Philip A. Verhoef

A committed precursor to innate lymphoid cells

Innate lymphoid cells (ILCs) specialize in the rapid secretion of polarized sets of cytokines and chemokines to combat infection and promote tissue repair at mucosal barriers. Their diversity and similarities with previously characterized natural killer (NK) cells and lymphoid tissue inducers (LTi) have prompted a provisional classification of all innate lymphocytes into groups 1, 2 and 3 solely on the basis of cytokine properties, but their developmental pathways and lineage relationships remain elusive. Here we identify and characterize a novel subset of lymphoid precursors in mouse fetal liver and adult bone marrow that transiently express high amounts of PLZF, a transcription factor previously associated with NK T cell development, by using lineage tracing and transfer studies. PLZFhigh cells were committed ILC progenitors with multiple ILC1, ILC2 and ILC3 potential at the clonal level. They excluded classical LTi and NK cells, but included a peculiar subset of NK1.1+DX5− ‘NK-like’ cells residing in the liver. Deletion of PLZF markedly altered the development of several ILC subsets, but not LTi or NK cells. PLZFhigh precursors also expressed high amounts of ID2 and GATA3, as well as TOX, a known regulator of PLZF-independent NK and LTi lineages. These findings establish novel lineage relationships between ILC, NK and LTi cells, and identify the common precursor to ILCs, termed ILCP. They also reveal the broad, defining role of PLZF in the differentiation of innate lymphocytes.

P2X7 Receptor-Dependent Blebbing and the Activation of Rho-Effector Kinases, Caspases, and IL-1β Release

In response to ATP binding, the P2X7R facilitates cation channel activation, nonspecific pore formation, rapid changes in plasma membrane morphology, and secretion of IL-1β from LPS-primed macrophages. To investigate the relationship between the P2X7R-dependent changes in plasma membrane organization and the release of IL-1β, we generated time-lapse movies of ATP-stimulated BAC1 murine macrophages in conjunction with biochemical analyses of IL-1β release. Similar image analyses in human embryonic kidney 293 cells expressing recombinant P2X7R (HEK-P2X7) permitted comparison of P2X7R-dependent effects in macrophage vs nonmacrophage backgrounds. Whereas HEK-P2X7 cells exhibit zeiotic blebbing within 5 min of ATP treatment, BAC1 macrophages initiated a distinct “tethered” blebbing 10 min after ATP addition. This blebbing was comparably induced by the P2X7R-selective agonist BzATP and was blocked by P2X7R inhibitors KN-62 and oxidized ATP. Blebbing was initiated at ATP concentrations ≥3 mM, but optimal IL-1β release occurred at 1 mM ATP. P2X7R-dependent blebbing was abrogated in the presence of Rho-effector kinase inhibitors Fasudil and Y-27632, but ATP-induced IL-1β release was unaffected. ATP-induced activation of RhoA could be detected in both HEK-P2X7 cells and BAC1 murine macrophages. Thus, P2X7R activation signals distinct, novel membrane blebbing events (dependent on RhoA activation and Rho-effector kinase activity) and simultaneously initiates release of IL-1β. Our observations that blebbing and IL-1β release are dissociable suggest these events occur via parallel rather than convergent signaling pathways.

PLZF expression maps the early stages of ILC1 lineage development

Significance Diverse populations of group 1 innate lymphocytes, which exert critical early cytolytic functions against virally infected cells, have recently been discovered, raising issues of lineage relationships. We used expression of the transcription factor promyelocytic leukaemia zinc finger (PLZF) to identify the developmental intermediates of innate lymphoid cells type 1 (ILC1s), a subset of innate lymphoid cells that are particularly abundant in the liver, and demonstrated that this lineage arises from a distinct precursor, but that its development partially overlaps with established classical NK stages. Using microarray analysis, we defined a set of PLZF-dependent genes that may contribute to lineage divergence between ILC1s and classical NK cells. Among the variety of tissue-resident NK-like populations recently distinguished from recirculating classical NK (cNK) cells, liver innate lymphoid cells (ILC) type 1 (ILC1s) have been shown to represent a distinct lineage that originates from a novel promyelocytic leukaemia zinc finger (PLZF)-expressing ILC precursor (ILCP) strictly committed to the ILC1, ILC2, and ILC3 lineages. Here, using PLZF-reporter mice and cell transfer assays, we studied the developmental progression of ILC1s and demonstrated substantial overlap with stages previously ascribed to the cNK lineage, including pre–pro-NK, pre-NK precursor (pre-NKP), refined NKP (rNKP), and immature NK (iNK). Although they originated from different precursors, the ILC1 and cNK lineages followed a parallel progression at early stages and diverged later at the iNK stage, with a striking predominance of ILC1s over cNKs early in ontogeny. Although a limited set of ILC1 genes depended on PLZF for expression, characteristically including Il7r, most of these genes were also differentially expressed between ILC1s and cNKs, indicating that PLZF together with other, yet to be defined, factors contribute to the divergence between these lineages.

Childhood Antecedents to Adult Cardiovascular Disease

Through research in the prevention and treatment of adult diseases, it has become clear that many adult diseases have their origins in childhood. As illustrated in this review, these antecedents are largely a function of the nutrition, physical activity, and habits of developing children. There is also increasing evidence that chronic and toxic levels of stress can play a significant role not only in the development of mental and behavioral conditions but in the developmental pathways that lead to a number of chronic physical health conditions. Internists, family medicine physicians, and medicine-pediatrics physicians generally are comfortable managing patients with a number of cardiovascular risk factors or conditions. Although pediatric clinical guidelines have recommended universal screening for hypertension since 1977 and targeted screening for dyslipidemia since 1992 and type 2 DM since 2000, this screening is not yet common practice in general pediatrics. As the population of children and youth with risk factors for metabolic syndrome--hypertension, dyslipidemia, and type 2 DM--increases as a result of the obesity epidemic, pediatricians will have to screen routinely, and diagnose and treat these conditions in the primary care setting. Pediatric residency programs and continuing medical education programs will have to provide knowledge and clinical training in the management of these conditions before primary care pediatricians are comfortable treating children and youth with multiple cardiovascular conditions.

Intrabronchial valves: a case series describing a minimally invasive approach to bronchopleural fistulas in medical intensive care unit patients.

Background:Bronchopleural fistulas (BPF) are conditions associated with prolonged hospital course, high morbidity, and possibly increased mortality. The presence of BPFs in critically ill patients may cause difficulty in ventilation and increased oxygen requirements. Intrabronchial valves (Spiration IBV) serve as a noninvasive therapeutic option for the closure of BPFs. Methods:This report is a retrospective description of 3 patients transferred to our medical intensive care unit (ICU) with BPFs and persistent air leaks (PAL). One patient required high levels of oxygen supplementation through a nonrebreather face mask, whereas 2 required mechanical ventilation because of respiratory failure. IBVs were placed in each patient with the intention of closing their BPF and weaning them from respiratory support. Results:The use of IBVs in ICU patients with BPFs and PALs resulted in 1 patient being weaned from the persistent need for a nonrebreather face mask to room air and also aided in the liberation from mechanical ventilation of 2 patients who had been failing spontaneous breathing trials. Conclusions:The use of IBVs is safe and well tolerated in ICU patients with BPFs and PALs. The placement of IBVs results in significant clinical improvement, allowing for either weaning from high levels of oxygen support or liberation from mechanical ventilation.

Unique challenges for appropriate management of a 16-year-old girl with superior mesenteric artery syndrome as a result of anorexia nervosa: a case report

IntroductionNausea and vomiting in an adolescent, though common presenting symptoms, often pose a diagnostic and therapeutic challenge to the physician. When the diagnosis involves both medical and psychiatric components, management can be complex, especially in the current healthcare system in the United States. To the best of our knowledge, there have been no previous publications detailing successful management of a patient with anorexia nervosa and superior mesenteric artery syndrome.Case presentationWe report the case of a 16-year-old Caucasian girl who presented to our emergency department with nausea, abdominal pain, diminished appetite and vomiting. Her history and examination were notable for a 15 kg weight loss and diffuse abdominal tenderness. A barium swallow X-ray with small bowel follow-through and computed tomography scan demonstrated remarkable duodenal narrowing between the superior mesenteric artery and the aorta, consistent with superior mesenteric artery syndrome. Initial management focused on relieving the obstruction and supporting the nutritional needs of the patient. Further history confirmed a diagnosis of anorexia nervosa, requiring intensive psychiatric and medical management, and necessitating a multifaceted approach to patient care involving social work, multiple primary care physicians and subspecialists, insurance company representatives, and the patients immediate family.ConclusionThis case illustrates important points regarding the pathogenesis of superior mesenteric artery syndrome in the setting of anorexia, and it highlights the complexities that arise when managing an adolescent with both medical and psychiatric needs, as well as outlining a viable solution. While superior mesenteric artery syndrome is an uncommon cause of small bowel obstruction, the general pediatrician and child psychiatrist should be aware of this complication of anorexia nervosa.

ICOS protects against mortality from acute lung injury through activation of IL-5 + ILC2s

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease causing irreversible lung scarring and loss of pulmonary function. IPF Patients suffer from a high rate of pulmonary infections and acute exacerbations of disease that further contribute to pulmonary decline. Low expression of the inducible T-cell costimulatory molecule (ICOS) in peripheral blood mononuclear cells predicts decreased survival of IPF patients, but the mechanisms by which ICOS protects are unclear. Using a model of bleomycin-induced lung injury and fibrosis, we now demonstrate that ICOS expression enhances survival from lung injury rather than regulating fibrogenesis. Of ICOS-expressing cells, type 2 innate lymphocytes (ILC2s) are the first to respond to bleomycin-induced injury, and this expansion is ICOS dependent. Interestingly, a similar decrease in ICOS+ ILCs was found in lung tissue from IPF patients. Interleukin (IL)-5, produced primarily by ILC2s, was significantly reduced after lung injury in ICOS−/− mice, and strikingly, treatment with IL-5 protected both ICOS−/− and wild-type mice from mortality. These results imply that low ICOS expression and decreased lung ILC2s in IPF patients may contribute to poor recovery from infections and acute exacerbation and that IL-5 treatment may be a novel therapeutic strategy to overcome these defects and protect against lung injury.

Preexisting Type 2 Immune Activation Protects against the Development of Sepsis.

to different qPCR master mix solutions has a negative effect on Ct values (Table E1). Finally, we compared the original and improved protocols with regard to both RNA isolation and RT-qPCR. A 7.4-fold increase in RNA yield was achieved by using the optimized RNA isolation protocol (Figure 1E). Moreover, optimization of the qPCR protocol resulted in significantly lower Ct values of GAPDH amplicons of all sizes compared with the original protocol (Table E2). In conclusion, we have developed a combined sputum processing/RNA extraction/RT-qPCR protocol to maximize the quantity and quality of RNA obtained from sputum, and to enhance detection of transcripts by qPCR. Successful elimination of bacterial DNA was shown to be a critical step in the optimization process. Our results should facilitate future gene-expression studies utilizing sputum. n

Distinct T-helper cell responses to Staphylococcus aureus bacteremia reflect immunologic comorbidities and correlate with mortality

BackgroundThe dysregulated host immune response that defines sepsis varies as a function of both the immune status of the host and the distinct nature of the pathogen. The degree to which immunocompromising comorbidities or immunosuppressive medications affect the immune response to infection is poorly understood because these patients are often excluded from studies about septic immunity. The objectives of this study were to determine the immune response to a single pathogen (Staphylococcus aureus) among a diverse case mix of patients and to determine whether comorbidities affect immune and clinical outcomes.MethodsBlood samples were drawn from 95 adult inpatients at multiple time points after the first positive S. aureus blood culture. Cox proportional hazards modeling was used to determine the associations between admission neutrophil counts, admission lymphocyte counts, cytokine levels, and 90-day mortality. A nested case-control flow cytometric analysis was conducted to determine T-helper type 1 (Th1), Th2, Th17, and regulatory T-cell (Treg) subsets among a subgroup of 28 patients. In a secondary analysis, we categorized patients as either having immunocompromising disorders (human immunodeficiency virus and hematologic malignancies), receiving immunosuppressive medications, or being not immunocompromised.ResultsHigher neutrophil-to-lymphocyte count ratios and higher Th17 cytokine responses relative to Th1 cytokine responses early after infection were independently associated with mortality and did not depend on the immune state of the patient (HR 1.93, 95% CI 1.17–3.17, p = 0.01; and HR 1.13, 95% CI 1.01–1.27, p = 0.03, respectively). On the basis of flow cytometric analysis of CD4 T-helper subsets, an increasing Th17/Treg response over the course of the infection was most strongly associated with increased mortality (HR 4.41, 95% CI 1.69–11.5, p < 0.01). This type of immune response was most common among patients who were not immunocompromised. In contrast, among immunocompromised patients who died, a decreasing Th1/Treg response was most common.ConclusionsThe association of both increased Th17 responses and increased neutrophil counts relative to lymphocyte counts with mortality suggests that an overwhelming inflammatory response is detrimental. However, the differential responses of patients according to immune state suggest that immune status is an important clinical indicator that should be accounted for in the management of septic patients, as well as in the development of novel immunomodulatory therapies.

Rebuttal From Drs Gaffney, Verhoef, and Hall.

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