(C) PLOS One This story was originally published by PLOS One and is unaltered. . . . . . . . . . . The antimicrobial peptide cathelicidin drives development of experimental autoimmune encephalomyelitis in mice by affecting Th17 differentiation [1] ['Katie J. Smith', 'Centre For Inflammation Research', 'University Of Edinburgh', 'Edinburgh', 'United Kingdom', 'Danielle Minns', 'Brian J. Mchugh', 'Rebecca K. Holloway', 'Centre For Reproductive Health', 'United Kingdom Dementia Research Institute At The University Of Edinburgh'] Date: 2022-08 Multiple sclerosis (MS) is a highly prevalent demyelinating autoimmune condition; the mechanisms regulating its severity and progression are unclear. The IL-17-producing Th17 subset of T cells has been widely implicated in MS and in the mouse model, experimental autoimmune encephalomyelitis (EAE). However, the differentiation and regulation of Th17 cells during EAE remain incompletely understood. Although evidence is mounting that the antimicrobial peptide cathelicidin profoundly affects early T cell differentiation, no studies have looked at its role in longer-term T cell responses. Now, we report that cathelicidin drives severe EAE disease. It is released from neutrophils, microglia, and endothelial cells throughout disease; its interaction with T cells potentiates Th17 differentiation in lymph nodes and Th17 to exTh17 plasticity and IFN-γ production in the spinal cord. As a consequence, mice lacking cathelicidin are protected from severe EAE. In addition, we show that cathelicidin is produced by the same cell types in the active brain lesions in human MS disease. We propose that cathelicidin exposure results in highly activated, cytokine-producing T cells, which drive autoimmunity; this is a mechanism through which neutrophils amplify inflammation in the central nervous system. Funding: This work was funded by a Royal Society Dorothy Hodgkin Fellowship (DH150175) and two Royal Society Fellows’ Enhancement Awards (RGF\EA\180049 and RGF\EA\201009) to EGF, an Action Medical Research grant to DJD (GN2703) and by Medical Research Council Senior Fellowships to DJD (G1002046) and VEM (V031260/1). Royal Society website - https://royalsociety.org/ ; Action Medical Research - https://action.org.uk/ ; MRC - https://mrc.ukri.org/ . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability: All relevant data are contained in the paper and supporting information files. The Nanostring data are available from the GEO database, accession number GSE188655. All other data are available at Figshare, DOI 10.6084/m9.figshare.20310363 . Copyright: © 2022 Smith et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. We now demonstrate that cathelicidin is not expressed in the healthy murine CNS or secondary lymphoid organs but is strongly produced during EAE and in the active demyelinated brain lesions of patients with MS. Cathelicidin production plays a fundamental role in disease pathogenesis. Mice lacking the peptide have reduced incidence of EAE as T cell production of proinflammatory cytokines is reduced. We propose that cathelicidin is critical for the development of pathogenic longer-term Th17 responses in inflammatory disease. We have previously shown that neutrophil release of the antimicrobial host defence peptide cathelicidin, which occurs during degranulation and release of extracellular traps, potentiates Th17 differentiation in vitro and in models of acute inflammation [ 31 ]. However, its role in longer-term inflammation or in inflammation of the CNS is not known. Neutrophils have a dynamic relationship with T cells and it is well understood that they can influence T cell activation and migration [ 23 , 24 ]. There is now substantial evidence that neutrophils are important and pathogenic in EAE and MS. Neutrophil populations expand during both diseases and move into the CNS [ 25 – 27 ]. The peripheral neutrophil populations are also dysregulated, with an activated phenotype [ 28 ]. Depletion of neutrophils abrogates EAE disease [ 4 , 29 , 30 ]. The precise mechanisms through which neutrophils worsen EAE and increase severity of autoimmune conditions have not, however, been described; and while Th17 cell impact on neutrophils in the CNS is known [ 4 ], the reverse—the impact of neutrophils on Th17 cell differentiation and survival—is still unclear. Like in other autoinflammatory conditions [ 9 ], the Th17 subset of cells is particularly important for driving disease in MS and EAE [ 10 – 14 ], through their ability to cross into the CNS following priming in the lymph nodes [ 15 – 18 ]; once there, Th17 cells also drive disease through contributing to blood–brain barrier (BBB) breakdown by attracting MMP-releasing neutrophils to the site [ 19 – 22 ]. In this project, we examined the differentiation of cytokine-producing T cells during multiple sclerosis (MS). MS is a demyelinating, neurodegenerative disease of the central nervous system (CNS) [ 1 ]. Through the modelling of MS using experimental autoimmune encephalomyelitis (EAE), we now understand that T cells play a central role in driving this disease [ 2 – 8 ]. However, the priming of pathogenic T cells during MS is complex and is still not fully understood. In particular, how other innate and adaptive immune cells affect longer-term T cell function in the CNS is unclear. Results Cathelicidin promotes severe disease in EAE Next, we examined whether expression of cathelicidin is required for disease, by inducing EAE in mice lacking cathelicidin (Camp−/−, knockout, KO), first produced in [47]. Observation of clinical signs of illness demonstrated that KO mice consistently showed significantly attenuated disease compared to WT mice (Fig 5A and 5B). PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 5. Mice lacking cathelicidin are resistant to EAE disease. EAE was induced in WT C57BL/6J, cathelicidin KO (Camp KO) mice and LysMCre conditional KO mice on day 0. (A) Clinical signs of illness were noted throughout the experiment and (B) area under the curve calculated for each individual experiment. (C-E) Conditional KO LysMCre mice lacking cathelicidin in myeloid-lineage cells were generated (see S2 Fig) and (C) immunofluorescence performed to confirm microglia and neutrophils lacked cathelicidin. (D) WT mice were culled on day 14 post-immunisation and spinal cord isolated. Spinal cord cross-sections were co-stained for neuronal marker NeuN (green) and cathelicidin (magenta), which showed that neurons did not co-localise with cathelicidin in the spinal cord. (E, F) Clinical signs of disease were tracked in conditional KO mice. Data shown are (A, E) mean and standard error. N values: A—WT 85, Camp−/− 71; E—WT 85, Camp−/− 71, LysMCre 8. Images in C are representative of 3 mice. Statistical tests used: A and E–one-way ANOVA, B–paired two-tailed t test. Data available at 10.6084/m9.figshare.20310363. Cath, cathelicidin; EAE, experimental autoimmune encephalomyelitis; KO, knockout; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001554.g005 Give the multiple cell types known to be capable of expressing cathelicidin, particularly in the context of inflammation, we wanted to start to define the key cellular compartment for cathelicidin production affecting EAE disease severity, so generated a new conditional cathelicidin KO mouse. This LysMCre mouse lacks cathelicidin in myeloid-lineage cells (generation described in S2 Fig). This mouse had no cathelicidin in microglia or neutrophils (Fig 5C). LysM can also be observed in neurons [48], but no cathelicidin was seen to be produced by WT neuronal cells in our system (Fig 5D). Observation of clinical signs of illness over time (Fig 5E) and incidence of disease (Fig 5F) showed the conditional KO mice phenocopied the full KO animals and had attenuated incidence of disease, demonstrating that myeloid cell production of cathelicidin is sufficient for full disease penetrance and therefore that the noted endothelial cell production is not essential for development of disease. [END] --- [1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001554 Published and (C) by PLOS One Content appears here under this condition or license: Creative Commons - Attribution BY 4.0. via Magical.Fish Gopher News Feeds: gopher://magical.fish/1/feeds/news/plosone/