Intestinal neuropathies have been described in a variety of functional and inflammatory gastrointestinal disorders. Although the degenerative alterations and neuronal are often associated to a lymphocytic inflammatory infiltrate, the underlying mechanisms remain obscure. Potential etiologic factors are neurotropic viruses, since they are able to specifically infect neurons and cause cell damage through direct or indirect mechanisms. Among the neurotrophic viruses the HSV-1 shows several interesting features. HSV-1 is most commonly known to latently infect the trigeminal ganglion of cranial nerve innervating the face and oral mucosa but latent HSV-1 has also been found in the nodose ganglia innervating the gastrointestinal tract in humans possibly as a consequence of swallowed viral particles.
The working hypothesis of my PhD work was that swallowed HSV-1 can infect the enteric nervous system and locally trigger an immune mediated response damaging neurons.
To assess the ability of α-herpesvirinae (HSV-1, HSV-2, VZV) to infect human ENS we performed a prospective cohort study using human surgical ileocolonic specimens of patients affected by colon carcinoma (CC), Crohn’s disease (CD), ulcerative colitis (UC) and diverticular disease (DD). A total of 121 patients were studies and presence of herpes simplex virus (HSV) types-1 and -2 and varicella-zoster virus (VZV) DNA was examined by RT-PCR in the muscle intestinal layer including the myenteric plexus. The α-herpesviruses DNA was detected in 44,5 % and 52 % of ileum and colon samples, respectively. No significant differences in the viral DNA positivity were observed among the different groups whereas the viral DNA load was significantly higher in samples of patients affected by Crohn’s disease. Moreover, HSV DNA was detected more frequently than VZV DNA, 46% vs 3%, respectively.
In order to mimic the human α-herpesvirinae infection and spread in ENS, my research group has established an original murine model of persistent ENS infection with HSV-1. Using this model we demonstrated that HSV-1 infection resulted in time-dependent intestinal neuromuscular abnormalities, providing the first in vivo evidence for the ability of neurotropic viruses to reach and damage the ENS.
Since significant intestinal motility anomalies and damage of enteric nerves were evident 8 - 10 weeks (W) post intragastric (IG) challenge with HSV-1, I attempted to characterize the adaptive immune response to HSV-1 and whether was causing the neuromuscular abnormalities.
In the longitudinal muscle myenteric plexus (LMMP) I observed an increase in CD3+ lymphocytes starting at 6 W and persisting up to 10 W after viral IG inoculum. At 8 weeks post IG viral inoculum, HSV-1 reactive CD3+CD4+IL4+ and CD3+CD8+INF-γ+ were detected, whereas at 10 W activated CD8+ T-cells infiltrated the LMMP. By immunohistochemistry CD3+ lymphocytes were demonstrated in the myenteric ganglia of HSV-1 infected mice 6-10 W post IG viral challenge.
To verify the involvement of CD8+ and CD4+ T-cells in HSV-1 induced dysmotility we performed depletion experiments by administration of monoclonal antibody. ENS damage and the neuromuscular anomalies were observed only after depletion of CD8 T cells at 8 and 10 W after viral IG inoculum.
To validate the role of lymphocytes in HSV-1 induced ENS dysfunction, CD3+CD4+ and CD+CD8+ cells were isolated from the LMMP of mice 8-10 W after viral IG inoculum, in vitro pulsed with HSV-1, and injected in recipient mice. The effects on isometric muscle tension were determined after one week. Adoptive transfer of CD8+ T cells isolated from LMMP at 8 W post IG HSV-1 inoculum caused significant neuromuscular anomalies (p<0.01 vs control) in recipient mice only if HSV-1 pulsed in vitro. Instead, the adoptive transfer of both CD4+ and CD8+ T cells isolated from 10 W infected mice resulted in dysmotility with or without in vitro exposure to viral antigens in recipient mice.
In conclusion, in this study we showed that α-herpesvirinae DNA is present in a large percentage of patients and the presence of HSV-1 in murine myenteric ganglia triggered a strong and specific immune response able to damage the ENS. We speculate that persistent HSV-1 infection in the ENS, in predisposed individuals, may contribute to alter neuronal functional integrity favouring the onset of bowel disorders
