungal community, particularly in twins, has not been addressed.

Aims & Methods

In this work, we investigated the fungal community in a twin cohort and explored its relationship with associated genetic and environmental factors through in-depth analysis.
The twin cohort consisted of 106 well-characterised and systematically studied monozygotic and dizygotic pairs (212 stool samples). DNA was extracted and PCR reactions were performed to sequence the lower gastrointestinal microbiome and the mycobiome community. The bacterial community was characterised using the V1-V2 region of the 16 rRNA gene and the fungal community was analysed using the ITS1-ITS2 region.

Results

The fungal community, represented by the sum of all fungal sequences, showed a significantly higher heterogeneity than the bacterial community. Several individual and genetic factors, such as the zygotic status of the twins (monozygotic vs. dizygotic), as well as sex, mode of birth, breast-feeding as newborns or living in the same household, were independent of the total number of fungal sequences. The bacterial community was significantly more abundant than the fungal community, the most common representatives of which were Candida and Geotrichum with sometimes very high abundances. Saccharomyces was also common in the cohort, but only in low abundance, other fungi with a wider distribution were Cladosporium or Penicillium. The environmental and individual factors mentioned above were also correlated with the individual fungal genera, but there were no significant correlations found here either. The next step was to correlate the bacterial and fungal communities with each other, and strong negative correlations were found with certain bacteria such as Prevotella.

Conclusion

Genetic factors in twins are not associated with dynamics in the faecal fungal community and were the subject of high inter-individual variability. However, there is a strong association between bacterial and mycobial interaction, which requires further in-depth understanding of microbial interactions.

Disclosure

No conflict of interest to declare