C. difficile intoxicates neurons and pericytes to drive neurogenic inflammation.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections1,2. The exaggerated colonic inflammation caused by C. difficile toxins such as toxin B (TcdB) damages tissues and promotes C. difficile colonization3-6, but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI.

Microbiota: a novel regulator of pain.

Among the various regulators of the nervous system, the gut microbiota has been recently described to have the potential to modulate neuronal cells activation. While bacteria-derived products can induce aversive responses and influence pain perception, recent work suggests that "abnormal" microbiota is associated with neurological diseases such as Alzheimer's, Parkinson's disease or autism spectrum disorder (ASD). Here we review how the gut microbiota modulates afferent sensory neurons function and pain, highlighting the role of the microbiota/gut/brain axis in the control of behaviors and neurological diseases. We outline the changes in gut microbiota, known as dysbiosis, and their influence on painful gastrointestinal disorders. Furthermore, both direct host/microbiota interaction that implicates activation of "pain-sensing" neurons by metabolites, or indirect communication via immune activation is discussed. Finally, treatment options targeting the gut microbiota, including pre- or probiotics, will be proposed. Further studies on microbiota/nervous system interaction should lead to the identification of novel microbial ligands and host receptor-targeted drugs, which could ultimately improve chronic pain management and well-being.

Bacterial modulation of visceral sensation: mediators and mechanisms.

The potential role of the intestinal microbiota in modulating visceral pain has received increasing attention during recent years. This has led to the identification of signaling pathways that have been implicated in communication between gut bacteria and peripheral pain pathways. In addition to the well-characterized impact of the microbiota on the immune system, which in turn affects nociceptor excitability, bacteria can modulate visceral afferent pathways by effects on enterocytes, enteroendocrine cells, and the neurons themselves. Proteases produced by bacteria, or by host cells in response to bacteria, can increase or decrease the excitability of nociceptive dorsal root ganglion (DRG) neurons depending on the receptor activated. Short-chain fatty acids generated by colonic bacteria are involved in gut-brain communication, and intracolonic short-chain fatty acids have pronociceptive effects in rodents but may be antinociceptive in humans. Gut bacteria modulate the synthesis and release of enteroendocrine cell mediators, including serotonin and glucagon-like peptide-1, which activate extrinsic afferent neurons. Deciphering the complex interactions between visceral afferent neurons and the gut microbiota may lead to the development of improved probiotic therapies for visceral pain.

Capsaicin-sensitive vagal afferent neurons contribute to the detection of pathogenic bacterial colonization in the gut.

Vagal activation can reduce inflammation and disease activity in various animal models of intestinal inflammation via the cholinergic anti-inflammatory pathway. In the current model of this pathway, activation of descending vagal efferents is dependent on a signal initiated by stimulation of vagal afferents. However, little is known about how vagal afferents are activated, especially in the context of subclinical or clinical pathogenic bacterial infection. To address this question, we first determined if selective lesions of capsaicin-sensitive vagal afferents altered c-Fos expression in the nucleus of the solitary tract (nTS) after mice were inoculated with either Campylobacter jejuni or Salmonella typhimurium. Our results demonstrate that the activation of nTS neurons by intraluminal pathogenic bacteria is dependent on intact, capsaicin sensitive vagal afferents. We next determined if inflammatory mediators could cause the observed increase in c-Fos expression in the nTS by a direct action on vagal afferents. This was tested by the use of single-cell calcium measurements in cultured vagal afferent neurons. We found that tumor necrosis factor alpha (TNFα) and lipopolysaccharide (LPS) directly activate cultured vagal afferent neurons and that almost all TNFα and LPS responsive neurons were sensitive to capsaicin. We conclude that activation of the afferent arm of the parasympathetic neuroimmune reflex by pathogenic bacteria in the gut is dependent on capsaicin sensitive vagal afferent neurons and that the release of inflammatory mediators into intestinal tissue can be directly sensed by these neurons.

Rat dorsal root ganglia neurons as a model for Listeria monocytogenes infections in culture.

Neurotropism of Listeria monocytogenes was studied in rat dorsal root ganglia (DRG) and hippocampal neurons in culture. Using a system in which the DRG neurons can grow relatively free from other cells, it was observed that such DRG neurons, in contrast to hippocampal neurons, can be effectively infected by L. monocytogenes. The bacteria aligned along DRG axons, but not along hippocampal neurites. A mutant deficient in internalin, a protein required for entry into E-cadherin-expressing cells, did not interact with DRG neurons. Axonal migration of bacteria was studied in the DRG neurons grown in a double-chamber system, where either the neurites or the nerve cell bodies were exposed to the bacteria. The data suggest that L. monocytogenes can infect both axons and DRG nerve cell bodies, and that the bacteria can migrate in a retrograde as well as anterograde direction. These results support the notion that L. monocytogenes can spread via primary sensory neurons to the central nervous system. Infection of DRG primary sensory neurons, as employed in the present study, provides a model for analysis of bacterial and neuronal factors of importance for neurovirulence of L. monocytogenes.

Quantitative analysis of herpes simplex virus DNA and transcriptional activity in ganglia of mice latently infected with wild-type and thymidine kinase-deficient viral strains.

The relationship between herpes simplex virus (HSV) DNA replication and establishment of latent infection was examined using an experimental model that makes use of the segmental sensory innervation of mouse flanks (T7 to T12). Ganglia from consecutive thoracic segments of C57BL/10 mice latently infected with a virulent strain of HSV-1 (SC16) were compared with respect to (i) HSV DNA levels, (ii) latency-associated transcripts (LATs) and (iii) numbers of LAT+ neurons. In concordance with previous results, two patterns of virus persistence were detected distinguished by either a low (10 to 23) or high (approx. 200) number of viral genomes/LAT+ neuron. The high copy pattern was associated, anatomically, with ganglia directly innervating inoculated skin (T7/8). Paradoxically, the highest number of LAT+ neurons and the highest concentrations of LATs were detected in spinal segments (e.g. T10) containing the lowest number of viral genomes, implying that most of the latent SC16 DNA detected at T7 and T8 was transcriptionally repressed. When neuronal amplification of HSV DNA during the establishment phase was prevented by infecting mice with a viral thymidine kinase deletion mutant (TKDM21), the high copy pattern was eliminated and each LAT+ neuron contained, on average, 22 TKDM21 genomes. We conclude that input (i.e. unamplified) and progeny (i.e. amplified) DNA sequences persist in the peripheral nervous systems of mice infected with SC16. Structurally, latent TKDM21 DNA lacked free genomic termini, consistent with persistence of input DNA in an integrated or circular episomal configuration.

Regulation of the herpes simplex virus latency-associated transcripts during establishment of latency in sensory neurons in vitro.

The temporal appearance of the major latency-associated transcript (LAT) of herpes simplex virus, type-1 (HSV-1) was examined in sensory neuronal cultures during the establishment of either a latent or a lytic infection. Under conditions that result in the establishment of a latent infection, a significant delay in LAT accumulation was observed. The delay in the appearance of LAT was reflected in both a reduced number of LAT-positive neurons detected by in situ hybridizations and by low levels of the major 2-kb LAT detected by Northern blot analysis at early times compared to later in the latent infection. The percentage of LAT-positive neurons shown by in situ hybridizations and the relative abundance of the major LAT by Northern blot analysis increased markedly by 14 days after inoculation with virus. In addition to the major LAT, a spliced 1.5-kb LAT species was detected in Northern blot analysis after establishment of latency in the neuronal cultures, similar to observations in vivo. In contrast to the latent infection, under conditions that produced lytic infections in the neuronal cultures, LAT and HSV-1-specific antigens were detected in the majority of neurons 24 hr after inoculation with virus. These results indicate that LAT expression during the establishment of latency is regulated differently than during the lytic infection: LAT expression appears to be inhibited initially during the establishment of latency, whereas LAT is readily expressed during the lytic infection.

Neuron-specific restriction of a herpes simplex virus recombinant maps to the UL5 gene.

We have previously shown that, when compared with either parent, a herpes simplex virus type 1/herpes simplex virus type 2 intertypic recombinant (R13-1) is attenuated by 10,000-fold with respect to neurovirulence in mice. Despite this, after intracranial inoculation, R13-1 replicated to titers of 10(5) PFU per brain. We present evidence that the restriction is specific for replication in neurons and have taken a three-step approach in determining the basis of the attenuation by (i) characterizing cellular tropism of the virus in both central and peripheral nervous systems, (ii) defining where in the viral replication cycle the restriction is manifest, and (iii) identifying the genetic basis of the restriction through marker rescue analysis. Following inoculation into the animal, R13-1 viral antigens predominate in nonneuronal cells, and the block to replication in neurons was found to be beyond the level of adsorption and penetration. Despite the restricted replication within neurons, the virus established a latent infection in spinal ganglia and could be reactivated by in vitro cocultivation of the ganglia. In studies carried out in cell culture, R13-1 was found to replicate normally in mouse embryo fibroblasts and primary mouse glial cells but was restricted by 1,000-fold in primary mouse neurons and PC12 cells. R13-1 appeared to produce normal levels of early RNA in these cells, but production of DNA and late RNA was less than that of the wild type. Marker rescue analysis localized the fragment responsible for restoring neurovirulence to UL5, a component of the origin-binding complex implicated in replication of the viral genome. Our results with this virus, with a cell-specific restriction, suggest that a neuron-specific component is involved in viral replication.

Restricted herpes simplex virus type 1 gene expression within sensory neurons in the absence of functional B and T lymphocytes.

Herpes simplex virus type I (HSV-1) establishes a latent infection in sensory ganglion neurons. During latency no viral-specific proteins are detected and virus gene expression is restricted to the latency-associated transcripts. We report here that trigeminal ganglia of mice with severe combined immunodeficiency contain individual sensory neurons exhibiting restricted viral gene expression characteristic of latency; this occurred during acute (4-6 days) infection with the wild-type HSV-1 strain 17+ and after prolonged (4 weeks) infection with the replication impaired HSV-1 mutant in 1814. These results indicate that T and B lymphocytes, while important for the recovery from viral infections, are not required for the establishment or maintenance of latency in neurons.

Latent infection can be established with drastically restricted transcription and replication of the HSV-1 genome.

Viral functions essential for the establishment of latent infection in murine sensory neurons in vivo were investigated by employing a herpes simplex virus type 1 (HSV-1) variant (KD6/B11) deleted for expression of the ICP4 gene and therefore unable to replicate. Since the viral DNA persisted in these cells, the latency-associated transcripts were expressed for prolonged periods of time, and the variant was biologically retrievable by superinfection with an ICP4-competent agent, we concluded that a latent infection had been established. In situ hybridization experiments designed to investigate gene expression during the acute phase of infection with the variant revealed a highly restricted pattern compared to that of the wild-type parent virus HSV-1 KOS(M). While latency-associated transcripts were detected in a large number of infected neurons, expression of other virus genes was limited to a subset of immediate-early and early genes (ICP0, ICP8, ICP27, and HSV-1 DNA polymerase genes). Expression was further limited to a small proportion of the infected neurons (approximately 1% of neurons expressing latency-associated transcripts). No hybridization was detected with probes specific for the viral TK gene and late genes VP5 and gC. Quantitative assays of viral DNA during the acute phase of infection indicated that the input viral DNA did not replicate. From these results we conclude that HSV-1 latent infection can be established in murine sensory neurons under conditions in which viral genetic expression and DNA replication are severely restricted.

Efficacy of the herpes simplex virus types 1 and 2 mutant viruses to confer protection against zosteriform spread in mice.

Two mutant viruses, HSV-2 XD192 and HSV-1 1716, failed to generate zosteriform lesions when injected in high dose into BALB/c and C3H mice. Mice exposed to mutant viruses were solidly immune to challenge by wild-type homologous or heterologous virus. However, at lower immunizing doses protection was evident against lethality, but not skin lesions, especially in the case of mutant XD192. Protection could be conferred with lymphoid cells from mutant virus immune mice and again, protection against lethality was more frequent than prevention of skin lesions. On the basis of cell fractionation studies, protection against lethality was assumed to be principally the function of CD8+ T lymphocytes. The implications of the results in terms of vaccine development were briefly discussed.

Relationship between polyadenylated and nonpolyadenylated herpes simplex virus type 1 latency-associated transcripts.

RNA from the region of the genome encoding herpes simplex virus type 1 latency-associated transcripts (LATs) expressed during lytic infection yields low abundances of both polyadenylated and nonpolyadenylated forms. As has been previously shown for latent infection (A. T. Dobson, F. Sedarati, G. Devi-Rao, W. M. Flanagan, M. J. Farrell, J. G. Stevens, E. K. Wagner, and L. T. Feldman. J. Virol. 63:3844-3851, 1989), all lytic-phase expression of such transcripts requires promoter elements situated approximately 600 bases 5' of the previously mapped 5' end of the poly(A)- forms of LAT. Transient expression experiments revealed no other clear promoter elements within this region, and relatively small amounts of latent-phase transcripts initiating at the same site as observed for lytic-phase LAT could be detected by RNase protection assays. In the lytic phase of infection, the most abundant forms of polyadenylated LAT extended 1,600 bases from the initiation site near the LAT promoter to a potential splice donor site. Poly(A)- LAT species were not recovered in significant amounts from lytically infected neuroblastoma cells, but such RNA from lytically infected rabbit skin cells comapped with poly(A)- LAT from latently infected sensory neurons. Both map between canonical 5' splice donor and 3' splice acceptor site 1,950 bases apart. Poly(A)- LAT cochromatographed with uncapped rRNA on m-aminophenyl boronate agarose under conditions in which capped mRNA was bound. All of these data confirm the previously presented scheme for the expression of poly(A)- LAT as a stable intron derived from the splicing of a large primary transcript; however, we were unable to detect the spliced polyadenylated product of this splicing reaction.

Rabies virus infection and transport in human sensory dorsal root ganglia neurons.

Cultured human sensory neurons are directly susceptible to CVS rabies virus infection and produce virus yields of 10(5) p.f.u./ml; infection can persist for more than 20 days without any sign of c.p.e. The use of a compartmentalized two-chamber culture system, with access to either the cell soma or neuritic extensions, permitted the study of viral retrograde transport, which occurs at between 50 and 100 mm/day. Neurons of human origin were more susceptible to virus infection than rat neurons and the axonal transport of rabies virus was more efficient. Electron microscopy allowed virus transport and infection of human dorsal root ganglia neurons to be observed.

Transneuronal transport of herpes simplex virus from the cervical vagus to brain neurons with axonal inputs to central vagal sensory nuclei in the rat.

The recent introduction of live viruses as intra-axonal tracing agents has raised questions concerning which central neurons are transneuronally labelled after application of the virus to peripheral organs or peripheral nerves. Since the central connections of the vagus nerve have been well described using conventional neuronal tracing agents, we chose to inject Herpes Simplex Virus Type 1 into the cervical vagus of the rat. After survival times of up to 3 days the rat brains were processed immunohistochemically using a polyclonal antiserum against herpes simplex virus. Two days after injection of the virus we observed viral antigen in the area postrema and in the nucleus tractus solitarius and the dorsal motor nucleus of the vagus (dorsal vagal complex), principally ipsilaterally. At this survival time the viral antigen in the dorsal vagal complex was largely confined to glial cells. After 3 days the viral antigen was localized both in glia and in nerve cells within the dorsal vagal complex and in brain regions previously demonstrated, using conventional tracing procedures, to contain neurons with axonal projections to the dorsal vagal complex. This was true for medullary, pontine, midbrain and hypothalamic regions and for telencephalic regions including the amygdala, the bed nucleus of the stria terminalis, and the insular and medial frontal cortices. Many of the nerve cells containing viral antigen were displayed in a Golgi-like manner, with excellent visualization of the dendritic tree. Axonal processes, in contrast, were not visualized. We used co-localization studies to confirm previous findings concerning monoamine neurotransmitter-related antigens present in medullary and pontine neurons projecting to the dorsal vagal complex. After 3 days there were many Herpes Simplex Virus Type 1-containing glial cells along the intra-medullary course of the vagal rootlets. However, no viral antigen was found in brain regions containing neurons whose axons pass through the region of glial cell-labelled rootlets. Glial cells containing viral antigen were particularly numerous in brain regions known to receive an input from neurons in the area postrema and the dorsal vagal complex. Taken together with our observation concerning the early appearance of viral antigen within glial cells in the dorsal vagal complex, this suggests that when the virus reaches the axon terminal portion it is transferred to nearby glial cells and possibly enters central neurons by way of these structures.

Cell lines derived from dorsal root ganglion neurons are nonpermissive for HSV and express only the latency-associated transcript following infection.

The effect of herpes simplex virus type 1 infection in a series of immortalized dorsal root ganglion cell lines has been investigated. Following infection of one of these lines, the viral immediate-early genes are not transcribed and the lytic cycle is aborted at an early stage. In contrast these cells do support transcription of the gene encoding the latency-associated transcripts which are the only viral RNAs present in latently infected ganglia in vivo. These cell lines are therefore a suitable model system for studies of the processes regulating the interaction of HSV with neuronal cell types and the establishment of latent infections in vivo.

Nerve growth factor-dependence of herpes simplex virus latency in peripheral sympathetic and sensory neurons in vitro.

Previously, we reported that nerve growth factor (NGF) is required to maintain herpes simplex virus (HSV) latency in cultures of rat sympathetic neurons (Wilcox and Johnson, 1987, 1988). Here, we extend these results by showing that NGF was also required to maintain HSV latency in cultures of sensory neurons obtained from dorsal root ganglia of rats, monkeys, and humans. The interruption of the neuronal supply of NGF for 1 hr reactivated HSV, indicating that the latent virus was exquisitely sensitive to perturbations in the concentration or binding of NGF. A species-specific monoclonal antibody directed against the human NGF-receptor, which blocks NGF binding, reactivated latent HSV in human, but not rat, sensory neurons. In contrast, a monoclonal antibody against the rat NGF-receptor, which binds the receptor without blocking NGF action, did not produce reactivation. These results indicate that the effects of NGF on HSV latency are mediated via NGF binding to the NGF receptor. In addition, treatments that interfere with specific steps in the transduction of the NGF signal, including treatment with 6-hydroxydopamine and colchicine, reactivated latent HSV. Further, in neurons harboring latent virus, interruption of protein synthesis or RNA transcription for 1 hr resulted in viral reactivation, suggesting that a short-lived factor may be present in neurons which represses viral reactivation.

Biological and molecular aspects on herpes simplex virus latency.

Latent reactivateable herpes simplex virus (HSV) infections of sensory neurons of peripheral ganglia are most plausibly the source of clinical herpetic recurrences. The establishment of latency is the result of a concert of both viral and cellular factors such as the neuroinvasiveness of the virus, the relative density of receptors binding virus to the nerve cell plasma membrane, the permissiveness of the infection, including the axonal transport of the viral nucleocapsids, and the restriction of virus replication. Several hypotheses have been presented suggesting various mechanisms for the restriction of the HSV infection of the neuron. Thus, for instance the the importance of thymidine kinase negative mutants, hypermethylation of viral DNA and the existence of latency-associated viral genes have been discussed. Activation of the latent infection to a virus-producing lytic infection by means of superinfection with a replication-incompetent mutant is probably a result of genetic complementation. Reactivation by means of superinfection with replication-competent virus seems dependent upon the multiplicity of the superinfecting virus and more than one copy of the virus genome is required for the initiation of the reactivating process. These observations would be consistent with the overcoming of a cellularly controlled restriction of the latent infection. The cellular control of latency which can be impaired mechanically and chemically seems particularly important for the maintenance of latent HSV infection. However, recent observations indicate that reactivation of latent HSV infection is also associated with the expression of a latency-related viral gene (LAT), whereas establishment of the latency apparently is influenced by other properties determined by the genome of the virus, as well as by the capacity of the cell to restrict the lytic infection.

Latent infections in spinal ganglia with thymidine kinase-deficient herpes simplex virus.

A herpes simplex virus type 1 variant [C239(TK-)] harboring a deletion in the thymidine kinase (TK) gene was assessed for capacity to establish latent infections. Outbred Swiss Webster mice were inoculated on both hind footpads, and numbers of neurons expressing latency-associated transcript and amounts of viral DNA in latently infected lumbosacral spinal ganglia were scored. C239(TK-) established levels of latent infection that were only slightly lower than those found for either a TK rescued variant of this agent or the parent wild-type KOS. However, in contrast to the TK+ viruses, C239(TK-) could not be reactivated when spinal ganglia were cultured in vitro. The results presented show that expression of the viral TK gene plays no major role in establishment of the latent state but that it functions during reactivation of latent virus from explanted ganglia maintained in vitro.

Herpes simplex virus type 1 latency-associated transcription plays no role in establishment or maintenance of a latent infection in murine sensory neurons.

Using herpes simplex viruses deleted and restored for the latency-associated transcripts (LATs), we have quantitatively assessed the role of the transcripts in establishment and maintenance of latent infection. Determination of the number of neurons latently infected and the copy number of viral genomes per latently infected ganglion indicated that there is no difference between viruses expressing and not expressing the transcripts. In addition, the amount of viral DNA present in ganglia latently infected with the LAT-negative virus KOS 8117 did not differ from the value for LAT+ counterparts over an 11-month period of analysis. From these results we conclude that LATs play no role in establishment or maintenance of a latent infection with herpes simplex virus type 1. If these transcripts play a role in latency, they must function during the reactivation step.