IL-10 Restrains IL-17 to Limit Lung Pathology Characteristics following Pulmonary Infection with Francisella tularensis Live Vaccine Strain

IL-10 production during intracellular bacterial infections is generally thought to be detrimental because of its role in suppressing protective T-helper cell 1 (Th1) responses. Francisella tularensis is a facultative intracellular bacterium that activates both Th1 and Th17 protective immune responses. Herein, we report that IL-10–deficient mice (Il10^−/−), despite having increased Th1 and Th17 responses, exhibit increased mortality after pulmonary infection with F. tularensis live vaccine strain. We demonstrate that the increased mortality observed in Il10^−/−-infected mice is due to exacerbated IL-17 production that causes increased neutrophil recruitment and associated lung pathology. Thus, although IL-17 is required for protective immunity against pulmonary infection with F. tularensis live vaccine strain, its production is tightly regulated by IL-10 to generate efficient induction of protective immunity without mediating pathology. These data suggest a critical role for IL-10 in maintaining the delicate balance between host immunity and pathology during pulmonary infection with F. tularensis live vaccine strain.Francisella tularensis, a facultative intracellular bacterium, because of its infectious nature and the severe disease caused by low doses of airborne bacteria, has been classified as a category A select bioterrorism agent.¹ Infection in humans is caused by two main subspecies, F. tularensis (type A) and Francisella holarctica (type B).² An F. tularensis live vaccine strain (LVS) has been developed from the F. tularensis B strain as an experimental vaccine, but is not licensed for use in humans.¹ F. tularensis LVS has been used as a representative attenuated model to address the immune requirements for protection against Francisella. By using this model, the importance of IL-12 in driving interferon γ (IFN-γ) and T-helper cell 1 (Th1) responses in immunity to F. tularensis LVS infection is well described.^3–5 In contrast, IL-17 is generally thought to play a role in protection against extracellular, but not intracellular, pathogens.⁶ However, we and others recently identified a protective role for IL-17 in the induction of cellular immunity to F. tularensis LVS pulmonary infection,^7–9 by driving the production of IFN-γ through IL-12 induction.⁷ IL-17 is a proinflammatory cytokine also known to induce chemokines, such as keratinocyte chemoattractant, macrophage inflammatory protein 2 (MIP-2), and granulocyte colony-stimulating factor (G-CSF), to mediate granulopoiesis, neutrophil recruitment, and inflammation.⁶ Accordingly, the absence of IL-17 during F. tularensis LVS pulmonary infection also results in decreased induction of G-CSF and MIP-2, as well as decreased accumulation of neutrophils and lung inflammation.⁷ Neutrophil depletion alone does not affect bacterial control after pulmonary infection with F. tularensis LVS,¹⁰ suggesting that the role for IL-17 in driving Th1 responses, and not neutrophil recruitment, was the primary immune mechanism mediating protection in this model.⁷ These data together suggest that both IL-17 and IFN-γ are required for generating protective immunity to pulmonary F. tularensis LVS infection.IL-10 is an anti-inflammatory cytokine best studied for its inhibitory effects on IL-12 production and down-regulation of Th1 responses.¹¹ Accordingly, IL-10–deficient mice show enhanced protection in models of intracellular bacterial infections, such as Mycobacterium tuberculosis¹² and Listeria monocytogenes.¹³ In addition, in a cutaneous model of F. tularensis LVS infection, IL-10–deficient mice exhibit increased protection, and this was reversed when IL-17 was depleted.¹⁴ In contrast to these published studies, in the current study, we report that after pulmonary infection with F. tularensis LVS, mice deficient in IL-10 (Il10^−/−) exhibit increased mortality. We clearly demonstrate that the increased mortality in the Il10^−/−-infected mice is not associated with loss of protective immunity, because bacterial burden between wild-type and Il10^−/− mice is similar, but is caused by exacerbated inflammation and increased lung pathology. We demonstrate that the exacerbated inflammation observed in Il10^−/−-infected mice is the result of unrestrained IL-17 production and IL-17–dependent recruitment of neutrophils and resulting lung pathology. These data together suggest that, although IL-17 is required for protective immunity against pulmonary infection with F. tularensis LVS,^7,9 IL-17 production is tightly regulated by anti-inflammatory cytokines, such as IL-10. Our studies highlight how inflammatory cytokines, such as IL-17, can be beneficial for host protection, but when produced unrestrained, can mediate host pathology.     

Role of Bile Acids in Liver Injury and Regeneration following Acetaminophen Overdose

Bile acids play a critical role in liver injury and regeneration, but their role in acetaminophen (APAP)–induced liver injury is not known. We tested the effect of bile acid modulation on APAP hepatotoxicity using C57BL/6 mice, which were fed a normal diet, a 2% cholestyramine (CSA)–containing diet for bile acid depletion, or a 0.2% cholic acid (CA)–containing diet for 1 week before treatment with 400 mg/kg APAP. CSA-mediated bile acid depletion resulted in significantly higher liver injury and delayed regeneration after APAP treatment. In contrast, 0.2% CA supplementation in the diet resulted in a moderate delay in progression of liver injury and significantly higher liver regeneration after APAP treatment. Either CSA-mediated bile acid depletion or CA supplementation did not affect hepatic CYP2E1 levels or glutathione depletion after APAP treatment. CSA-fed mice exhibited significantly higher activation of c-Jun N-terminal protein kinases and a significant decrease in intestinal fibroblast growth factor 15 mRNA after APAP treatment. In contrast, mice fed a 0.2% CA diet had significantly lower c-Jun N-terminal protein kinase activation and 12-fold higher fibroblast growth factor 15 mRNA in the intestines. Liver regeneration after APAP treatment was significantly faster in CA diet–fed mice after APAP administration secondary to rapid cyclin D1 induction. Taken together, these data indicate that bile acids play a critical role in both initiation and recovery of APAP-induced liver injury.Bile acids are versatile biological molecules that regulate energy homeostasis, activate nuclear receptors and cell signaling pathways, and control cell proliferation and inflammatory processes in the liver and gastrointestinal tract.^1,2 Bile acids maintain their own homeostasis by activating a complex signaling network involving hepatic and intestinal farnesoid X receptor (FXR), small heterodimer partner, and intestinal fibroblast growth factor (FGF) 15 (FGF19 in human) expression, culminating in inhibition of the primary bile acid–synthesizing enzyme, CYP7A1.^3–6 Although bile acids are potent signaling molecules at pathophysiological concentrations, they cause apoptosis, necrosis, and oxidative stress.^3,7–10 Bile acids have also been implicated in stimulation of liver regeneration.^11–14 Studies in recent years indicate that the bile acid–mediated gut-liver signaling axis may play a critical role in regulation of liver homeostasis.^6,15,16Acetaminophen (APAP) is the most commonly used analgesic and antipyretic agent.¹⁷ An overdose of APAP is the major cause of acute liver failure in the United States.^18,19 The mechanisms of APAP-induced liver injury and subsequent liver regeneration are the focus of intense investigation.^20–22 In an overdose situation, excess APAP is mainly metabolized by CYP2E1 to a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI). In hepatocytes, conjugation of NAPQI to GSH is the key mechanism for detoxification of NAPQI. Once the GSH is depleted, NAPQI attacks cellular proteins, especially mitochondrial proteins, to form protein adducts. This triggers a cascade of intracellular signaling events involving c-Jun N-terminal protein kinase (JNK) activation and mitochondrial permeability transition, finally culminating in necrotic cell death.²⁰ Liver injury is followed by compensatory liver regeneration, which is a critical determinant of final outcome of liver injury.²³ Despite decades of research, how these intracellular events are affected by extracellular signaling is not known.The current study was designed to explore the role of bile acids in initiation of liver injury and stimulation of liver regeneration after APAP overdose. These studies are highly significant because the data reveal a novel role of bile acids in cellular protection and liver regeneration after APAP overdose, and these studies investigate the effect of resin-mediated bile acid depletion, a commonly used therapy, on APAP toxicity.     

The influence of patient-centered communication during radiotherapy education sessions on post-consultation patient outcomes

Objective

To adapt an observational tool for assessing patient-centeredness of radiotherapy consultations and to assess whether scores for this tool and an existing tool assessing patient-perceived patient-centeredness predict patient outcomes.

Methods

The Measure of Patient-Centered Communication (MPCC), an observational coding system that assesses depth of discussion during a consultation, was adapted to the radiotherapy context. Fifty-six radiotherapy patients (from 10 radiation therapists) had their psycho-education sessions recorded and coded using the MPCC. Patients also completed instruments assessing their perception of patient-centeredness, trust in the radiation therapist, satisfaction with the consultation, authentic self-representation (ASR) and state anxiety.

Results

The MPCC correlated weakly with patient-perceived patient-centeredness. The Feelings subcomponent of the MPCC predicted one aspect of ASR and trust, and interacted with level of therapist experience to predict trust. Patient-perceived patient-centeredness, which exhibited a ceiling effect, predicted satisfaction.

Conclusion

Patient-centered communication is an important predictor of patient outcomes in radiotherapy and obviates some negative aspects of radiation therapists’ experience on patient trust. As in other studies, there is a weak association between self-reported and observational coding of PCC.

Practice implications

Radiation therapists have both technical and supportive roles to play in patient care, and may benefit from training in their supportive role.     

Blood–spinal cord barrier disruption contributes to early motor-neuron degeneration in ALS-model mice

Humans with ALS and transgenic rodents expressing ALS-associated superoxide dismutase (SOD1) mutations develop spontaneous blood–spinal cord barrier (BSCB) breakdown, causing microvascular spinal-cord lesions. The role of BSCB breakdown in ALS disease pathogenesis in humans and mice remains, however, unclear, although chronic blood–brain barrier opening has been shown to facilitate accumulation of toxic blood-derived products in the central nervous system, resulting in secondary neurodegenerative changes. By repairing the BSCB and/or removing the BSCB-derived injurious stimuli, we now identify that accumulation of blood-derived neurotoxic hemoglobin and iron in the spinal cord leads to early motor-neuron degeneration in SOD1^G93A mice at least in part through iron-dependent oxidant stress. Using spontaneous or warfarin-accelerated microvascular lesions, motor-neuron dysfunction and injury were found to be proportional to the degree of BSCB disruption at early disease stages in SOD1^G93A mice. Early treatment with an activated protein C analog restored BSCB integrity that developed from spontaneous or warfarin-accelerated microvascular lesions in SOD1^G93A mice and eliminated neurotoxic hemoglobin and iron deposits. Restoration of BSCB integrity delayed onset of motor-neuron impairment and degeneration. Early chelation of blood-derived iron and antioxidant treatment mitigated early motor-neuronal injury. Our data suggest that BSCB breakdown contributes to early motor-neuron degeneration in ALS mice and that restoring BSCB integrity during an early disease phase retards the disease process.The blood–brain barrier (BBB) and blood–spinal cord barrier (BSCB) prevent entry of toxic circulating molecules and cells into the central nervous system (CNS) (1). Amyotrophic lateral sclerosis (ALS) is the most prominent adult motor-neuron disorder resulting in progressive motor-neuron loss in the spinal cord, brainstem, and motor cortex (2). Most ALS cases are sporadic (90%) whereas 10% are familial ALS. Over twenty independent studies in postmortem human tissue and cerebrospinal fluid (CSF) sampling from living ALS patients have established that the BBB and BSCB are damaged in familial and sporadic ALS, as reviewed elsewhere (1, 3). This BBB and BSCB disruption has been shown by spinal-cord and/or motor-cortex accumulation of different plasma proteins (e.g., IgG, fibrin, thrombin), erythrocytes, erythrocyte-derived hemoglobin and iron-containing hemosiderin, elevated CSF/serum albumin ratios, and diminished expression or degradation of the BSCB tight-junction proteins (1, 3–5). Deposition of hemoglobin-derived iron within the CNS has also been shown in ALS patients (3, 6, 7). Because human postmortem studies reflect, however, end-stage disease, it has remained unclear as to which stage of disease is enhanced by BSCB disruption. Longitudinal CSF or BSCB imaging studies have yet to be performed in living ALS patients (3) to clarify whether spinal-cord vascular dysfunction contributes to early- or late-stage disease.Transgenic rodents expressing human ALS-associated Cu/Zn superoxide dismutase (SOD1) mutations that represent 20% of all familial cases also develop a spontaneous BBB/BSCB disruption (8–12) similar to vascular pathology reported in humans (1, 3–7). Mice with a chronic BBB disruption due to aberrant signal transduction between the central nervous system endothelial cells and pericytes or astrocytes and pericytes develop a chronic BBB opening accompanied by accumulation of toxic blood-derived products in the central nervous system and secondary functional and structural neuronal changes (13–15).To determine whether BSCB disruption contributes to fatal paralytic disease caused by expression of an ALS-causing mutant, we now report how perturbing the BSCB, repairing the BSCB, and/or removing the BSCB-derived injurious stimuli influence development of disease in SOD1^G93A mice that develop a spontaneous BSCB breakdown (8, 9, 12).     

Spatially robust estimates of biological nitrogen (N) fixation imply substantial human alteration of the tropical N cycle

Biological nitrogen fixation (BNF) is the largest natural source of exogenous nitrogen (N) to unmanaged ecosystems and also the primary baseline against which anthropogenic changes to the N cycle are measured. Rates of BNF in tropical rainforest are thought to be among the highest on Earth, but they are notoriously difficult to quantify and are based on little empirical data. We adapted a sampling strategy from community ecology to generate spatial estimates of symbiotic and free-living BNF in secondary and primary forest sites that span a typical range of tropical forest legume abundance. Although total BNF was higher in secondary than primary forest, overall rates were roughly five times lower than previous estimates for the tropical forest biome. We found strong correlations between symbiotic BNF and legume abundance, but we also show that spatially free-living BNF often exceeds symbiotic inputs. Our results suggest that BNF in tropical forest has been overestimated, and our data are consistent with a recent top-down estimate of global BNF that implied but did not measure low tropical BNF rates. Finally, comparing tropical BNF within the historical area of tropical rainforest with current anthropogenic N inputs indicates that humans have already at least doubled reactive N inputs to the tropical forest biome, a far greater change than previously thought. Because N inputs are increasing faster in the tropics than anywhere on Earth, both the proportion and the effects of human N enrichment are likely to grow in the future.Over the last few decades, humans have dramatically altered the global nitrogen (N) cycle (1–3). Three main processes—Haber–Bosch fixation of atmospheric N₂, widespread cultivation of leguminous N-fixing crops, and incidental N fixation during fossil fuel combustion—collectively add more reactive N to the biosphere each year than all natural processes combined (2). Although human perturbation of the N cycle has brought substantial benefits to society (most notably, an increase in crop production) (4), it has also had a number of negative effects on both ecosystems (5, 6) and people (7).Although humanity’s large imprint on the global N cycle is clear, quantifying the extent of anthropogenic changes depends, in large part, on establishing baseline estimates of nonanthropogenic N inputs (1, 8, 9). Before recent human activities, biological N fixation (BNF) was the largest source of new N to the biosphere (9). Terrestrial BNF has been particularly challenging to quantify, because it displays high spatial and temporal heterogeneity at local scales, it arises from both symbiotic associations between bacteria and plants as well as free-living microorganisms (e.g., in leaf litter and soil) (10), and high atmospheric concentrations of N₂ make direct flux measurements unfeasible. Consequently, spatial estimates of BNF have always been highly uncertain (11), and global rate estimates have fallen precipitously in the last 15 y (from 100–290 to ∼44 Tg N y⁻¹) (9). This decline in BNF implies an increase in the relative magnitude of anthropogenic N inputs from 100–150% to 190–470% of BNF (9).Historically, the largest anthropogenic changes to the N cycle have occurred in the northern temperate zone: first throughout the United States and western Europe and more recently, in China (12, 13). Large-scale estimates of BNF in natural ecosystems in these regions are consistently low (11), leading some to conclude that anthropogenic N inputs in the northern temperate zone exceed naturally occurring BNF and preindustrial atmospheric N deposition by an order of magnitude or more (1, 14). By contrast, the highest rates of naturally occurring BNF have been thought to occur in the evergreen lowland tropical rainforest biome (11), implying that, on a regional basis, human alteration of the tropical N cycle has been comparatively modest. However, in recent years, the tropics have seen some of the most dramatic increases in anthropogenic N inputs of any region on Earth—a trend that is likely to continue (2, 6, 13). Anthropogenic N inputs are increasing in tropical regions, primarily because of increasing fossil fuel combustion (13) and expanding high-N-input agriculture for both food and biofuels (6). These anthropogenic N inputs are having a measurable effect on tropical ecosystems (15). However, understanding and forecasting the effects of anthropogenic N depend, in part, on accurate estimates of BNF in lowland tropical rainforest.Unfortunately, the paradigm that the tropics have high rates of BNF is based on a paucity of evidence and several tenuous assumptions. For example, an early global synthesis of terrestrial BNF (11)—which included contributions from both symbiotic and free-living sources—included only one measured estimate of symbiotic BNF from tropical forest (16 kg N ha⁻¹ y⁻¹) (16). That single estimate, scaled over thousands of square kilometers, represented the only direct evidence of high tropical BNF rates available at that time (Fig. 1). Subsequent modeled estimates (17) that indirectly estimated BNF have reinforced the notion that tropical BNF rates are high and dominated by the symbiotic form of fixation (Fig. 1). Such high estimates of symbiotic BNF are consistent with the large number of leguminous trees in tropical forest (18–20). However, many legume species do not form N-fixing nodules (21), and of those species that do, nodulation in individuals varies with soil nutrient status, N demand, and tree age (22). Several recent analyses (10, 22–24) indicate lower tropical forest BNF and suggest that symbiotic BNF may not be as important to total BNF as previously thought (Fig. 1), although few studies have simultaneously measured symbiotic and free-living BNF.Open in a separate windowFig. 1.Previous estimates of BNF in tropical rainforest and BNF measured in this study. Percentages indicate the proportion of total BNF from symbiotic BNF. Cleveland et al. 1999 A (11) is a literature database-derived estimate of tropical forest BNF; Cleveland et al. 1999 B (11) is a modeled estimate of BNF based on the correlation between net primary productivity (NPP) and BNF derived with remotely sensed NPP and evergreen broadleaved forest (EBF) land cover classification. Central estimates and variance for Cleveland et al., 1999 A (11) and Reed et al. 2011 (10) represent the low, central, and high data-based estimates of BNF assuming 5%, 15%, and 15% legume cover, respectively. Central estimates and variance for Wang and Houlton 2009 (17) represent the modeled mean and SD of BNF predicted for the EBF biome. Central estimates and variance for Cleveland et al. 2010 (23) represent the low, central, and high estimates of symbiotic BNF plus free-living BNF or modeled BNF plus free-living BNF. Central estimates and variance for BNF in the four forest ages measured here (primary, 5–15 y, 15–30 y, and 30–50 y) represent means ± 1 SD (n = 3). Our estimate of BNF in a dynamic primary forest (gap dynamics) lacks SD, because it consisted of only two measurements: low and high estimates of forest turnover times equal to 150 and 75 y, respectively.There is also a sound theoretical basis for questioning high estimates of BNF in tropical forest. Namely, high concentrations of soil N in the legume-rich tropics create something of a paradox. Although BNF could create N-rich conditions, the substantial energetic cost of BNF means—and some data show—that BNF should be suppressed under high N availability in primary forests (25). Because of high rates of net primary productivity and high N demand in secondary forests (26, 27), regenerating canopy gaps or abandoned agricultural land may have higher rates of BNF than late-successional forest ecosystems (26).Resolving the uncertainty in the tropical (and global) N cycle requires that we overcome the enduring challenge of quantifying BNF in any ecosystem. How do we estimate large-scale rates of a process that displays extreme spatial heterogeneity at local scales? Whether using acetylene reduction assays, ¹⁵N tracer incubations, or the ¹⁵N natural abundance method, most past approaches to empirically estimate symbiotic BNF have relied on spatial extrapolations of BNF rates measured at the level of individual trees. Typically, such extrapolations are based on legume abundance (e.g., percent cover) and make species- or genera-level assumptions about nodulation status of putative N fixers. Here, we applied a method commonly used by community ecologists to measure rare species abundances—stratified adaptive cluster sampling (SACS) (28)—to measure symbiotic BNF. This approach could be used in any ecosystem, and in contrast to other methods, SACS generates unbiased estimates of mean symbiotic BNF (independent of legume abundance) and can more robustly capture the irregular distribution of nodules on the landscape. We simultaneously measured symbiotic and free-living BNF multiple times over the course of 1 y to generate spatially explicit rates of BNF inputs in primary and secondary (5–50 y old) lowland tropical forest in Costa Rica and then used the understanding gained from those estimates to revisit estimates of BNF and anthropogenic N inputs in the tropical forest biome.     

Error-prone mammalian female meiosis from silencing the spindle assembly checkpoint without normal interkinetochore tension

It is well established that chromosome segregation in female meiosis I (MI) is error-prone. The acentrosomal meiotic spindle poles do not have centrioles and are not anchored to the cortex via astral microtubules. By Cre recombinase-mediated removal in oocytes of the microtubule binding site of nuclear mitotic apparatus protein (NuMA), which is implicated in anchoring microtubules at poles, we determine that without functional NuMA, microtubules lose connection to MI spindle poles, resulting in highly disorganized early spindle assembly. Subsequently, very long spindles form with hyperfocused poles. The kinetochores of homologs make attachments to microtubules in these spindles but with reduced tension between them and accompanied by alignment defects. Despite this, the spindle assembly checkpoint is normally silenced and the advance to anaphase I and first polar body extrusion takes place without delay. Females without functional NuMA in oocytes are sterile, producing aneuploid eggs with altered chromosome number. These findings establish that in mammalian MI, the spindle assembly checkpoint is unable to sustain meiotic arrest in the presence of one or few misaligned and/or misattached kinetochores with reduced interkinetochore tension, thereby offering an explanation for why MI in mammals is so error-prone.