The antimicrobial peptide cathelicidin drives development of experimental autoimmune encephalomyelitis in mice by affecting Th17 differentiation.

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.

High Purity Isolation of Low Density Neutrophils Casts Doubt on Their Exceptionality in Health and Disease.

Low density neutrophils (LDNs) are described in a number of inflammatory conditions, cancers and infections and associated with immunopathology, and a mechanistic role in disease. The role of LDNs at homeostasis in healthy individuals has not been investigated. We have developed an isolation protocol that generates high purity LDNs from healthy donors. Healthy LDNs were identical to healthy normal density neutrophils (NDNs), aside from reduced neutrophil extracellular trap formation. CD66b, CD16, CD15, CD10, CD54, CD62L, CXCR2, CD47 and CD11b were expressed at equivalent levels in healthy LDNs and NDNs and underwent apoptosis and ROS production interchangeably. Healthy LDNs had no differential effect on CD4+ or CD8+ T cell proliferation or IFNγ production compared with NDNs. LDNs were generated from healthy NDNs in vitro by activation with TNF, LPS or fMLF, suggesting a mechanism of LDN generation in disease however, we show neutrophilia in people with Cystic Fibrosis (CF) was not due to increased LDNs. LDNs are present in the neutrophil pool at homeostasis and have limited functional differences to NDNs. We conclude that increased LDN numbers in disease reflect the specific pathology or inflammatory environment and that neutrophil density alone is inadequate to classify discrete functional populations of neutrophils.

The Outcome of Neutrophil-T Cell Contact Differs Depending on Activation Status of Both Cell Types.

Neutrophils and T cells exist in close proximity in lymph nodes and inflamed tissues during health and disease. They are able to form stable interactions, with profound effects on the phenotype and function of the T cells. However, the outcome of these effects are frequently contradictory; in some systems neutrophils suppress T cell proliferation, in others they are activatory or present antigen directly. Published protocols modelling these interactions in vitro do not reflect the full range of interactions found in vivo; they do not examine how activated and naïve T cells differentially respond to neutrophils, or whether de-granulating or resting neutrophils induce different outcomes. Here, we established a culture protocol to ask these questions with human T cells and autologous neutrophils. We find that resting neutrophils suppress T cell proliferation, activation and cytokine production but that de-granulating neutrophils do not, and neutrophil-released intracellular contents enhance proliferation. Strikingly, we also demonstrate that T cells early in the activation process are susceptible to suppression by neutrophils, while later-stage T cells are not, and naïve T cells do not respond at all. Our protocol therefore allows nuanced analysis of the outcome of interaction of these cells and may explain the contradictory results observed previously.

The neutrophil antimicrobial peptide cathelicidin promotes Th17 differentiation.

The host defence peptide cathelicidin (LL-37 in humans, mCRAMP in mice) is released from neutrophils by de-granulation, NETosis and necrotic death; it has potent anti-pathogen activity as well as being a broad immunomodulator. Here we report that cathelicidin is a powerful Th17 potentiator which enhances aryl hydrocarbon receptor (AHR) and RORγt expression, in a TGF-ß1-dependent manner. In the presence of TGF-ß1, cathelicidin enhanced SMAD2/3 and STAT3 phosphorylation, and profoundly suppressed IL-2 and T-bet, directing T cells away from Th1 and into a Th17 phenotype. Strikingly, Th17, but not Th1, cells were protected from apoptosis by cathelicidin. We show that cathelicidin is released by neutrophils in mouse lymph nodes and that cathelicidin-deficient mice display suppressed Th17 responses during inflammation, but not at steady state. We propose that the neutrophil cathelicidin is required for maximal Th17 differentiation, and that this is one method by which early neutrophilia directs subsequent adaptive immune responses.

Interleukin-22 orchestrates a pathological endoplasmic reticulum stress response transcriptional programme in colonic epithelial cells.

OBJECTIVE: The functional role of interleukin-22 (IL22) in chronic inflammation is controversial, and mechanistic insights into how it regulates target tissue are lacking. In this study, we evaluated the functional role of IL22 in chronic colitis and probed mechanisms of IL22-mediated regulation of colonic epithelial cells. DESIGN: To investigate the functional role of IL22 in chronic colitis and how it regulates colonic epithelial cells, we employed a three-dimentional mini-gut epithelial organoid system, in vivo disease models and transcriptomic datasets in human IBD. RESULTS: As well as inducing transcriptional modules implicated in antimicrobial responses, IL22 also coordinated an endoplasmic reticulum (ER) stress response transcriptional programme in colonic epithelial cells. In the colon of patients with active colonic Crohn's disease (CD), there was enrichment of IL22-responsive transcriptional modules and ER stress response modules. Strikingly, in an IL22-dependent model of chronic colitis, targeting IL22 alleviated colonic epithelial ER stress and attenuated colitis. Pharmacological modulation of the ER stress response similarly impacted the severity of colitis. In patients with colonic CD, antibody blockade of IL12p40, which simultaneously blocks IL12 and IL23, the key upstream regulator of IL22 production, alleviated the colonic epithelial ER stress response. CONCLUSIONS: Our data challenge perceptions of IL22 as a predominantly beneficial cytokine in IBD and provide novel insights into the molecular mechanisms of IL22-mediated pathogenicity in chronic colitis. Targeting IL22-regulated pathways and alleviating colonic epithelial ER stress may represent promising therapeutic strategies in patients with colitis. TRIAL REGISTRATION NUMBER: NCT02749630.

Orchestration of Adaptive T Cell Responses by Neutrophil Granule Contents.

Neutrophils are the most abundant leukocytes in peripheral blood and respond rapidly to danger, infiltrating tissues within minutes of infectious or sterile injury. Neutrophils were long thought of as simple killers, but now we recognise them as responsive cells able to adapt to inflammation and orchestrate subsequent events with some sophistication. Here, we discuss how these rapid responders release mediators which influence later adaptive T cell immunity through influences on DC priming and directly on the T cells themselves. We consider how the release of granule contents by neutrophils-through NETosis or degranulation-is one way in which the innate immune system directs the phenotype of the adaptive immune response.