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Neutrophils release neutrophil extracellular traps (NETs) which ensnare pathogens and have pathogenic functions in diverse diseases. We examined the NETosis pathways induced by five stimuli; PMA, the calcium ionophore A23187, nigericin, Candida albicans and Group B Streptococcus. We studied NET production in neutrophils from healthy donors with inhibitors of molecules crucial to PMA-induced NETs including protein kinase C, calcium, reactive oxygen species, the enzymes myeloperoxidase (MPO) and neutrophil elastase. Additionally, neutrophils from chronic granulomatous disease patients, carrying mutations in the NADPH oxidase complex or a MPO-deficient patient were examined. We show that PMA, C. albicans and GBS use a related pathway for NET induction, whereas ionophores require an alternative pathway but that NETs produced by all stimuli are proteolytically active, kill bacteria and composed mainly of chromosomal DNA. Thus, we demonstrate that NETosis occurs through several signalling mechanisms, suggesting that extrusion of NETs is important in host defence. The immune system protects the body against microorganisms that can cause infections and diseases. Neutrophils are a type of immune cell that patrol the blood in search of germs. Once they encounter potentially harmful microbes, neutrophils eradicate them in different ways. One way to catch the germs is by using ‘neutrophil extracellular traps’, or NETs for short, to confine and kill the invaders. NETs are web-like structures made up of anti-microbial proteins and the neutrophil’s own DNA. The process of making NETs kills the neutrophil, as it eventually explodes to release the NETs. NETs play a key role in disease prevention, but producing too many NETs or producing them at the wrong time or in the wrong place can actually make certain diseases worse. Therefore, it is important to fully understand the signaling pathways and molecules the neutrophils use to make NETs. Kenny et al. exposed neutrophils from healthy people to five different compounds known to cause the cells to make NETs, including some harmful molecules, a fungus and a bacterium. Microscopy was then used to count how many neutrophils made NETs in response to each of the five stimuli. Further experiments showed that neutrophils from patients with an immune system disorder produced fewer NETs when stimulated with some of the compounds, while the other stimuli caused neutrophils to produce the same levels of NETs as healthy individuals. Kenny et al. also revealed that neutrophils use several different ways to produce and release NETs, depending on the stimulus used. Some of the ways required reactive oxygen species, such as hydrogen peroxide and enzymes, while others produced NETs without the need for these molecules. Lastly, Kenny et al. showed that the way the cells die after the NET is released is unique from other pathways that are known to kill cells. Future work will aim to identify a single molecule that can block neutrophils from releasing NETs at the wrong time and place, without affecting the important role NETs play in fighting germs. Such a molecule could be developed into a drug for people with diseases like lupus or rheumatoid arthritis, where the release of NETs makes the disease worse not better.

Fingolimod, a sphingosine-1-phosphate receptor (S1PR) modulator, was the first oral disease-modifying therapy approved for relapsing forms of multiple sclerosis; it reduces autoreactive lymphocytes’ egress from lymphoid tissues by down-regulating S1PRs. Sphingosine-1-phosphate signaling is implicated in a range of physiologic functions, and S1PRs are expressed differentially in various tissues, including the cardiovascular system. Modulation of S1PRs on cardiac cells provides an explanation for the transient effects of fingolimod on heart rate and atrioventricular conduction at initiation of fingolimod therapy, and for the mild but more persistent effects on blood pressure observed in some patients on long-term treatment. This review describes the nontherapeutic actions of fingolimod in the context of sphingosine-1-phosphate signaling in the cardiovascular system, as well as providing a summary of the associated clinical implications useful to physicians considering initiation of fingolimod therapy in patients. A transient reduction in heart rate (mean decrease of 8 beats per minute) and, less commonly, a temporary delay in atrioventricular conduction observed in some patients when initiating fingolimod therapy are both due to activation of S1PR subtype 1 on cardiac myocytes. These effects are a reflection of fingolimod first acting as a full S1PR agonist and thereafter functioning as an S1PR antagonist after down-regulation of S1PR subtype 1 at the cell surface. For most individuals, first-dose effects of fingolimod are asymptomatic, but all patients need to be monitored for at least 6 hours after the first dose, in accordance with the label recommendations.

The transition zones of the squamous and columnar epithelia constitute hotspots for the emergence of cancer, often preceded by metaplasia, in which one epithelial type is replaced by another. It remains unclear how the epithelial spatial organization is maintained and how the transition zone niche is remodelled during metaplasia. Here we used single-cell RNA sequencing to characterize epithelial subpopulations and the underlying stromal compartment of endo- and ectocervix, encompassing the transition zone. Mouse lineage tracing, organoid culture and single-molecule RNA in situ hybridizations revealed that the two epithelia derive from separate cervix-resident lineage-specific stem cell populations regulated by opposing Wnt signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodelling and increases expression of the Wnt inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Our data indicate that homeostasis at the transition zone results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages. Chumduri, Gurumurthy et al. show that cervical squamous and columnar epithelia derive from two stem cell populations, regulated by opposing Wnt signals, and that a Wnt-repressive environment can induce metaplasia.

Arturo Zychlinsky and Volker Brinkman recall the discovery of neutrophil extracellular traps.

Coinfections with pathogenic microbes continually confront cervical mucosa, yet their implications in pathogenesis remain unclear. Lack of in-vitro models recapitulating cervical epithelium has been a bottleneck to study coinfections. Using patient-derived ectocervical organoids, we systematically modeled individual and coinfection dynamics of Human papillomavirus (HPV)16 E6E7 and Chlamydia, associated with carcinogenesis. The ectocervical stem cells were genetically manipulated to introduce E6E7 oncogenes to mimic HPV16 integration. Organoids from these stem cells develop the characteristics of precancerous lesions while retaining the self-renewal capacity and organize into mature stratified epithelium similar to healthy organoids. HPV16 E6E7 interferes with Chlamydia development and induces persistence. Unique transcriptional and post-translational responses induced by Chlamydia and HPV lead to distinct reprogramming of host cell processes. Strikingly, Chlamydia impedes HPV-induced mechanisms that maintain cellular and genome integrity, including mismatch repair in the stem cells. Together, our study employing organoids demonstrates the hazard of multiple infections and the unique cellular microenvironment they create, potentially contributing to neoplastic progression. Here, Koster et al ., model human papillomavirus and Chlamydia coinfection dynamics in patient-derived ectocervical organoids, and characterize the effects of multiple infections in the cellular microenvironment, potentially contributing to neoplasia.

Chronic infections of the fallopian tubes with Chlamydia trachomatis ( Ctr ) cause scarring and can lead to infertility. Here we use human fallopian tube organoids and genital Ctr serovars D, K and E for long-term in vitro analysis. The epithelial monolayer responds with active expulsion of the bacteria into the lumen and with compensatory cellular proliferation—demonstrating a role of epithelial homeostasis in the defense against this pathogen. In addition, Ctr infection activates LIF signaling, which we find to be an essential regulator of stemness in the organoids. Infected organoids exhibit a less differentiated phenotype with higher stemness potential, as confirmed by increased organoid forming efficiency. Moreover, Ctr increases hypermethylation of DNA, which is an indicator of accelerated molecular aging. Thus, the chronic organoid infection model suggests that Ctr has a long-term impact on the epithelium. These heritable changes might be a contributing factor in the development of tubal pathologies, including the initiation of high grade serous ovarian cancer. Chronic infections of the fallopian tubes with Chlamydia trachomatis can cause scarring and infertility. Here, the authors show that the pathogen alters stem cell differentiation and DNA methylation in human fallopian tube organoids, suggesting a potential link to cellular ageing and malignant transformation.

The epithelial lining of the fallopian tube is of critical importance for human reproduction and has been implicated as a site of origin of high-grade serous ovarian cancer. Here we report on the establishment of long-term, stable 3D organoid cultures from human fallopian tubes, indicative of the presence of adult stem cells. We show that single epithelial stem cells in vitro can give rise to differentiated organoids containing ciliated and secretory cells. Continuous growth and differentiation of organoids depend on both Wnt and Notch paracrine signalling. Microarray analysis reveals that inhibition of Notch signalling causes downregulation of stem cell-associated genes in parallel with decreased proliferation and increased numbers of ciliated cells and that organoids also respond to oestradiol and progesterone treatment in a physiological manner. Thus, our organoid model provides a much-needed basis for future investigations of signalling routes involved in health and disease of the fallopian tube. The mechanisms underlying fallopian tube epithelial renewal are unclear. Here, Kessler et al. isolate adult stem cells from the human fallopian tube epithelium and generate 3D organoids from these cells in vitro that have a similar architecture to that of the fallopian tube.

Key Points Multiple sclerosis (MS) is a chronic, autoimmune disorder of the central nervous system (CNS) that is characterized by inflammation leading to astrogliosis, demyelination and oligodendrocyte and neuronal loss. Current treatment strategies in MS involve management of symptoms and use of disease-modifying drugs, all of which must be injected. Oral fingolimod (FTY720/Gilenya; Novartis) is a first-in-class sphingosine 1-phosphate (S1P) receptor modulator that, in relapsing–remitting MS, has demonstrated improved efficacy compared to placebo and one of the first-line interferon (IFN) products in terms of relapses and magnetic resonance imaging measures. Fingolimod has also shown an effect on disability and reduces brain atrophy compared to placebo and IFN-β on intent-to-treat analysis over the full duration of the studies. The therapeutic activity of the drug requires phosphorylation in vivo by sphingosine kinases to form the active moiety fingolimod phosphate. Fingolimod phosphate binds to lymphocytic S1P 1 receptors, causing internalization and degradation of the receptors. This reduces S1P–S1P 1 -dependent egress of lymphocytes from lymph nodes and reduces the recirculation of autoaggressive T cells via lymph and blood to the CNS. Fingolimod retains central but not effector memory T cells in lymph nodes; this leads to a preferential reduction of MS-pathogenic immune responses and spares large parts of protective immunity. Based on its lipophilic nature, fingolimod crosses the blood–brain barrier, where the drug may down-modulate S1P receptors on neural cells, particularly astrocytes, to reduce astrogliosis, a phenomenon associated with neurodegeneration in MS. This may help restore gap-junctional communication of astrocytes with neurons and cells of the blood–brain barrier. Fingolimod may act through immune-based and central mechanisms to reduce inflammation and to support structural restoration of the CNS parenchyma. In September 2010, 0.5 mg fingolimod was approved by the US Food and Drug Administration as an oral first-line treatment for relapsing–remitting MS. In September 2010, fingolimod (FTY720/Gilenya; Novartis) became the first oral disease-modifying therapy to be approved by the US Food and Drug Administration for relapsing–remitting multiple sclerosis. Brinkmann and colleagues describe its discovery and development, and how elucidation of its effects on sphingosine 1-phosphate receptors has improved the understanding of the biology of these receptors. The discovery of fingolimod (FTY720/Gilenya; Novartis), an orally active immunomodulatory drug, has opened up new approaches to the treatment of multiple sclerosis, the most common inflammatory disorder of the central nervous system. Elucidation of the effects of fingolimod — mediated by the modulation of sphingosine 1-phosphate (S1P) receptors — has indicated that its therapeutic activity could be due to regulation of the migration of selected lymphocyte subsets into the central nervous system and direct effects on neural cells, particularly astrocytes. An improved understanding of the biology of S1P receptors has also been gained. This article describes the discovery and development of fingolimod, which was approved by the US Food and Drug Administration in September 2010 as a first-line treatment for relapsing forms of multiple sclerosis, thereby becoming the first oral disease-modifying therapy to be approved for multiple sclerosis in the United States.

Neutrophils are critical to host defence, executing diverse strategies to perform their antimicrobial and regulatory functions. One tactic is the production of neutrophil extracellular traps (NETs). In response to certain stimuli, neutrophils decondense their lobulated nucleus and release chromatin into the extracellular space through a process called NETosis. However, NETosis, and the subsequent degradation of NETs, can become dysregulated. NETs are proposed to play a role in infectious as well as many non-infection related diseases including cancer, thrombosis, autoimmunity and neurological disease. Consequently, there is a need to develop specific tools for the study of these structures in disease contexts. In this study, we identified a NET-specific histone H3 cleavage event and harnessed this to develop a cleavage site-specific antibody for the detection of human NETs. By microscopy, this antibody distinguishes NETs from chromatin in purified and mixed cell samples. It also detects NETs in tissue sections. We propose this antibody as a new tool to detect and quantify NETs.

Systemic lupus erythematosus (SLE) is an autoimmune disease in which patients develop autoantibodies to DNA, histones, and often to neutrophil proteins. These form immune complexes that are pathogenic and may cause lupus nephritis. In SLE patients, infections can initiate flares and are a major cause of mortality. Neutrophils respond to infections and release extracellular traps (NETs), which are antimicrobial and are made of DNA, histones, and neutrophil proteins. The timely removal of NETs may be crucial for tissue homeostasis to avoid presentation of self-antigens. We tested the hypothesis that SLE patients cannot clear NETs, contributing to the pathogenesis of lupus nephritis. Here we show that serum endonuclease DNase1 is essential for disassembly of NETs. Interestingly, a subset of SLE patients' sera degraded NETs poorly. Two mechanisms caused this impaired NET degradation: (i) the presence of DNase1 inhibitors or (ii) anti-NET antibodies prevented DNase1 access to NETs. Impairment of DNase1 function and failure to dismantle NETs correlated with kidney involvement. Hence, identification of SLE patients who cannot dismantle NETs might be a useful indicator of renal involvement. Moreover, NETs might represent a therapeutic target in SLE.