Human-relevant near-organ neuromodulation of the immune system via the splenic nerve

Neuromodulation of immune function by stimulating the autonomic connections to the spleen has been demonstrated in rodent models. Consequently, neuroimmune modulation has been proposed as a new therapeutic strategy for the treatment of inflammatory conditions. However, demonstration of the translation of these immunomodulatory mechanisms in anatomically and physiologically relevant models is still lacking. Additionally, translational models are required to identify stimulation parameters that can be transferred to clinical applications of bioelectronic medicines. Here, we performed neuroanatomical and functional comparison of the mouse, rat, pig, and human splenic nerve using in vivo and ex vivo preparations. The pig was identified as a more suitable model of the human splenic innervation. Using functional electrophysiology, we developed a clinically relevant marker of splenic nerve engagement through stimulation-dependent reversible reduction in local blood flow. Translation of immunomodulatory mechanisms were then assessed using pig splenocytes and two models of acute inflammation in anesthetized pigs. The pig splenic nerve was shown to locally release noradrenaline upon stimulation, which was able to modulate cytokine production by pig splenocytes. Splenic nerve stimulation was found to promote cardiovascular protection as well as cytokine modulation in a high- and a low-dose lipopolysaccharide model, respectively. Importantly, splenic nerve–induced cytokine modulation was reproduced by stimulating the efferent trunk of the cervical vagus nerve. This work demonstrates that immune responses can be modulated by stimulation of spleen-targeted autonomic nerves in translational species and identifies splenic nerve stimulation parameters and biomarkers that are directly applicable to humans due to anatomical and electrophysiological similarities.

The inflammatory status of the body is monitored and regulated through the neuroimmune axis, connecting the brain to the immune system via both humoral and neural pathways (1–3). In particular, the inflammatory reflex (3) controls systemic immune responses; detection of inflammatory stimuli in the periphery is communicated to the brain that activates outflow of neural signals to promote peripheral immune responses proportional to the threat. Studies in rodent models have identified the cholinergic anti-inflammatory pathway (CAIP) as the brain’s efferent response to infection and inflammation through peripheral neurotransmitters released in lymphoid organs, mainly the spleen (4, 5). Within this pathway, the peripheral connection between the vagus nerve (VN), the splenic nerve (SpN), and its terminal release of noradrenaline (NA) into the spleen have been identified as crucial components of this neural circuit (6–8) (SI Appendix, Fig. S1A).Importantly, the CAIP can be harnessed to promote immune control. Activation of the cervical VN by electrical stimulation (vagus nerve stimulation—VNS; SI Appendix, Fig. S1A) has been shown to be effective in reducing lipopolysaccharide (LPS)-induced levels of tumor necrosis factor alpha (TNF-α) (4, 6, 7) and in preclinical rodent models of chronic inflammatory diseases (9, 10). Murine models have generally been used to demonstrate biological proof of concepts of novel neuromodulation therapies in this and other contexts. However, the development of clinical bioelectronic medicines requires the accurate estimation and validation of stimulation parameters in a histologically, surgically, and anatomically relevant model to define device and therapy requirements. The translation of stimulation parameters from rodent to human is hampered by anatomical (e.g., size of nerves), histological (e.g., number of axons, connective tissue thickness, proportion of adipose tissue), and physiological (e.g., immunological) differences. Therefore, it is suggested that the use of large animal models, human tissues, and in silico modeling are more appropriate for the optimization and scaling of human-relevant parameters (11, 12).Although early clinical feasibility studies have provided preliminary evidence of immunomodulatory effects of VNS in patients (13, 14), clear demonstration of the translation of the splenic anti-inflammatory pathway in clinically relevant species is currently lacking in the literature. The VN has a functionally and anatomically complex composition. In animals and humans, the VN contains both afferent and efferent axons of varying size (large, medium, and small) and degree of myelination (heavily myelinated, lightly myelinated, and unmyelinated axons) innervating multiple organs and muscles (15). As a consequence, currently used VNS results in activation of off-target circuits (SI Appendix, Fig. S1A) that can cause dysphonia, coughing, hoarseness, pain, and dyspnea (16–18); in some patients, these can be managed and can also improve over time (18). Further, it remains unclear which axons (efferent versus afferent, myelinated versus unmyelinated) within the VN relay immunomodulatory signals to peripheral organs (19, 20). As a result, it is difficult to optimize the stimulation parameters necessary to activate axons within the VN which carry signals to the spleen. Typically, clinical parameters are selected based on the individual patient’s tolerance of off-target effects (13, 21) without direct evidence of activation of the anti-inflammatory pathway because of a lack of an organ-specific biomarker. Since the SpN directly transmits neural signals to the spleen and is the fundamental nodal circuit in mediating the anti-inflammatory response (22), SpN stimulation (SpNS) may represent an alternative modality providing the opportunity for near-organ modulation of the immune system (SI Appendix, Fig. S1 B and C). Proof of concept experiments in rodents have shown that immune responses can indeed be modulated by stimulation of the SpN with comparable cytokine suppressive effects to VNS (7, 8, 23).Here, we anatomically, histologically, and functionally compared the mouse, rat, pig, and human SpN, demonstrating the superiority of the pig as a translational model of the human SpN. We then performed functional in vivo pig electrophysiological studies to identify organ-specific physiological biomarkers that can be used to assess nerve engagement and to refine stimulation parameters. Finally, we assessed the large animal translation of the spleen-dependent anti-inflammatory pathway in the pig using in vitro splenocyte preparations together with two in vivo models of acute inflammation.