Inflammasome activation occurs in CD4+ and CD8+ T cells during graft-versus-host disease.

A severe complication of hematopoietic stem cell transplantation is graft-versus-host disease (GvHD), a reaction that occurs following the transfer of donor immune cells (the graft) into an allogeneic host. Transplanted cells recognize host alloantigens as foreign, resulting in the activation of donor T cells and migration of these pathological cells into host tissues. In this study, we found that caspase-1 is activated in alloreactive murine and human CD4+ and CD8+ T cells early during acute GvHD (aGvHD). The presence of inflammasome-bound active caspase-1 (p33) and ASC-speck formation confirmed inflammasome activation in these cells. We further measured gasdermin D (GSDMD) cleavage and IL-18 secretion from alloreactive T cells ex vivo. Isolated T cells with high levels of active caspase-1 had a strong inflammatory transcriptional signature and a metabolic phenotype similar to inflammatory myeloid cells, including the upregulation of proinflammatory cytokines and metabolic switch from oxidative phosphorylation to aerobic glycolysis. We also observed oxidative stress, mitochondrial dysfunction, and cell death phenotypes consistent with inflammatory cell death in alloreactive T cells. For the first time, this study characterizes caspase-1 activation in transplanted T cells during aGvHD, using mouse and human models, adding to a body of literature supporting inflammasome function in cells of the adaptive immune system.

Cross-talk between cancer and Pseudomonas aeruginosa mediates tumor suppression.

Microorganisms living at many sites in the human body compose a complex and dynamic community. Accumulating evidence suggests a significant role for microorganisms in cancer, and therapies that incorporate bacteria have been tried in various types of cancer. We previously demonstrated that cupredoxin azurin secreted by the opportunistic pathogen Pseudomonas aeruginosa, enters human cancer cells and induces apoptotic death1-4. However, the physiological interactions between P. aeruginosa and humans and their role in tumor homeostasis are largely unknown. Here, we show that P. aeruginosa upregulated azurin secretion in response to increasing numbers of and proximity to cancer cells. Conversely, cancer cells upregulated aldolase A secretion in response to increasing proximity to P. aeruginosa, which also correlated with enhanced P. aeruginosa adherence to cancer cells. Additionally, we show that cancer patients had detectable P. aeruginosa and azurin in their tumors and exhibited increased overall survival when they did, and that azurin administration reduced tumor growth in transgenic mice. Our results suggest host-bacterial symbiotic mutualism acting as a diverse adjunct to the host defense system via inter-kingdom communication mediated by the evolutionarily conserved proteins azurin and human aldolase A. This improved understanding of the symbiotic relationship of bacteria with humans indicates the potential contribution to tumor homeostasis.

Detection of Bacteria in Bladder Mucosa of Adult Females.

PURPOSE: Interstitial cystitis/bladder pain syndrome is a chronic urological condition diagnosed in nearly 8 million females in the United States. Whether urinary microbiota play an etiological role remains controversial. Most studies assessed the microbiota of interstitial cystitis/bladder pain syndrome patients with voided or catheterized urine as a proxy for bladder urothelium; however, urine may not be a true reflection of the bladder microbiota. Bladder biopsy tissue may provide a more accurate, and thus more clinically relevant, picture of bladder microbiota. MATERIALS AND METHODS: Bladder biopsy tissues were obtained from: (1) 30 females with interstitial cystitis/bladder pain syndrome (18-80 years old) via cystoscopically guided cold-cup biopsy following therapeutic bladder hydrodistention, and (2) 10 non-interstitial cystitis/bladder pain syndrome females undergoing pelvic organ prolapse repair. To detect bacteria, technical duplicates of each RNAlater-preserved biopsy were subjected to 16S rRNA gene sequencing. To visualize bacteria, paraformaldehyde-fixed, paraffin-embedded biopsies were subjected to a combined multiplexed fluorescence in situ hybridization and fluorescence immunohistochemistry assay and confocal microscopy. RESULTS: Bacteria were detected by 16S rRNA gene sequencing in at least 1 technical duplicate of most biopsies. The most abundant genus was Staphylococcus, followed by Lactobacillus; Escherichia was common but not abundant. There was no significant difference between interstitial cystitis/bladder pain syndrome patients and controls (P > .05). Combined fluorescence in situ hybridization and immunohistochemistry reproducibly detected 16S rRNA in epithelial cells and shed cells in the urothelium and lesioned areas and capillary walls in the lamina propria of human bladder biopsy tissue. CONCLUSIONS: We conclude that urothelial and urinary microbiota are similar but not identical in adult females.

Polyamine Depletion Inhibits Bunyavirus Infection via Generation of Noninfectious Interfering Virions.

Several host and viral processes contribute to forming infectious virions. Polyamines are small host molecules that play diverse roles in viral replication. We previously demonstrated that polyamines are crucial for RNA viruses; however, the mechanisms by which polyamines function remain unknown. Here, we investigated the role of polyamines in the replication of the bunyaviruses Rift Valley fever virus (vaccine strain MP-12) and La Crosse virus (LACV). We found that polyamine depletion did not impact viral RNA or protein accumulation, despite significant decreases in titer. Viral particles demonstrated no change in morphology, size, or density. Thus, polyamine depletion promotes the formation of noninfectious particles. These particles interfere with virus replication and stimulate innate immune responses. We extended this phenotype to Zika virus; however, coxsackievirus did not similarly produce noninfectious particles. In sum, polyamine depletion results in the accumulation of noninfectious particles that interfere with replication and stimulate immune signaling, with important implications for targeting polyamines therapeutically, as well as for vaccine strategies.IMPORTANCE Bunyaviruses are emerging viral pathogens that cause encephalitis, hemorrhagic fevers, and meningitis. We have uncovered that diverse bunyaviruses require polyamines for productive infection. Polyamines are small, positively charged host-derived molecules that play diverse roles in human cells and in infection. In polyamine-depleted cells, bunyaviruses produce an overabundance of noninfectious particles that are indistinguishable from infectious particles. However, these particles interfere with productive infection and stimulate antiviral signaling pathways. We further find that additional enveloped viruses are similarly sensitive to polyamine depletion but that a nonenveloped enterovirus is not. We posit that polyamines are required to maintain bunyavirus infectivity and that polyamine depletion results in the accumulation of interfering noninfectious particles that limit infectivity. These results highlight a novel means by which bunyaviruses use polyamines for replication and suggest promising means to target host polyamines to reduce virus replication.

Interrogating the aged striatum: robust survival of grafted dopamine neurons in aging rats produces inferior behavioral recovery and evidence of impaired integration.

Advanced age is the primary risk factor for Parkinson's disease (PD). In PD patients and rodent models of PD, advanced age is associated with inferior symptomatic benefit following intrastriatal grafting of embryonic dopamine (DA) neurons, a pattern believed to result from decreased survival and reinnervation provided by grafted neurons in the aged host. To help understand the capacity of the aged, parkinsonian striatum to be remodeled with new DA terminals, we used a grafting model and examined whether increasing the number of grafted DA neurons in aged rats would translate to enhanced behavioral recovery. Young (3months), middle-aged (15months), and aged (22months) parkinsonian rats were grafted with proportionately increasing numbers of embryonic ventral mesencephalic (VM) cells to evaluate whether the limitations of the graft environment in subjects of advancing age can be offset by increased numbers of transplanted neurons. Despite robust survival of grafted neurons in aged rats, reinnervation of striatal neurons remained inferior and amelioration of levodopa-induced dyskinesias (LID) was delayed or absent. This study demonstrates that: 1) counter to previous evidence, under certain conditions the aged striatum can support robust survival of grafted DA neurons; and 2) unknown factors associated with the aged striatum result in inferior integration of graft and host, and continue to present obstacles to full therapeutic efficacy of DA cell-based therapy in this model of aging.

Context modulates the expression of conditioned motor sensitization, cellular activation and synaptophysin immunoreactivity.

We tested the hypothesis that amphetamine (AMPH)-induced conditioned motor sensitization is accompanied by cellular activation (measured by Fos immunoreactivity) and synaptophysin immunoreactivity in reward-related brain areas. Forty-eight rats were tested for conditioned motor sensitization using a conditioning paradigm that was performed in a three-chambered apparatus. Rats underwent two drug pairings with 1.0 mg/kg AMPH in one outer chamber and, on alternate days, were paired with saline in the other. On the fifth day, relative to the first AMPH treatment, AMPH administration increased motor activity in the AMPH-paired context but not in the saline-paired context. Relative to the first saline treatment, saline on the fifth day produced a conditioned increase in motor activity when given in the chamber previously paired with AMPH, and saline given in the saline-paired context produced a conditioned decrease in motor activity. AMPH administered in the AMPH-paired context increased the density of both Fos and synaptophysin immunoreactivity in the dentate gyrus, cornu ammonis (CA)1, CA3, basolateral amygdala and dorsolateral striatum. This pairing between context and drug increased Fos but not synaptophysin immunoreactivity in the nucleus accumbens core and shell. Saline administered in the AMPH-paired context increased the density of Fos immunoreactivity in the basolateral amygdala and nucleus accumbens core. These data indicate that the basolateral amygdala-nucleus accumbens core pathway is necessary for the context-elicited conditioned motor responses, while the hippocampus encodes the spatial context.

The neural substrates of amphetamine conditioned place preference: implications for the formation of conditioned stimulus-reward associations.

Associations formed between conditioned stimuli and drug reward are major contributors in human drug addiction. To better understand the brain changes that accompany this process, we used immunohistochemistry for c-Fos (a neuronal activity marker), synaptophysin (a marker for synaptogenesis) and tyrosine kinase B receptor (a neurotrophic factor receptor that mediates synaptic plasticity) to investigate the neural substrates of amphetamine-induced conditioned place preference in rats. Conditioned place preference was induced by both 1.0 mg/kg and 0.3 mg/kg doses of amphetamine. Furthermore, amphetamine conditioning increased the density of c-Fos-immunoreactive cells and these cells were fully colocalized with the tyrosine kinase B receptor in the dentate gyrus, CA1 field and basolateral amygdala. Amphetamine conditioning increased the density of synaptophysin-immunoreactive varicosities in all brain regions studied, except the nucleus accumbens shell and dorsolateral striatum. The degree of conditioned place preference was highly correlated with c-Fos-immunoreactive cell density in the basolateral amygdala and with the density of synaptophysin-immunoreactive varicosities in all mesolimbic regions studied. The latter correlation was particularly impressive for the ventral pallidum and basolateral amygdala. The formation of conditioned stimulus-amphetamine reward associations is accompanied by tyrosine kinase B receptor expression in the basolateral amygdala and dentate gyrus, CA1 and CA3 fields of the hippocampus. These data therefore suggest that the formation of conditioned stimulus-reward associations requires, at least in part, activation of amygdalar-hippocampal circuits.

Inhibition of restraint stress-induced neural and behavioural activation by endogenous cannabinoid signalling.

The role of endocannabinoid (eCB) signalling in restraint stress-induced neuronal activation was studied. Male mice exposed to 30 min of restraint exhibit increased Fos protein within prefrontal cortex (PFC), lateral septum (LS), nucleus accumbens (Acb) and medial amygdala. SR141716 (2 mg/kg) itself had no effect on Fos but pretreatment with SR141716 significantly potentiated restraint-induced Fos expression in cingulate, LS and Acb. SR141716 also significantly increased the time spent in active escape behaviours during the restraint. In restraint-habituated mice (mice exposed to four previous restraint episodes), the fifth restraint exposure resulted in decreased expression of active escape behaviours compared to the first exposure and only induced Fos protein in the central and medial amygdala. Administration of SR141716 prior to the fifth restraint episode resulted in greater potentiation of restraint-induced Fos induction than the first; significant increases occurred within all regions of PFC examined, LS and Acb. Brain regional eCB content was measured immediately after restraint. N-arachidonylethanolamine content within the amygdala was significantly decreased after both restraint episodes. 2-Arachidonylglycerol content was significantly increased in both the limbic forebrain and amygdala after the fifth restraint but not the first. Restraint had no effect on cerebellar eCB content. These data suggest that eCB activation of CB(1) receptors opposes the behavioural and neuronal responses to aversive stimuli. Because repeated homotypic stress increased both limbic 2-AG and resulted in a greater effect of SR141716 on limbic Fos expression, we hypothesize that increased CB(1) receptor activity contributes to the expression of habituation to homotypic stress.

Endocannabinoid signaling negatively modulates stress-induced activation of the hypothalamic-pituitary-adrenal axis.

Activation of the hypothalamic-pituitary-adrenal (HPA) axis is critical for the adaptation and survival of animals upon exposure to stressful stimuli, and data suggest that endocannabinoid (eCB) signaling modulates neuroendocrine function. We have explored the role of eCB signaling in the modulation of stress-induced HPA axis activation. Administration of the CB1 receptor antagonist/inverse agonist SR141716 (0.01, 0.1, 1, and 5 mg/kg, i.p.) to male mice produced a small, dose-dependent increase in the serum corticosterone (CORT) concentration. Despite this effect, the highest dose of SR141716 did not significantly increase neuronal activity within the paraventricular nucleus of the hypothalamus, as measured by the induction of Fos protein. Similarly, exposure of mice to 30 min of restraint increased serum CORT concentrations, but did not produce a consistent, statistically significant increase in Fos expression within the PVN. However, pretreatment of mice with SR141716 before restraint stress robustly potentiated restraint-induced CORT release and Fos expression within the PVN. Pretreatment of mice with either the CB1 receptor agonist CP55940, the eCB transport inhibitor AM404, or the fatty acid amide hydrolase inhibitor URB597 significantly decreased or eliminated restraint-induced CORT release. Upon exposure to acute restraint, hypothalamic 2-arachidonylglycerol content was reduced compared with the control value; however, after 5 d of restraint exposure (which resulted in an attenuated CORT response), the hypothalamic 2-arachidonylglycerol content was increased compared with the control value. These data indicate that eCB signaling negatively modulates HPA axis function in a context-dependent manner and suggest that pharmacological augmentation of eCB signaling could serve as a novel approach to the treatment of anxiety-related disorders.

Production of hydroxyeicosatetraenoic acids and prostaglandins by a novel rat microglial cell line.

We have established a clonal cell line derived from rat microglia that proliferates in response to macrophage-colony stimulating factor (CSF-1). Like primary neonatal microglia, these cells (named RTMGL1) exhibit a ramified morphology, bind isolectin B4, express CD68 and are weakly positive for CD11b and MHC class II. CSF-1-dependent proliferation requires intact signal transduction through several pathways. RTMGL1 synthesize multiple cyclooxygenase (COX) products including 11- and 15-hydroxyeicosatetraenoic acid (HETE) and express COX-2. RTMGL1 synthesize 5-HETE from arachidonic acid (AA) likely via a 5-lipoxygenase (LO). Thus, RTMGL1 have morphological and histological characteristics of primary microglia and metabolize AA via both COX and LO pathways.