HMGB1 released from nociceptors mediates inflammation Huan Yanga,1,2, Qiong Zenga,1, Harold A. Silvermana, Manojkumar Gunasekarana, Sam J. Georgea, Alex Devarajana, Meghan E. Addorisioa, Jianhua Lia, Téa Tsaavaa, Vivek Shaha, Timothy R. Billiarb, Haichao Wangc, Michael Brinesa, Ulf Anderssond, Valentin A. Pavlova,e,f, Eric H. Changa,e,f, Sangeeta S. Chavana,e,f,2, and Kevin J. Traceya,e,f,2 aLaboratory of Biomedical Sciences, Institute of Bioelectronic Medicine, Feinstein Institutes for Medical Research, Northwell Health, Manhasset, NY 11030; bDepartment of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213; cInstitute of Molecular Medicine, Feinstein Institutes for Medical Research, Northwell Health, Manhasset, NY 11030; dDepartment of Women’s and Children’s Health, Karolinska Institute, Karolinska University Hospital, 17176 Stockholm, Sweden; eThe Elmezzi Graduate School of Molecular Medicine, Manhasset, NY 11030; and fDonald and Barbara Zucker School of Medicine at Hofstra University, Hempstead, NY 11549 Edited by Lawrence Steinman, Stanford University School of Medicine, Stanford, CA, and approved June 17, 2021 (received for review February 1, 2021) Inflammation, the body’s primary defensive response system to homeostatic mechanisms at the intersection of the nervous sys- injury and infection, is triggered by molecular signatures of mi- tem and immune system have a fundamental role in health by crobes and tissue injury. These molecules also stimulate specialized inhibiting inflammation (15–17). sensory neurons, termed nociceptors. Activation of nociceptors me- The nervous system inhibits inflammation by transmitting diates inflammation through antidromic release of neuropeptides antiinflammatory signals in the vagus nerve, which arises in the into infected or injured tissue, producing neurogenic inflammation. brainstem and sends efferent projections to the organs (15–18). In Because HMGB1 is an important inflammatory mediator that is syn- the inflammatory reflex, signals descend from the brain to the ab- thesized by neurons, we reasoned nociceptor release of HMGB1 dominal celiac mesenteric ganglia, the origin of the splenic nerve (19, might be a component of the neuroinflammatory response. In sup- 20). In the spleen, splenic nerve signals stimulate a subset of T port of this possibility, we show here that transgenic nociceptors lymphocytes to secrete acetylcholine, which interacts with alpha 7 expressing channelrhodopsin-2 (ChR2) directly release HMGB1 in re- nicotinic acetylcholine receptors to inhibit macrophage production sponse to light stimulation. Additionally, HMGB1 expression in neu- of TNF and other inflammatory mediators (21–23). Detailed IMMUNOLOGY AND INFLAMMATION rons was silenced by crossing synapsin-Cre (Syn-Cre) mice with mechanistic insight into the neuroscience, functional immunology, f/f floxed HMGB1 mice (HMGB1 ). When these mice undergo sciatic and molecular mechanisms of the inflammatory reflex enabled nerve injury to activate neurogenic inflammation, they are pro- therapeutic trials using vagus nerve stimulation to inhibit inflam- tected from the development of cutaneous inflammation and allo- fl/fl mation in humans. This strategy to stimulate the inflammatory reflex dynia as compared to wild-type controls. Syn-Cre/HMGB1 mice inhibits cytokine release and suppresses inflammation in patients – subjected to experimental collagen antibody induced arthritis, a with inflammatory bowel disease and rheumatoid arthritis (24–26). disease model in which nociceptor-dependent inflammation plays a significant pathological role, are protected from the development Significance of allodynia and joint inflammation. Thus, nociceptor HMGB1 is re- quired to mediate pain and inflammation during sciatic nerve injury and collagen antibody–induced arthritis. Nociceptors are sensory neurons that detect changes in the body’s internal and external milieu. Although occupying a HMGB1 | cytokine | arthritis | DAMP | sciatic nerve injury primary role in signaling these changes to the nervous system, nociceptors also initiate neurogenic inflammation by sending antidromic signals back into the tissue. Because HMGB1 is a he dual threat of infection and injury exerted a significant well-characterized endogenous mediator, which stimulates in- Tinfluence on the evolution of the mammalian immune and flammation and is expressed by neurons, we reasoned HMGB1 nervous systems. The nervous system detects changes in the release may be an important component of neurogenic inflam- environment, generates reflexive responses to those changes, and mation. Here, by combining optogenetics, neuronal-specific ab- integrates these responses across space and time to establish lation, nerve-injury, and inflammatory disease models, with adaptive memories of events (1, 2). Sensory neurons, termed direct assessment of inflammation and neuropathic pain, we nociceptors, innervate tissues subjected to infection and injury show that nociceptor HMGB1 is required for an inflamma- and are activated by the molecular products of microbes and tory response. These results provide direct evidence that tissue injury (3–6). Nociceptor signaling stimulates reflexive nociceptor-related pain and inflammation can be prevented neural circuits to coordinate defensive behavior, including in- by targeting HMGB1. flammation (3–9). Reflex neural signaling arising in the brain and spinal cord is capable of integrating divergent stimulating Author contributions: H.Y., U.A., S.S.C., and K.J.T. designed research; H.Y., Q.Z., M.G., and inhibitory inputs, because neural signaling circuits act in S.J.G., A.D., M.E.A., T.T., and V.S. performed research; J.L. and T.R.B. contributed new reagents/analytic tools; H.Y. and U.A. analyzed data; and H.Y., H.A.S., H.W., M.B., U.A., opposition (10, 11). This balance of opposing signals enables fine V.A.P., E.H.C., S.S.C., and K.J.T. wrote the paper. tuning of physiological responses by smoothing gradients of The authors declare no competing interest. corrective action in response to a changing environment (12). This article is a PNAS Direct Submission. Evolutionary pressure from infection and injury also molded the This open access article is distributed under Creative Commons Attribution-NonCommercial- mammalian immune system to detect changes in the environ- NoDerivatives License 4.0 (CC BY-NC-ND). ment, mobilize coordinated defensive reactions, and establish 1H.Y. and Q.Z. contributed equally to this work. memory (13). The acute onset of infection and injury produces 2To whom correspondence may be addressed. Email: [email protected], schavan@ inflammation, defined by pain, edema, heat, redness, and loss of northwell.edu, or [email protected]. function mediated by immune cells producing cytokines and This article contains supporting information online at https://www.pnas.org/lookup/suppl/ other inflammatory mediators (6, 14). Because uninhibited in- doi:10.1073/pnas.2102034118/-/DCSupplemental. flammation also causes dangerous shock and lethal tissue injury, Published August 12, 2021. PNAS 2021 Vol. 118 No. 33 e2102034118 https://doi.org/10.1073/pnas.2102034118 | 1of9 Downloaded by guest on September 25, 2021 Since evolution selects for reflexively controlled homeostatic systems ChR2, fails to induce HMGB1 release (Fig. 1B,control).Lactate acting antagonistically, here we reasoned it likely also selected for dehydrogenase (LDH), a soluble cytoplasmic enzyme released upon neural reflex circuits which stimulate inflammation and enhance membrane disruption or cell death (47), is not released during light immunity. exposure (Fig. 1C), further confirming that HMGB1 is actively se- High mobility group box 1 protein (HMGB1), a ubiquitous creted by stimulated nociceptors. nuclear protein conserved across evolution from archaea to mammals, is a necessary and sufficient mediator of sterile injury- Generation and Characterization of Syn-Cre/HMGB1fl/fl Mice. To in- and infection-elicited inflammation and immunity (27, 28). It is vestigate the functional role of nociceptors and HMGB1 in in- passively released by cells undergoing necrosis and secreted by flammation, we selectively ablated HMGB1 expression in neurons innate immune cells activated by inflammation (27, 29). HMGB1 by crossing synapsin-Cre (Syn-Cre) mice with floxed Hmgb1 mice stimulates inflammation by activating the receptor for advanced (HMGB1f/f)(SI Appendix,Fig.S1A). Although global deletion of glycation end products (RAGE) and Toll-like receptor 2, 4, and 9 Hmgb1 leads to early postnatal death (48), mice lacking HMGB1 (TLR 2, 4, and 9) expressed by many different cell types (30–32). expression in neurons (Syn-Cre/HMGB1fl/fl) develop normally. No Signal transduction via these receptors culminates in increased significant differences are observed in body weights of 8- to expression of cytokines, and in recruiting and stimulating mono- 10-wk-old Syn-Cre/HMGB1fl/fl mice as compared to age- and sex- cytes, lymphocytes, and neutrophils. HMGB1 also stimulates matched wild-type (WT) and HMGB1fl/fl control mice (SI Ap- dendritic cell maturation and enhances antibody responses to pendix,Fig.S1B). Specific genetic ablation of HMGB1 expression antigen, an important early stage in memory formation (33, 34). in neurons significantly depletes HMGB1 protein levels in DRG Administration of HMGB1 antagonists significantly attenuates the sensory neurons in Syn-Cre/HMGB1fl/fl mice as compared to WT severity of inflammation in preclinical models of sepsis, inflam- and HMGB1fl/fl