Merkel cell carcinoma (MCC) is an aggressive skin cancer of neuroendocrine origin with a high propensity of recurrence and metastasis. Merkel cell polyomavirus (MCPyV) causes the majority of MCC cases due to the expression of the MCPyV Small and Large Tumour (ST and LT) antigens. Although a number of molecular mechanisms have been attributed to MCPyV tumour antigen-mediated cellular transformation or replication, to date, no studies have investigated any potential link between MCPyV T antigen expression and the highly metastatic nature of MCC. Here we use a quantitative proteomic approach to show that MCPyV ST promotes differential expression of cellular proteins implicated in microtubule-associated cytoskeletal organisation and dynamics. Intriguingly, we demonstrate that MCPyV ST expression promotes microtubule destabilisation leading to a motile and migratory phenotype. We further highlight the essential role of the microtubule-associated protein stathmin in MCPyV ST-mediated microtubule destabilisation and cell motility and implicate the cellular phosphatase catalytic subunit PP4C in the regulation of this process. These findings suggest a possible molecular mechanism for the highly metastatic phenotype associated with MCC.
Helminth parasites remain one of the most common causes of infections worldwide, yet little is still known about the immune signaling pathways that control their expulsion. C57BL/6 mice are chronically susceptible to infection with the gastrointestinal helminth parasite Heligmosomoides polygyrus. In this article, we report that C57BL/6 mice lacking the adapter protein MyD88, which mediates signaling by TLRs and IL-1 family members, showed enhanced immunity to H. polygyrus infection. Alongside increased parasite expulsion, MyD88-deficient mice showed heightened IL-4 and IL-17A production from mesenteric lymph node CD4(+) cells. In addition, MyD88(-/-) mice developed substantial numbers of intestinal granulomas around the site of infection, which were not seen in MyD88-sufficient C57BL/6 mice, nor when signaling through the adapter protein TRIF (TIR domain-containing adapter-inducing IFN-? adapter protein) was also ablated. Mice deficient solely in TLR2, TLR4, TLR5, or TLR9 did not show enhanced parasite expulsion, suggesting that these TLRs signal redundantly to maintain H. polygyrus susceptibility in wild-type mice. To further investigate signaling pathways that are MyD88 dependent, we infected IL-1R1(-/-) mice with H. polygyrus. This genotype displayed heightened granuloma numbers compared with wild-type mice, but without increased parasite expulsion. Thus, the IL-1R-MyD88 pathway is implicated in inhibiting granuloma formation; however, protective immunity in MyD88-deficient mice appears to be granuloma independent. Like IL-1R1(-/-) and MyD88(-/-) mice, animals lacking signaling through the type 1 IFN receptor (i.e., IFNAR1(-/-)) also developed intestinal granulomas. Hence, IL-1R1, MyD88, and type 1 IFN receptor signaling may provide pathways to impede granuloma formation in vivo, but additional MyD88-mediated signals are associated with inhibition of protective immunity in susceptible C57BL/6 mice.
The expansion of human settlement into wildland areas, including forests in the eastern United States, has resulted in fragmented forest habitat that has been shown to drive higher entomological risk for Lyme disease. We investigated an alternative pathway between fragmentation and Lyme disease, namely whether increased risk of Lyme disease results in a reduced propensity to settle in high-risk areas at the interface of developed and undeveloped lands. We used longitudinal data analyses at the county level to determine whether Lyme disease incidence (LDI) influences the proportion of the population residing in the wildland-urban interface in 12 high LDI states in the eastern United States. We found robust evidence that a higher LDI reduces the proportion of a county's population residing in the wildland-urban interface in high-LDI states. This study provides some of the first evidence of human behavioral responses to Lyme disease risk via settlement decisions.
The actin cytoskeleton has been reported to restrict signalling in resting immune cells. Beta2-integrins, which mediate adhesion and cytoskeletal organization, are emerging as negative regulators of myeloid cell-mediated immune responses, but the molecular mechanisms involved are poorly understood. Here, we show that loss of the interaction between beta2-integrins and kindlin-3 abolishes the actin-linkage of integrins and the GM-CSF receptor in dendritic cells. This leads to increased GM-CSF receptor/Syk signalling, and to the induction of a transcriptional programme characteristic of mature, migratory dendritic cells, accumulation of migratory dendritic cells in lymphoid organs, and increased Th1 immune responses in vivo. We observe increased GM-CSF responses and increased survival in neutrophils where the interaction between integrin and the cytoskeleton is disrupted. Thus, ligand-reinforced beta2-integrin tail interactions restrict cytokine receptor signalling, survival, maturation and migration in myeloid cells and thereby contribute to immune homeostasis in vivo.
The larval stage of the cestode parasite Echinococcus granulosus causes hydatid disease in humans and livestock. This infection is characterized by the growth in internal organ parenchymae of fluid-filled structures (hydatids) that elicit surprisingly little inflammation in spite of their massive size and persistence. Hydatids are protected by a millimeter-thick layer of mucin-based extracellular matrix, termed the laminated layer (LL), which is thought to be a major factor determining the host response to the infection. Host cells can interact both with the LL surface and with materials that are shed from it to allow parasite growth. In this work, we analyzed the response of dendritic cells (DCs) to microscopic pieces of the native mucin-based gel of the LL (pLL). In vitro, this material induced an unusual activation state characterized by upregulation of CD86 without concomitant upregulation of CD40 or secretion of cytokines (interleukin 12 [IL-12], IL-10, tumor necrosis factor alpha [TNF-?], and IL-6). When added to Toll-like receptor (TLR) agonists, pLL-potentiated CD86 upregulation and IL-10 secretion while inhibiting CD40 upregulation and IL-12 secretion. In vivo, pLL also caused upregulation of CD86 and inhibited CD40 upregulation in DCs. Contrary to expectations, oxidation of the mucin glycans in pLL with periodate did not abrogate the effects on cells. Reduction of disulfide bonds, which are known to be important for LL structure, strongly diminished the impact of pLL on DCs without altering the particulate nature of the material. In summary, DCs respond to the LL mucin meshwork with a "semimature" activation phenotype, both in vitro and in vivo.
A 'genes-to-ecosystems' approach has been proposed as a novel avenue for integrating the consequences of intraspecific genetic variation with the underlying genetic architecture of a species to shed light on the relationships among hierarchies of ecological organization (genes ? individuals ? communities ? ecosystems). However, attempts to identify genes with major effect on the structure of communities and/or ecosystem processes have been limited and a comprehensive test of this approach has yet to emerge. Here, we present an interdisciplinary field study that integrated a common garden containing different genotypes of a dominant, riparian tree, Populus trichocarpa, and aquatic mesocosms to determine how intraspecific variation in leaf litter alters both terrestrial and aquatic communities and ecosystem functioning. Moreover, we incorporate data from extensive trait screening and genome-wide association studies estimating the heritability and genes associated with litter characteristics. We found that tree genotypes varied considerably in the quality and production of leaf litter, which contributed to variation in phytoplankton abundances, as well as nutrient dynamics and light availability in aquatic mesocosms. These 'after-life' effects of litter from different genotypes were comparable to the responses of terrestrial communities associated with the living foliage. We found that multiple litter traits corresponding with aquatic community and ecosystem responses differed in their heritability. Moreover, the underlying genetic architecture of these traits was complex, and many genes contributed only a small proportion to phenotypic variation. Our results provide further evidence that genetic variation is a key component of aquatic-terrestrial linkages, but challenge the ability to predict community or ecosystem responses based on the actions of one or a few genes.
A fifth of worldwide cancer cases have an infectious origin, with viral infection being the foremost. One such cancer is Merkel cell carcinoma (MCC), a rare but aggressive skin malignancy. In 2008, Merkel cell polyomavirus (MCPyV) was discovered as the causative agent of MCC. It is found clonally integrated into the majority of MCC tumours, which require MCPyV oncoproteins to survive. Since its discovery, research has begun to reveal the molecular virology of MCPyV, as well as how it induces tumourigenesis. It is thought to be a common skin commensal, found at low levels in healthy individuals. Upon loss of immunosurveillance, MCPyV reactivates, and a heavy viral load is associated with MCC pathogenesis. Although MCPyV is in many ways similar to classical oncogenic polyomaviruses, such as SV40, subtle differences are beginning to emerge. These unique features highlight the singular position MCPyV has as the only human oncogenic polyomavirus, and open up new avenues for therapies against MCC.
Dural arteriovenous fistulae (DAVFs) are a rare form of intracranial arteriovenous malformation. We present the case of a patient who presented in a previously undescribed manner with facial swelling and bruising initially misdiagnosed as cellulitis. He was found subsequently to have DAVFs of the sphenoparietal sinus that had hemorrhaged. The rarity of DAVFs at this location may account for this unique presentation. Successful treatment was achieved by transarterial embolization.
Dural arteriovenous fistulae (DAVFs) are a rare form of intracranial arteriovenous malformation. We present the case of a patient who presented in a previously undescribed manner with facial swelling and bruising initially misdiagnosed as cellulitis. He was found subsequently to have DAVFs of the sphenoparietal sinus that had hemorrhaged. The rarity of DAVFs at this location may account for this unique presentation. Successful treatment was achieved by transarterial embolization.
The monitoring of molecular systems usually requires sophisticated technologies to interpret nanoscale events into electronic-decipherable signals. We demonstrate a new method for obtaining read-outs of molecular states that uses graphics processing units made from molecular circuits. Because they are made from molecules, the units are able to directly interact with molecular systems. We developed deoxyribozyme-based graphics processing units able to monitor nucleic acids and output alphanumerical read-outs via a fluorescent display. Using this design we created a molecular 7-segment display, a molecular calculator able to add and multiply small numbers, and a molecular automaton able to diagnose Ebola and Marburg virus sequences. These molecular graphics processing units provide insight for the construction of autonomous biosensing devices, and are essential components for the development of molecular computing platforms devoid of electronics.
Th1 and Th2 cell fates are traditionally viewed as mutually exclusive, but recent work suggests that these lineages may be more plastic than previously thought. When isolating splenic CD4(+) T cells from mice infected with the parasitic helminth Schistosoma mansoni, we observed a defined population of IFN-?/IL-4 double-positive cells. These IFN-?(+) IL-4(+) cells showed differences in DNA methylation at the Ifng and Il4 loci when compared with IFN-?(+) IL-4(-) (Th1) and IFN-?(-) IL-4(+) (Th2) cells, demonstrating that they represent a distinct effector cell population. IFN-?(+) IL-4(+) cells also displayed a discrete DNA methylation pattern at a CpG island within the body of the Gata3 gene, which encodes the master regulator of Th2 identity. DNA methylation at this region correlated with decreased Gata3 levels, suggesting a possible role in controlling Gata3 expression. These data provide important insight into the molecular mechanisms behind the co-existence of Th1 and Th2 characteristics.
The sensing of nucleic acids by receptors of the innate immune system is a key component of antimicrobial immunity. RNA:DNA hybrids, as essential intracellular replication intermediates generated during infection, could therefore represent a class of previously uncharacterised pathogen-associated molecular patterns sensed by pattern recognition receptors. Here we establish that RNA:DNA hybrids containing viral-derived sequences efficiently induce pro-inflammatory cytokine and antiviral type I interferon production in dendritic cells. We demonstrate that MyD88-dependent signalling is essential for this cytokine response and identify TLR9 as a specific sensor of RNA:DNA hybrids. Hybrids therefore represent a novel molecular pattern sensed by the innate immune system and so could play an important role in host response to viruses and the pathogenesis of autoimmune disease.
MicroRNAs (miRNAs) are a class of short non-coding RNA that play important roles in disease processes in animals and are present in a highly stable cell-free form in body fluids. Here, we examine the capacity of host and parasite miRNAs to serve as tissue or serum biomarkers of Schistosoma mansoni infection.
Human papillomavirus (HPV) can successfully evade the host immune response to establish a persistent infection. We show here that expression of the E7 oncoprotein in primary human keratinocytes results in increased production of interleukin-18 (IL-18) binding protein (IL-18BP). This anti-inflammatory cytokine binding protein is a natural antagonist of IL-18 and is necessary for skin homeostasis. We map increased IL-18BP production to the CR3 region of E7 and demonstrate that this ability is shared among E7 proteins from different HPV types. Furthermore, mutagenesis shows that increased IL-18BP production is mediated by a gamma-activated sequence (GAS) in the IL-18BP promoter. Importantly, the increased IL-18BP levels seen in E7-expressing keratinocytes are capable of diminishing IL-18-mediated CD4 lymphocyte activation. This study provides the first evidence for a virus protein that targets IL-18BP and further validates E7 as a key component of the HPV immune evasion armor.
A 24-year-old Nepalese man presented with intermittent confusion, fever and weight loss over a 2-month period. Lumbar puncture was performed demonstrating acid-fast bacilli, consistent with Mycobacterium tuberculosis meningitis.
Merkel cell carcinoma (MCC) is a highly aggressive nonmelanoma skin cancer arising from epidermal mechanoreceptor Merkel cells. In 2008, a novel human polyomavirus, Merkel cell polyomavirus (MCPyV), was identified and is strongly implicated in MCC pathogenesis. Currently, little is known regarding the virus-host cell interactions which support virus replication and virus-induced mechanisms in cellular transformation and metastasis. Here we identify a new function of MCPyV small T antigen (ST) as an inhibitor of NF-?B-mediated transcription. This effect is due to an interaction between MCPyV ST and the NF-?B essential modulator (NEMO) adaptor protein. MCPyV ST expression inhibits I?B kinase ? (IKK?)/IKK?-mediated I?B phosphorylation, which limits translocation of the NF-?B heterodimer to the nucleus. Regulation of this process involves a previously undescribed interaction between MCPyV ST and the cellular phosphatase subunits, protein phosphatase 4C (PP4C) and/or protein phosphatase 2A (PP2A) A?, but not PP2A A?. Together, these results highlight a novel function of MCPyV ST to subvert the innate immune response, allowing establishment of early or persistent infection within the host cell.
Oculodentodigital dysplasia (ODDD) is mainly an autosomal dominant human disease caused by mutations in the GJA1 gene, which encodes the gap junction protein connexin43 (Cx43). Surprisingly, there have been two autosomal recessive mutations reported that cause ODDD: a single amino acid substitution (R76H) and a premature truncation mutation (R33X). When expressed in either gap junctional intercellular communication (GJIC)-deficient HeLa cells or Cx43-expressing NRK cells, the R76H mutant trafficked to the plasma membrane to form gap junction-like plaques, whereas the R33X mutant remained diffusely localized throughout the cell, including the nucleus. As expected, the R33X mutant failed to form functional channels. In the case of the R76H mutant, dye transfer studies in HeLa cells and electrical conductance analysis in GJIC-deficient N2a cells revealed that this mutant could form functional gap junction channels, albeit with reduced macroscopic and single channel conductance. Alexa 350 dye transfer studies further revealed that the R76H mutant had no detectable negative effect on the function of co-expressed Cx26, Cx32, Cx37 or Cx40, whereas the R33X mutant exhibited significant dominant or trans-dominant effects on Cx43 and Cx40 as manifested by a reduction in wild-type connexin gap junction plaques. Taken together, our results suggest that the trans-dominant effect of R33X together with its complete inability to form a functional channel may explain why patients harboring this autosomal recessive R33X mutant exhibit greater disease burden than patients harboring the R76H mutant.
Foxp3(+) regulatory T (Treg) cells are key immune regulators during helminth infections, and identifying the mechanisms governing their induction is of principal importance for the design of treatments for helminth infections, allergies and autoimmunity. Little is yet known regarding the co-stimulatory environment that favours the development of Foxp3(+) Treg-cell responses during helminth infections. As recent evidence implicates the co-stimulatory receptor ICOS in defining Foxp3(+) Treg-cell functions, we investigated the role of ICOS in helminth-induced Foxp3(+) Treg-cell responses. Infection of ICOS(-/-) mice with Heligmosomoides polygyrus or Schistosoma mansoni led to a reduced expansion and maintenance of Foxp3(+) Treg cells. Moreover, during H. polygyrus infection, ICOS deficiency resulted in increased Foxp3(+) Treg-cell apoptosis, a Foxp3(+) Treg-cell specific impairment in IL-10 production, and a failure to mount putatively adaptive Helios(-) Foxp3(+) Treg-cell responses within the intestinal lamina propria. Impaired lamina propria Foxp3(+) Treg-cell responses were associated with increased production of IL-4 and IL-13 by CD4(+) T cells, demonstrating that ICOS dominantly downregulates Type 2 responses at the infection site, sharply contrasting with its Type 2-promoting effects within lymphoid tissue. Thus, ICOS regulates Type 2 immunity in a tissue-specific manner, and plays a key role in driving Foxp3(+) Treg-cell expansion and function during helminth infections.
miR-132 and miR-212 are two closely related miRNAs encoded in the same intron of a small non-coding gene, which have been suggested to play roles in both immune and neuronal function. We describe here the generation and initial characterisation of a miR-132/212 double knockout mouse. These mice were viable and fertile with no overt adverse phenotype. Analysis of innate immune responses, including TLR-induced cytokine production and IFN? induction in response to viral infection of primary fibroblasts did not reveal any phenotype in the knockouts. In contrast, the loss of miR-132 and miR-212, while not overtly affecting neuronal morphology, did affect synaptic function. In both hippocampal and neocortical slices miR-132/212 knockout reduced basal synaptic transmission, without affecting paired-pulse facilitation. Hippocampal long-term potentiation (LTP) induced by tetanic stimulation was not affected by miR-132/212 deletion, whilst theta burst LTP was enhanced. In contrast, neocortical theta burst-induced LTP was inhibited by loss of miR-132/212. Together these results indicate that miR-132 and/or miR-212 play a significant role in synaptic function, possibly by regulating the number of postsynaptic AMPA receptors under basal conditions and during activity-dependent synaptic plasticity.
Human respiratory syncytial virus (HRSV) is a member of the family Paramyxoviridae, and is responsible for serious respiratory illness in infants, the elderly and the immunocompromised. HRSV exists as two distinct lineages known as subgroups A and B, which represent two lines of divergent evolution with extensive genetic and serologic differences. While both subgroup A and B viruses contribute to overall HRSV disease, subgroup A isolates are associated with both increased frequency and morbidity of infections, and reasons for this are unclear. HRSV disease is characterized by virus-mediated cell destruction in combination with extensive inflammatory and immune modulatory responses, and for HRSV subgroup A isolates, several of these signaling pathways are regulated through activation of the transcription factor NF-?B. In contrast, the NF-?B activation characteristics of HRSV subgroup B infection remain untested. Here, we performed a quantitative and comparative analysis of NF-?B activation in response to infection of both continuous and primary cell cultures with HRSV subgroup A and B isolates. Our results showed the model HRSV subgroup A isolate consistently induced increased NF-?B activation compared to its HRSV subgroup B counterpart. The differential NF-?B activation characteristics of HRSV subgroup A and B viruses may contribute to differences in their pathogenesis.
Dendritic cells (DCs) play a pivotal role in polarising Th lymphocyte subsets but it is unclear what molecular events occur when DCs generate Th2-type responses. Here, we analysed plasma membrane-enriched fractions from immature, pro-Th1 and pro-Th2 DCs and used a combination of iTRAQ labelling and LC-MS/MS to quantify changes in the proteomes. Analysis was performed on triplicate biological samples and changes verified by flow cytometry. MHC class II molecules and CD29 were up-regulated in pro-Th1 DCs whilst CD18 and CD44 were up-regulated in pro-Th2 DCs. One of the most down-regulated molecules in pro-Th1 DCs was YM-1 whilst the greatest decrease in pro-Th2 DCs was NAP-22. Other molecules up-regulated in pro-Th2 DC compared to pro-Th1 DCs included some potentially involved in protein folding during antigen processing (clathrin and Rab-7), whilst other non-membrane proteins such as enzymes/transporters related to cell metabolism (malate dehydrogenase, pyruvate kinase, and ATPase Na(+)/K(+)) were also recorded. This suggests that pro-Th2 DCs are more metabolically active while pro-Th1 DCs have a mature end state. Overall, although several molecules were preferentially expressed on pro-Th2 DCs, our proteomics data support the view of a limited maturation of pro-Th2 DCs compared to pro-Th1 DCs.
The immune suppression that characterizes human helminth infections can hinder the development of protective immunity or help to reduce pathogenic inflammation. Signaling through the T cell costimulator glucocorticoid-induced TNFR-related protein (GITR) counteracts immune downregulation by augmenting effector T cell responses and abrogating suppression by Foxp3(+) regulatory T cells. Thus, superphysiological Ab-mediated GITR costimulation represents a novel therapy for promoting protective immunity toward parasitic helminths, whereas blocking physiological GITR-GITR ligand (GITRL) interactions may provide a mechanism for dampening pathogenic Th2 inflammation. We investigated the superphysiological and physiological roles of the GITR-GITRL pathway in the development of protective and pathogenic Th2 responses in murine infection models of filariasis (Litomosoides sigmodontis) and schistosomiasis (Schistosoma mansoni). Providing superphysiological GITR costimulation using an agonistic anti-GITR mAb over the first 12 d of L. sigmodontis infection initially increased the quantity of Th2 cells, as well as their ability to produce Th2 cytokines. However, as infection progressed, the Th2 responses reverted to normal infection levels, and parasite killing remained unaffected. Despite the Th2-promoting role of superphysiological GITR costimulation, Ab-mediated blockade of the GITR-GITRL pathway did not affect Th2 cell priming or maintenance during L. sigmodontis infection. Blockade of GITR-GITRL interactions during the acute egg phase of S. mansoni infection resulted in reduced Th2 responses, but this effect was confined to the spleen and did not lead to changes in liver pathology. Thus, although superphysiological GITR costimulation can therapeutically enhance Th2 responses, physiological GITR-GITRL interactions are not required for the development of Th2-mediated resistance or pathology in murine models of filariasis and schistosomiasis.
Tregs play a central role in modulating Fc?RI-dependent MC effector functions in the course of the allergic response. Cellular interaction depends on the constitutive expression of OX40 on Tregs and the OX40L counterpart on MCs. Study of OX40L signaling on MCs is hampered by the need of a highly purified molecule, which triggers OX40L specifically. We now report that sOX40 mimics the physiological activity of Treg interaction by binding to activated MCs. When treated with sOX40, activated MCs showed decreased degranulation and Ca(++) influx, whereas PLC-?2 phosphorylation remained unaffected. Once injected into experimental animals, sOX40 not only located within the endothelium but also in parenchyma, where it could be found in close proximity and apparently bound to MCs. This soluble molecule triggers MC-OX40L without the requirement of Tregs, thus allowing study of OX40L signaling pathways in MCs and in other OX40L-expressing cell populations. Importantly, as sOX40 inhibits MC degranulation, it may provide an in vivo therapeutic tool in allergic disease.
We examined the sensitivity and accuracy of measuring osteolysis around total knee arthroplasty (TKA) on radiographs, computed tomography (CT), and magnetic resonance imaging (MRI) in a cadaver model. Fifty-four simulated osteolytic defects ranging from 0.7 to 14 cm(3) were created in 6 cadaver knees implanted with either a cemented or an uncemented TKA. Three blinded investigators assessed the presence, location, and volume of defects on radiographs and CT and MRI scans with metal reduction protocols. Both CT and MRI had significantly higher sensitivities and specificities than did plain radiographs (P < .005). Overall, there was no difference in the accuracy of defect volume measurements between CT and MRI (P = .574). This study demonstrates the limitations of radiographs and the high sensitivity and specificity of both CT and MRI in assessing osteolysis around TKA.
Gastrointestinal helminth infections are extremely prevalent in many human populations and are associated with downmodulated immune responsiveness. In the experimental model system of Heligmosomoides polygyrus, a chronic infection establishes in mice, accompanied by a modulated Th2 response and increased regulatory T cell (Treg) activity. To determine if dendritic cell (DC) populations in the lymph nodes draining the intestine are responsible for the regulatory effects of chronic infection, we first identified a population of CD11c(lo) nonplasmacytoid DCs that expand after chronic H. polygyrus infection. The CD11c(lo) DCs are underrepresented in magnetic bead-sorted preparations and spared from deletion in CD11c-diptheria toxin receptor mice. After infection, CD11c(lo) DCs did not express CD8, CD103, PDCA, or Siglec-H and were poorly responsive to TLR stimuli. In DC/T cell cocultures, CD11c(lo) DCs from naive and H. polygyrus-infected mice could process and present protein Ag, but induced lower levels of Ag-specific CD4(+) T cell proliferation and effector cytokine production, and generated higher percentages of Foxp3(+) T cells in the presence of TGF-?. Treg generation was also dependent on retinoic acid receptor signaling. In vivo, depletion of CD11c(hi) DCs further favored the dominance of the CD11c(lo) DC phenotype. After CD11c(hi) DC depletion, effector responses were inhibited dramatically, but the expansion in Treg numbers after H. polygyrus infection was barely compromised, showing a significantly higher regulatory/effector CD4(+) T cell ratio compared with that of CD11c(hi) DC-intact animals. Thus, the proregulatory environment of chronic intestinal helminth infection is associated with the in vivo predominance of a newly defined phenotype of CD11c(lo) tolerogenic DCs.
A defining feature of inflammation is the accumulation of innate immune cells in the tissue that are thought to be recruited from the blood. We reveal that a distinct process exists in which tissue macrophages undergo rapid in situ proliferation in order to increase population density. This inflammatory mechanism occurred during T helper 2 (T(H)2)-related pathologies under the control of the archetypal T(H)2 cytokine interleukin-4 (IL-4) and was a fundamental component of T(H)2 inflammation because exogenous IL-4 was sufficient to drive accumulation of tissue macrophages through self-renewal. Thus, expansion of innate cells necessary for pathogen control or wound repair can occur without recruitment of potentially tissue-destructive inflammatory cells.
Small RNA viruses have evolved many mechanisms to increase the capacity of their short genomes. Here we describe the identification and characterization of a novel open reading frame (ORF4) encoded by the murine norovirus (MNV) subgenomic RNA, in an alternative reading frame overlapping the VP1 coding region. ORF4 is translated during virus infection and the resultant protein localizes predominantly to the mitochondria. Using reverse genetics we demonstrated that expression of ORF4 is not required for virus replication in tissue culture but its loss results in a fitness cost since viruses lacking the ability to express ORF4 restore expression upon repeated passage in tissue culture. Functional analysis indicated that the protein produced from ORF4 antagonizes the innate immune response to infection by delaying the upregulation of a number of cellular genes activated by the innate pathway, including IFN-Beta. Apoptosis in the RAW264.7 macrophage cell line was also increased during virus infection in the absence of ORF4 expression. In vivo analysis of the WT and mutant virus lacking the ability to express ORF4 demonstrated an important role for ORF4 expression in infection and virulence. STAT1-/- mice infected with a virus lacking the ability to express ORF4 showed a delay in the onset of clinical signs when compared to mice infected with WT virus. Quantitative PCR and histopathological analysis of samples from these infected mice demonstrated that infection with a virus not expressing ORF4 results in a delayed infection in this system. In light of these findings we propose the name virulence factor 1, VF1 for this protein. The identification of VF1 represents the first characterization of an alternative open reading frame protein for the calicivirus family. The immune regulatory function of the MNV VF1 protein provide important perspectives for future research into norovirus biology and pathogenesis.
We examine the onset of classical topological order in a nearest neighbour kagome ice model. Using Monte Carlo simulations, we characterize the topological sectors of the ground state using a nonlocal cut measure which circumscribes the toroidal geometry of the simulation cell. We demonstrate that simulations which employ global loop updates that are allowed to wind around the periodic boundaries cause the topological sector to fluctuate, while restricted local loop updates freeze the simulation into one topological sector. The freezing into one topological sector can also be observed in the susceptibility of the real magnetic spin vectors projected onto the kagome plane. The ability of the susceptibility to distinguish between fluctuating and non-fluctuating topological sectors should motivate its use as a local probe of topological order in a variety of related systems.
Type I IFNs (IFN?/?) are essential anti-viral cytokines produced in response to the detection of viral components by host pattern recognition receptors. IFN?/? production is transient, and aberrant activation can be hazardous to the host. In this article, we review our current understanding of host negative regulatory mechanisms that control IFN?/? production.
Although dendritic cells (DCs) are adept initiators of CD4(+) T cell responses, their fundamental importance in this regard in Th2 settings remains to be demonstrated. We have used CD11c-diphtheria toxin (DTx) receptor mice to deplete CD11c(+) cells during the priming stage of the CD4(+) Th2 response against the parasitic helminth Schistosoma mansoni. DTx treatment significantly depleted CD11c(+) DCs from all tissues tested, with 70-80% efficacy. Even this incomplete depletion resulted in dramatically impaired CD4(+) T cell production of Th2 cytokines, altering the balance of the immune response and causing a shift toward IFN-? production. In contrast, basophil depletion using Mar-1 antibody had no measurable effect on Th2 induction in this system. These data underline the vital role that CD11c(+) antigen-presenting cells can play in orchestrating Th2 development against helminth infection in vivo, a response that is ordinarily balanced so as to prevent the potentially damaging production of inflammatory cytokines.
Virus-host interactions involve complex interplay between viral and host factors, rendering them an ideal target for proteomic analysis. Here we detail a high throughput quantitative proteomics analysis of Vero cells infected with the coronavirus infectious bronchitis virus (IBV), a positive strand RNA virus that replicates in the cytoplasm. Stable isotope labeling with amino acids in cell culture (SILAC) was used in conjunction with LC-MS/MS to identify and quantify 1830 cellular and two viral proteins from IBV-infected cells. Fractionation of cells into cytoplasmic, nuclear, and nucleolar extracts was used to reduce sample complexity and provide information on the trafficking of proteins between the different compartments. Each fraction showed a proportion of proteins exhibiting >or=2-fold changes in abundance. Ingenuity Pathway Analysis revealed that proteins that changed in response to infection could be grouped into different functional categories. These included proteins regulated by NF-kappaB- and AP-1-dependent pathways and proteins involved in the cytoskeleton and molecular motors. A luciferase-based reporter gene assay was used to validate the up-regulation of AP-1- and NF-kappaB-dependent transcription in IBV-infected cells and confirmed using immunofluorescence. Immunofluorescence was used to validate changes in the subcellular localization of vimentin and myosin VI in IBV-infected cells. The proteomics analysis also confirmed the presence of the viral nucleocapsid protein as localizing in the cytoplasm, nucleus, and nucleolus and the viral membrane protein in the cytoplasmic fraction. This research is the first application of SILAC to study total host cell proteome changes in response to positive sense RNA virus infection and illustrates the versatility of this technique as applied to infectious disease research.
The innate immune response provides a critical defense against microbial infections, including viruses. These are recognised by pattern recognition receptors including Toll-like receptors (TLRs) and RIG-I like helicases (RLHs). Detection of virus triggers signalling cascades that induce transcription of type I interferons including IFNbeta, which are pivotal for the initiation of an anti-viral state. Despite the essential role of IFNbeta in the anti-viral response, there is an incomplete understanding of the negative regulation of IFNbeta induction. Here we provide evidence that expression of the Nemo-related protein, optineurin (NRP/FIP2), has a role in the inhibition of virus-triggered IFNbeta induction. Over-expression of optineurin inhibited Sendai-virus (SeV) and dsRNA triggered induction of IFNbeta, whereas depletion of optineurin with siRNA promoted virus-induced IFNbeta production and decreased RNA virus replication. Immunoprecipitation and immunofluorescence studies identified optineurin in a protein complex containing the antiviral protein kinase TBK1 and the ubiquitin ligase TRAF3. Furthermore, mutagenesis studies determined that binding of ubiquitin was essential for both the correct sub-cellular localisation and the inhibitory function of optineurin. This work identifies optineurin as a critical regulator of antiviral signalling and potential target for future antiviral therapy.
Common ragweed (Ambrosia artemisiifolia) is an abundant weed in its native North America, despite supporting a wide range of natural enemies. Here, we tested whether these enemies have significant impacts on the performance of this plant in its native range. We excluded enemies from the three principal life-history stages (seed, seedling, and adult) of this annual in a series of field experiments; at the adult stage, we also manipulated soil disturbance and conspecific density. We then measured the consequences of these treatments for growth, survival, and reproduction. Excluding fungi and vertebrate granivores from seeds on the soil surface did not increase germination relative to control plots. Seedling survivorship was only slightly increased by the exclusion of molluscs and other herbivores. Insecticide reduced damage to leaves of adult plants, but did not improve growth or reproduction. Growth and survivorship of adults were strongly increased by disturbance, while higher conspecific density reduced performance in disturbed plots. These results indicate ragweed is insensitive to attack by many of its natural enemies, helping to explain its native-range success. In addition, they suggest that even though ragweed lost most of its insect folivores while invading Europe, escape from these enemies is unlikely to have provided a significant demographic advantage; instead, disturbance is likely to have been a much more important factor in its invasion. Escape from enemies should not be assumed to explain the success of exotic species unless improved performance also can be demonstrated; native-range studies can help achieve this goal.
Bovine viral diarrhea virus (BVDV; genus Pestivirus) can exist as two biotypes, cytopathogenic (CP) and non-cytopathogenic (NCP). The CP form differs from NCP by the continual expression of free non-structural protein 3 (NS3). CP BVDV infection of cultured cells induces apoptosis, whereas NCP BVDV infection has been reported to block the induction of beta interferon (IFN-beta). To investigate the viral mechanisms underlying these effects, NS3 or NS2-3 proteins of NCP and CP BVDV biotypes, together with the cognate NS3 co-factor NS4A, were expressed in cells, and their effect on apoptosis and induction of IFN-beta was investigated. Expression of NS3/4A resulted in increased activity of caspase-9 and caspase-3, indicating induction of the intrinsic apoptosis pathway. Mutational analysis revealed that a protease-inactive NS3/4A was unable to induce apoptosis, suggesting that NS3 protease activity is required for initiation of apoptosis during CP BVDV infection. The ability of NS2-3 to modulate activation of the IFN-beta promoter was also investigated. These studies confirmed that, unlike the related hepatitis C virus and GB virus-B, BVDV proteases are unable to inhibit TLR3- and RIG-I-dependent activation of the IFN-beta promoter. These data suggest that BVDV NS3/4A is responsible for regulating the levels of cellular apoptosis and provide new insights regarding the viral elements associated with CP biotype pathogenesis.
An estimated 3% of the global population are infected with hepatitis C virus (HCV), and the majority of these individuals will develop chronic liver disease. As with other chronic viruses, establishment of persistent infection requires that HCV-infected cells must be refractory to a range of pro-apoptotic stimuli. In response to oxidative stress, amplification of an outward K(+) current mediated by the Kv2.1 channel, precedes the onset of apoptosis. We show here that in human hepatoma cells either infected with HCV or harboring an HCV subgenomic replicon, oxidative stress failed to initiate apoptosis via Kv2.1. The HCV NS5A protein mediated this effect by inhibiting oxidative stress-induced p38 MAPK phosphorylation of Kv2.1. The inhibition of a host cell K(+) channel by a viral protein is a hitherto undescribed viral anti-apoptotic mechanism and represents a potential target for antiviral therapy.
IL-18 belongs to the IL-1 family of cytokines and has recently regained interest in the context of inflammasome activation. The inflammasome dependent caspase 1 cleaves pro-IL-18 into the active form - similar to what is known for IL-1ss. Still, the action and importance of IL-18 are not completely understood. There are several indications that it plays a pathogenetically important role in chronic inflammatory conditions of epithelial organs (such as skin, gut, kidney) and importantly also in responses against self. Here, we summarise current knowledge on the role of IL-18 in human skin inflammation with a focus on its role in Cutaneous Lupus Erythematosus (CLE). There is evidence that IL-18 plays a role in CLE upstream of TNFalpha. In CLE but not normal keratinocytes IL-18 strongly induces TNFalpha release, which then results in apoptosis. Blocking TNFalpha in vitro prevents apoptosis of keratinocytes but anti-TNFalpha therapy is not applicable in LE conditions. We will discuss potential approaches to control IL-18 in skin inflammation.
The costimulatory requirements for Th17 development remain to be defined. In this study, we show that CD40-deficient animals immunized with the gram-positive bacterium Propionibacterium acnes were specifically impaired in their ability to mount an IL-17 response, but not that of IFN-gamma. The same cytokine imbalance resulted from in vivo priming with pathogen-pulsed, CD40-deficient dendritic cells (DC). Engagement of CD40 on P. acnes-conditioned DC stimulated the release of IL-12, IL-23, and IL-6, of which IL-6 alone proved essential for Th17 differentiation. Compared with wild-type DC, priming with those lacking expression of CD40 resulted in reduced disease severity during experimental autoimmune encephalomyelitis, coincident with reduced IL-17 production. Our data delineate sequential requirements for DC expression of CD40 and production of IL-6 during Th17 polarization in vitro and in vivo, and reveal distinct costimulatory requirements for Th1 vs Th17 generation.
Human cytomegalovirus (HCMV) regulates NF-kappaB during infection by a variety of mechanisms. For example, the HCMV gene product, UL144, is known to activate NF-kappaB in a tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6)-dependent manner, causing the upregulation of the chemokine CCL22 (MDC). Viral UL144 is expressed from the UL/b region of the HCMV genome at early times postinfection and is a TNFR1-like homologue. Despite this homology to the TNFR1 receptor superfamily, UL144 does not bind to members of the TNF ligand superfamily. We show here that the upregulation of NF-kappaB by UL144 is dependent upon cellular tripartite motif 23 (TRIM23) protein. We propose a mechanism by which UL144 activates NF-kappaB through a direct interaction with the cellular protein TRIM23 in a complex containing TRAF6. In contrast, TRIM23 is not involved in conventional double-stranded RNA signaling via NF-kappaB. Therefore, we present a novel role for TRIM23 that is specific to UL144-mediated activation of NF-kappaB during the course of virus infection.
Experimental autoimmune encephalomyelitis (EAE) depends on the initial activation of CD4(+) T cells responsive to myelin autoantigens. The key antigen presenting cell (APC) population that drives the activation of naïve T cells most efficiently is the dendritic cell (DC). As such, we should be able to trigger EAE by transfer of DC that can present the relevant autoantigen(s). Despite some sporadic reports, however, models of DC-driven EAE have not been widely adopted. We sought to test the feasibility of this approach and whether activation of the DC by toll-like receptor (TLR)-4 ligation was a sufficient stimulus to drive EAE.
Alternative macrophage activation is largely defined by IL-4R? stimulation but the contribution of Toll-like receptor (TLR) signaling to this phenotype is not currently known. We have investigated macrophage activation status under Th2 conditions in the absence of the core TLR adaptor molecule, MyD88. No impairment was observed in the ability of MyD88-deficient bone marrow derived macrophages to produce or express alternative activation markers, including arginase, RELM-? or Ym1, in response to IL-4 treatment in vitro. Further, we observed no difference in the ability of peritoneal exudate cells from nematode implanted wild type (WT) or MyD88-deficient mice to produce arginase or express the alternative activation markers RELM-? or Ym1. Therefore, MyD88 is not a fundamental requirement for Th2-driven macrophage alternative activation, either in vitro or in vivo.
Infection with gastrointestinal helminths generates a dominant type 2 response among both adaptive (Th2) and innate (macrophage, eosinophil, and innate lymphoid) immune cell types. Two additional innate cell types, CD11c(high) dendritic cells (DCs) and basophils, have been implicated in the genesis of type 2 immunity. Investigating the type 2 response to intestinal nematode parasites, including Heligmosomoides polygyrus and Nippostrongylus brasiliensis, we first confirmed the requirement for DCs in stimulating Th2 adaptive immunity against these helminths through depletion of CD11c(high) cells by administration of diphtheria toxin to CD11c.DOG mice. In contrast, responsiveness was intact in mice depleted of basophils by antibody treatment. Th2 responses can be induced by adoptive transfer of DCs, but not basophils, exposed to soluble excretory-secretory products from these helminths. However, innate type 2 responses arose equally strongly in the presence or absence of CD11c(high) cells or basophils; thus, in CD11c.DOG mice, the alternative activation of macrophages, as measured by expression of arginase-1, RELM-?, and Ym-1 (Chi3L3) in the intestine following H. polygyrus infection or in the lung following N. brasiliensis infection, was unaltered by depletion of CD11c-expressing DCs and alveolar macrophages or by antibody-mediated basophil depletion. Similarly, goblet cell-associated RELM-? in lung and intestinal tissues, lung eosinophilia, and expansion of innate lymphoid ("nuocyte") populations all proceeded irrespective of depletion of CD11c(high) cells or basophils. Thus, while CD11c(high) DCs initiate helminth-specific adaptive immunity, innate type 2 cells are able to mount an autonomous response to the challenge of parasite infection.
Transient knock-down of the gap junction protein Cx43 by antisense and siRNA, or gap junction block with mimetic peptides, have been shown to enhance epidermal wound healing. However, patients with oculodentodigital dysplasia (ODDD) express mutant Cx43 that leads to a chronic reduction in gap junctional intercellular communication. To determine whether mutant Cx43 in keratinocytes would impact upon the wound healing process, we localized Cx43 in human and mouse skin tissue expressing mutant Cx43 and assessed the ability of primary keratinocytes derived from a mouse model of ODDD to proliferate, migrate and differentiate. In the epidermis from an ODDD patient and in the epidermis of mice expressing the G60S mutant or in keratinocytes obtained from mutant mice, Cx43 was frequently found within intracellular compartments and rarely localized to punctate sites of cell-cell apposition. Primary keratinocytes derived from G60S mutant mice proliferated faster but migrated similarly to keratinocytes derived from wild-type control mice. Keratinocytes derived from mutant mice expressed abundant Cx43 and higher levels of involucrin and loricrin under low calcium conditions. However, after calcium-induced differentiation, similar levels of Cx43, involucrin and loricrin were observed. Thus, we conclude that during wound healing, mutant Cx43 may enhance keratinocyte proliferation and promote early differentiation of keratinocytes.
Current research on macronutrient cycling in UK agricultural systems aims to optimise soil and nutrient management for improved agricultural production and minimise effects on the environment and provision of ecosystem services. Nutrient use inefficiencies can cause environmental pollution through the release of greenhouse gases into the atmosphere and of soluble and particulate forms of N, P and carbon (C) in leachate and run-off into watercourses. Improving nutrient use efficiencies in agriculture calls for the development of sustainable nutrient management strategies: more efficient use of mineral fertilisers, increased recovery and recycling of waste nutrients, and, better exploitation of the substantial inorganic and organic reserves of nutrients in the soil. Long-term field experimentation in the UK has provided key knowledge of the main nutrient transformations in agricultural soils. Emerging analytical technologies, especially stable isotope labelling, that better characterise macronutrient forms and bioavailability and improve the quantification of the complex relationships between the macronutrients in soils at the molecular scale, are augmenting this knowledge by revealing the underlying processes. The challenge for the future is to determine the relationships between the dynamics of N, P and C across scales, which will require both new modelling approaches and integrated approaches to macronutrient cycling.
Interleukin-4 is a cytokine widely known for its role in CD4(+) T cell polarization and its ability to alternatively activate macrophage populations. In contrast, the impact of IL-4 on the activation and function of dendritic cells (DCs) is poorly understood. We report here that DCs respond to IL-4 both in vitro and in vivo by expression of multiple alternative activation markers with a different expression pattern to that of macrophages. We further demonstrate a central role for DC IL-4R? expression in the optimal induction of IFN? responses in vivo in both Th1 and Th2 settings, through a feedback loop in which IL-4 promotes DC secretion of IL-12. Finally, we reveal a central role for RELM? during T-cell priming, establishing that its expression by DCs is critical for optimal IL-10 and IL-13 promotion in vitro and in vivo. Together, these data highlight the significant impact that IL-4 and RELM? can have on DC activation and function in the context of either bacterial or helminth pathogens.
Phylogenetic diversity (PD) describes the total amount of phylogenetic distance among species in a community. Although there has been substantial research on the factors that determine community PD, exploration of the consequences of PD for ecosystem functioning is just beginning. We argue that PD may be useful in predicting ecosystem functions in a range of communities, from single-trophic to complex networks. Many traits show a phylogenetic signal, suggesting that PD can estimate the functional trait space of a community, and thus ecosystem functioning. Phylogeny also determines interactions among species, and so could help predict how extinctions cascade through ecological networks and thus impact ecosystem functions. Although the initial evidence available suggests patterns consistent with these predictions, we caution that the utility of PD depends critically on the strength of phylogenetic signals to both traits and interactions. We advocate for a synthetic approach that incorporates a deeper understanding of how traits and interactions are shaped by evolution, and outline key areas for future research. If these complexities can be incorporated into future studies, relationships between PD and ecosystem function bear promise in conceptually unifying evolutionary biology with ecosystem ecology.
This is an essay highlighting the fundamental importance of agriculture (historical and present) in the agro-socioeconomic evolution of human societies, from the times of the hunter/gatherers to the modern day. Attention is drawn in the text to the importance of deforestation in relation to micro and macro climate changes, and the vital role of carbon dioxide to plant and animal life. The essay also relates the worlds natural resources to the present unsustainable population pressures.
Certain parasites have evolved to evade the immune response and establish chronic infections that may persist for many years. T cell responses in these conditions become muted despite ongoing infection. Upregulation of surface receptors with inhibitory properties provides an immune cell-intrinsic mechanism that, under conditions of chronic infection, regulates immune responses and limits cellular activation and associated pathology. The negative regulator, CD200 receptor, and its ligand, CD200, have been shown to regulate macrophage activation and reduce pathology following infection. We show that CD4 T cells also increase expression of inhibitory CD200 receptors (CD200R) in response to chronic infection. CD200R was upregulated on murine effector T cells in response to infection with bacterial, Salmonella enterica, or helminth, Schistosoma mansoni, pathogens that respectively drive predominant Th1- or Th2-responses. In vitro chronic and prolonged stimuli were required for the sustained upregulation of CD200R, and its expression coincided with loss of multifunctional potential in T effector cells during infection. Importantly, we show an association between IL-4 production and CD200R expression on T effector cells from humans infected with Schistosoma haematobium that correlated effectively with egg burden and, thus infection intensity. Our results indicate a role of CD200R:CD200 in T cell responses to helminths which has diagnostic and prognostic relevance as a marker of infection for chronic schistosomiasis in mouse and man.
High-risk human papillomavirus type 16 (HPV16) is the primary causative agent of cervical cancer and therefore is responsible for significant morbidity and mortality worldwide. Cellular transformation is mediated directly by the expression of viral oncogenes, the least characterized of which, E5, subverts cellular proliferation and immune recognition processes. Despite a growing catalogue of E5-specific host interactions, little is understood regarding the molecular basis of its function. Here we describe a novel function for HPV16 E5 as an oligomeric channel-forming protein, placing it within the virus-encoded "viroporin" family. The development of a novel recombinant E5 expression system showed that E5 formed oligomeric assemblies of a defined luminal diameter and stoichiometry in membranous environments and that such channels mediated fluorescent dye release from liposomes. Hexameric E5 channel stoichiometry was suggested by native PAGE studies. In lieu of high-resolution structural information, established de novo molecular modeling and design methods permitted the development of the first specific small-molecule E5 inhibitor, capable of both abrogating channel activity in vitro and reducing E5-mediated effects on cell signaling pathways. The identification of channel activity should enhance the future understanding of the physiological function of E5 and could represent an important target for antiviral intervention.
Infection with schistosome helminths is associated with granulomatous inflammation that forms around parasite eggs trapped in host tissues. In severe cases, the resulting fibrosis can lead to organ failure, portal hypertension, and fatal bleeding. Murine studies identified IL-17 as a critical mediator of this immunopathology, and mouse strains that produce high levels of IL-17 in response to schistosome infection show increased mortality. In this article, we demonstrate that schistosome-specific IL-17 induction by dendritic cells from low-pathology C57BL/6 mice is normally regulated by their concomitant induction of IL-10. Simultaneous stimulation of schistosome-exposed C57BL/6 dendritic cells with a heat-killed bacterium enabled these cells to overcome IL-10 regulation and induce IL-17, even in wild-type C57BL/6 recipients. This schistosome-specific IL-17 was dependent on IL-6 production by the copulsed dendritic cells. Coimmunization of C57BL/6 animals with bacterial and schistosome Ags also resulted in schistosome-specific IL-17, and this response was enhanced in the absence of IL-10-mediated immune regulation. Together, our data suggest that the balance of pro- and anti-inflammatory cytokines that determines the severity of pathology during schistosome infection can be influenced not only by host and parasite, but also by concurrent bacterial stimulation.
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