Spleen vagal denervation inhibits the production of antibodies to circulating antigens

Abstract

Background: Recently the vagal output of the central nervous system has been shown to suppress the innate immune defense to pathogens. Here we investigated by anatomical and physiological techniques the communication of the brain with the spleen and provided evidence that the brain has the capacity to stimulate the production of antigen specific antibodies by its parasympathetic autonomic output.

Methodology/principal findings: This conclusion was reached by successively demonstrating that: 1. The spleen receives not only sympathetic input but also parasympathetic input. 2. Intravenous trinitrophenyl-ovalbumin (TNP-OVA) does not activate the brain and does not induce an immune response. 3. Intravenous TNP-OVA with an inducer of inflammation; lipopolysaccharide (LPS), activates the brain and induces TNP-specific IgM. 4. LPS activated neurons are in the same areas of the brain as those that provide parasympathetic autonomic information to the spleen, suggesting a feed back circuit between brain and immune system. Consequently we investigated the interaction of the brain with the spleen and observed that specific parasympathetic denervation but not sympathetic denervation of the spleen eliminates the LPS-induced antibody response to TNP-OVA.

Conclusions/significance: These findings not only show that the brain can stimulate antibody production by its autonomic output, it also suggests that the power of LPS as adjuvant to stimulate antibody production may also depend on its capacity to activate the brain. The role of the autonomic nervous system in the stimulation of the adaptive immune response may explain why mood and sleep have an influence on antibody production.

Figure 1. PRV labeling from spleen shows parasympathetic and sympathetic autonomic neurons in brain stem (A) and spinal cord (B).

After injection of PRV into the spleen, the origin of the parasympathetic respectively sympathetic input to the spleen is revealed. A) PRV positive neurons are present in the neurons of the dorsal motor nucleus of the vagus (DMV), at this stage of infection there are no labeled neurons in the area postrema(AP) or nucleus tractus solitarius (NTS).B) PRV positive neurons are present in the sympathetic intermediolateral column (IML) or in the area of the central canal. (Bar = 50 µm).

Figure 2. Distribution of pseudo rabies virus (PRV) labeled neurons in different areas of the brain following injection of PRV into the spleen after sympathetic A) or parasympathetic denervation B).

Following different survival times these procedures revealed respectively the first (red) or second order (blue) or third order (brown) neurons that project to the spleen. The upper graph illustrates the areas in the brain that have parasympathetic (pre)autonomic neurons and the lower illustrates those areas that have sympathetic (pre)autonomic neurons.

Figure 3. Specific IgM antibodies to TNP-OVA are only detected in the circulation after an i.v. injection of TNP-OVA with a prior injection of LPS.

The antibody response was only present when LPS preceded the injection of TNP-OVA(uninterrupted line). Dashed line shows the IgM levels in plasma after injection of TNP-OVA only. (Two way ANOVA indicated difference between TNP-OVA saline and TNP-OVA + LPS p<0.01, Tukey post hoc test only showed significant difference between D0 and D3 and D7 after TNP-OVA+LPS injection p<0.001).

Figure 4. The same area in the brain contains pre-autonomic neurons projecting to the spleen and neurons that are activated after LPS injection.

The presence of pre-autonomic spleen projecting neurons in the subfornical organ (A) is detected after PRV injection in the spleen. After i.v.injection of LPS, activated neurons are detected by c-Fos immunocytochemistry in the SFO (B) Bar  = 50 µm.

Figure 5. Shows the response of corticosterone after an injection of TNP-OVA alone (dashed lines) or TNP-OVA+LPS in sham (blue) or SSX (yellow) or PSX (red) animals.

Clearly LPS induces the secretion of corticosterone but no difference can be observed between denervated or intact animals, illustrating that after i.v. injection of LPS the corticosterone response is not dependent on sensory autonomic information from the spleen. (Two way ANOVA indicated difference between the LPS treated groups with all other groups without LPS p<0.01. Tukey post hoc test showed significant difference of day 7 and 10 of the sham and sympathetic denervation as compared to all other time points p<0.001.)

Figure 6. Illustrates neurons in the organum vasculosum lamina terminalis (OVLT) (A-B) and bed nucleus of the stria terminalis (BNST) (C-D) containing c-Fos 3 hours after injection of LPS in the general circulation (B,D) or PRV 4 days after injection in sympathetic denervated animals (A,C).

These comparable sections of the same areas show that these areas not only are activated after LPS injection, but that they also have the capacity to communicate to the spleen via the vagus Bar  = 50 µm.

Figure 7. Illustrates absence of TNP-OVA IgM antibody production after parasympathetic denervation of the spleen.

TNP-OVA IgM antibody production after i.v. injection of saline and TNP-OVA (dashed lines) or LPS and TNP-OVA(uninterrupted lines) in animals that sustained a parasympathetic denervation (red) or a sympathetic denervation (yellow) of the spleen or were sham operated (blue). Only parasympathetic denervation results in a nearly complete loss of antibody production with a similar response as if no LPS were given. (Two way ANOVA indicated difference between the sham and sympathetic denervation + LPS with the other groups without LPS and the parasympathetic denervated group with LPS p<0.01. Tukey post hoc test showed significant difference of day 7 and 10 of the sham and sympathetic denervation as compared to all other time points p<0.001.)

Figure 8. Schematic representation of the innervation of the spleen.

The sympathetic input (red) reaches the spleen via the arteries, the parasympathetic input (blue) reaches the spleen via both tips of the spleen.