Aec1Aec2 mice developed an earlier onset of sialadenitis with a higher composition of CD3+ T cells and a 10-fold increase in glandular infiltration of Th17 cells at the onset of clinical disease compared to male mice. Inflammatory Th17 cells of female mice exhibited a stronger proliferation in response to disease-specific antigen than their male counterpart. At the clinical disease stage, altered autoantibody patterns can be detected in females whereas they are seldom observed in male C57BL/6.NOD-Aec1Aec2 mice. Interestingly, male C57BL/6.NOD-Aec1Aec2 mice developed an earlier loss of secretory function, despite the fact that female C57BL/6.NOD-Aec1Aec2 mice exhibited a more rapid secretory loss. This data indicates the strong sexual dimorphism in the SjS-susceptible C57BL/6.NOD-Aec1Aec2 animal model, making it an appropriate animal model to examine human disease.Sjögren's syndrome is a complex autoimmune disease with an array of diverse immunological, genetic and environmental etiologies, making identification of the precise autoimmune mechanism difficult to define. One of the most distinctive aspects of Sjögren's syndrome is the high sexual dimorphism with women affected 10-20 times more than men. It is nearly impossible to study the sexual dimorphic development of Sjögren's syndrome in human patients; therefore it is pertinent to develop an appropriate animal model which resembles human disease. The data indicated that female C57BL/6.NOD- Summary: Sjogren's syndrome (SjS) is the one of the most common sexual dimorphic autoimmune diseases. This study demonstrates that female C57BL/6.NOD-Aec1Aec2 mice develop earlier onset and more severe symptoms of SjS than male counterparts. Therefore, the C57BL/6.NOD-Aec1Aec2 mouse model could be an appropriate model to study sexual dimorphism in SjS. Sjögren's syndrome (SjS) is a chronic, systemic autoimmune disease characterized most notably by development of dry eyes and dry mouth manifestations, indicative of secretory dysfunction of the lacrimal and salivary glands . The patSullivan, 1997h4 model, females exhibited more severe sialadenitis with a higher number of CD4+ T cell infiltration in the salivary glands and elevated levels of autoantibodies compared to males (s) NOD and C57BL/6.NOD-Aec1Aec2 mouse models, we have postulated that the development of SjS progresses through three distinct, but continuous phases  of age, female C57BL/6.NOD-Aec1Aec2 mice began to develop lymphocytic foci, while male C57BL/6.NOD-Aec1Aec2 mice showed little or no infiltration at a similar age. Interestingly, minor infiltrates can be seen in normal female C57BL/6 mice at 16 weeks of age with 26% frequency (data not shown). At the clinical- disease phase at approximately 31 weeks of age, salivary glands of both male and female C57BL/6.NOD-Aec1Aec2 mice were positive for lymphocytic foci with 100% frequency (data not shown). Area analysis using regions of interest, illustrated in Aec1Aec2 developed larger infiltrates at 16 weeks of age in comparison to female C57BL/6. At 31 weeks of age, both male and female C57BL/6.NOD-Aec1Aec2 mice developed similar size infiltrates but three to four times larger in area compared to both sexes of C57BL/6 mice. These data suggest that severe lymphocytic infiltration or sialadenitis occurs earlier in female C57BL/6.NOD-Aec1Aec2 mice than the male counterpart during the adaptive phase with progressive severity during the clinical-disease phase.Previous studies have demonstrated that aberrant pathophysiological changes are detected in 4 weeks old C57BL/6.NOD-ec2 mice . Startin+ T cells with larger foci occupied by a higher number of CD20+ B cells than CD3+ T cells. Larger histological lesions are indicative of disease severity, longer duration and more advanced clinical symptoms. In our animal model of SjS, similar observations were found in which early infiltrations are composed mainly of CD3+ T cells with equal number of CD3+T and B220+ B cells during the adaptive stage and higher number of B220+ B cells compared to CD3+ T cells at the clinical-disease stage. However, there is no data, which compares the changes within lymphocytic composition, specifically the temporal change over the course of the autoimmune reaction, in male and female human patients or animal models of SjS. As presented in Aec1Aec2 and C57BL/6 mice were stained and examined for B220+ B cells and CD3+ T cells at 4, 16 and 31 weeks of age. As expected, no B220+ B cells and CD3+ T cells were detected at 4 weeks of age. Female C57BL/6.NOD-Aec1Aec2 developed sialadenitis at 16 weeks of age with a higher number of B220+ B cells and CD3+ T cells compared to female C57BL/6 and none were detected in male C57BL/6.NOD-Aec1Aec2 mice. At 31 weeks of age, both male and female C57BL/6.NOD-Aec1Aec2 mice showed a significant increase in B220+ B cells and CD3+ T cells in comparison to C57BL/6 mice. Interestingly, as presented in Aec1Aec2 mice exhibited a marked increase in both B220+ B cells and CD3+ T cells in comparison to male C57BL/6.NOD-Aec1Aec2 mice at the adaptive and clinical-disease stages. Whereas all of the other strains exhibited a ratio of approximately 2:1 (T/B cells), female C57BL/6.NOD-Aec1Aec2 mice at 16 weeks displayed at least five times the area of T cells composing these infiltrates . Cells were labeled with CFDA and stimulated with anti-CD3/anti-CD28 (antigen-non-specific activation) and the third cytoplasmic loop peptide of M3R (antigen-specific activation). CFDA-labeled proteins are inherited by daughter cells after cell division; therefore, the loss of fluorescent intensity by flow cytometric analysis is indicative of cell proliferation. As presented in +CD4+IL17+) of lymph nodes from male and female C57BL/6 and C57BL/6.NOD-Aec1Aec2 mice responded equally with anti-CD3/anti-CD28. Interestingly, Th17 cells in salivary glands of female C57BL/6.NOD-Aec1Aec2 mice exhibited higher proliferation with M3R peptide stimulation compared to male C57BL/6.NOD-Aec1Aec2 mice and both sexes of control mice , while female C57BL/6.NOD-Aec1Aec2 mice displayed a rapid loss of 70% within a 8-week period (12 weeks-20 weeks). C57BL/6 mice appeared to fluctuate saliva secretion at similar time frame with a higher contrast at 20 weeks of age; however there were no overall significant differences in male and female mice. The data provide a clear indication that although male C57BL/6.NOD-Aec1Aec2 mice appear to exhibit an early onset of saliva loss, female C57BL/6.NOD-Aec1Aec2 mice show a more rapid loss of secretory function once the disease is established.Secretory dysfunction could be contributed by acinar cell death mediated by the intrinsic nature of the autoimmune-susceptible background or an inflammatory response initiated from the glands and/or peripheral lymphoid organs. As indicated in Aec1Aec2 animal model, we have shown that female C57BL/6.NOD-Aec1Aec2 mice developed an earlier and more severe onset of sialadenitis. Infiltrates were composed of a significantly higher number of CD3+T cells with a 10-fold increase in glandular infiltration of CD4+IL17+ Th17 cells in female compared to male mice at the onset of the clinical disease. In addition, gland specific Th17 cells exhibited a robust proliferative capacity in female C57BL/6.NOD-Aec1Aec2 mice. Infiltrated B cells were significantly elevated in females compared to males with a concomitant altered ANA pattern observed solely in female C57BL/6.NOD-Aec1Aec2 mice. Secretory dysfunction was discerned in both sexes in which females developed a later and more rapid onset of secretory dysfunction once the disease is established. This data indicates significant differences at various stages of the autoimmune process in males and females, suggesting a clear sexual dimorphism in the C57BL/6.NOD-Aec1Aec2 animal model.SjS is a systemic autoimmune disease with one of the highest female to male disparities. The precise biological mechanism governing this sexual dimorphism remains elusive. One of the major challenges is studying the covert autoimmune process of the disease prior to the overt clinical disease. The other challenge is the lack of male cohorts to perform a proper and essential comparative analysis between male and female patients. As a result, identifying an appropriate animal model will allow us to examine both the covert and overt autoimmune process, and more importantly better facilitate the comparison between males and females. Using the C57BL/6.NOD-bH2 and NOD.H2h4, have been used to examine the role of MHC and non-MHC association in SjS. Sex differences were observed in female NOD.H2h4 mice in which female mice developed increased severity of sialadenitis with higher CD4+ T cells producing Th2 and Th17 cytokines. Females developed enhanced B cell responses with higher levels of serum anti-SSA, anti-SSB and B cell activation factor  region of NOD-derived C57BL/6.NOD-Aec1Aec2 mice revealed that a sub-region between 2.4 and 19.2 cM is associated with stomatitis sicca in the absence of detectable keratoconjunctivitis sicca in female mice, whereas males maintained the full SjS disease . The potent activation of autoreactive B and T cells, which were found to be highly upregulated at the clinical-disease stage might contribute to a more rapid loss of salivary gland function in female C57BL/6.NOD-Aec1Aec2 mice.The impact of androgen on the immune response is still controversial but generally it is thought to exert suppressive potential . Studies changes . InteresAec1Aec2 mice exhibited sexual dimorphic disease development. There are a number of factors that require further investigation. The focus of this study is limited to the salivary gland function; however, lacrimal gland function is the other key manifestation of SjS. Additional work is needed to completely profile the possible sexual dimorphism in the lacrimal glands. Moreover, the precise stage of the disease in which estrogen and androgen are involved needs to be defined. Presently, it is thought that sex hormones have a limited role in glandular development; however, it is possible that defective estrogen/androgen signaling can perturb glandular development leading to the onset of SjS. We need to define the precise mechanism(s) on which sex hormones modulate the differentiation and function of autoreactive lymphocytes in particular pathogenic Th17 cells and autoantibody-producing B cells. Lastly, the immune regulatory component mediated by sex hormones on immune cells and salivary gland epithelium needs to be examined to fully understand the autoimmune process of SjS.The current study has demonstrated that C57BL/6.NOD-s C57BL/6J.NOD/ShiLtJ-Aec1Aec2 (C57BL/6.NOD-Aec1Aec2) and non-SjSs C57BL/6J (C57BL/6) control mice were bred and maintained under specific pathogen free conditions in the animal facility of Animal Care Services at the University of Florida. The breeding and the use of animals described herein were approved by the University of Florida's Institutional Animal Care and Use Committee. Development of the C57BL/6.NOD-Aec1Aec2 mouse and its SjS-like disease phenotype are described elsewhere  and the second on chromosome 3 (designated Aec1) derived from the NOD/LtJ mouse into the C57BL/6 mouse. All animals were maintained on a 12 h light-dark schedule and provided food and acidified water ad libitum. At times indicated in the study, mice were euthanized by cervical dislocation after deep anesthetization with isoflurane and their organs and tissues freshly harvested for analyses.SjSlsewhere . BrieflySalivary glands were fixed in 10% phosphate-buffered formalin for 24 h. Fixed tissues were embedded in paraffin and sectioned at a thickness of 5 μm. Paraffin-embedded sections were deparaffinized by immersing in xylene, followed by dehydration in ethanol. The prepared tissue sections were stained with hematoxylin and eosin (H&E) dye . Stained sections were observed at 200× magnification by using Nikon Eclipse Ti-E inverted microscope. To detect and determine leukocytic infiltrations in salivary glands, a single histological section per gland per mouse was examined by a blinded examiner. Lymphocytic infiltrations were defined as aggregates of >50 leukocytes.Paraffin-embedded tissues of the salivary glands were sectioned and mounted onto microscope slides. Slides were deparaffinized and dehydrated by pressure-cooking in Trilogy  according to manufacturer's instructions. Following three 5-min washes with phosphate buffered saline with Tween-20 (PBS-T) at 25°C, the sections were incubated one hour with blocking solution containing donkey serum diluted 1:50 in PBS-T. Each section was incubated with purified rat anti-mouse CD45R  diluted 1:25 and goat polyclonal IgG anti-mouse CD3ε  diluted 1:50 in antibody diluent  for one hour at 25°C. The slides were washed three times with PBS-T, followed by a one hour incubation with Alexa Fluor (AF) 488 donkey anti-goat IgG (H+L) diluted 1:100 and AF 594 donkey anti-rat IgG (H+L)  diluted 1:25 at 25°C. The slides were washed thoroughly with PBS-T, treated with VectashieldDAPI -mounting medium  and overlaid with glass coverslips. Stained sections were visualized at 200× magnification on NikonTi-E fluorescent microscope. Infiltrate size and composition were calculated using Nikon NIS-Elements software by a blinded examiner. Areas of infiltrates were determined using the region of interest (ROI) established around the infiltrates and the composition based on the threshold intensities with background subtraction for individual fluorescent channels. The threshold intensities were kept consistent throughout the experiment.+ T cells were selected first, followed by the IL-17+ within the CD4+ positive region, providing the area of the double positive signal. The threshold intensities were kept consistent throughout the experiment.Salivary glands were digested in a digest buffer  and 1 mg/ml Collagenase Type 4  in RPMI  complete media containing 10% FBS, 2 mM L-glutamine, 0.05 mM β-mercaptoethanol) and placed in a MACS C tube  for desiccation on GentleMACS V1.02 for a pulse of 38 s, twice. After a 10-min incubation at 37°C the digest buffer was removed and placed into 4°C RPMI complete media and replaced with another digest buffer to repeat the process twice more. Single-cell suspensions were centrifuged  and resuspended in PBS for filtration through a 70-μm sterile cell strainer . After a wash with PBS, cells were resuspended again in PBS for lymphocyte isolation with Lympholyte-M cell separation media  per manufacturer's instructions. Single-cell suspensions were stained for surface markers, Brilliant Violet 605 anti-mouse CD3  and APC-Alexa Fluor 750 Rat anti-mouse CD4 . The membranes were permeabilized using Cytofix/Cytoperm Fixation/Permeabilization kit and intracellularly stained for Pacific Blue anti-mouse IFN-γ , PerCP/Cy5.5 anti-mouse IL-17A , and PE/Cy7 anti-mouse IL-4  per manufacturer's instruction . The samples were analyzed using BD Fortessa Flow Cytometer  and analysis was performed using FlowJo VX software . Immunofluorescent staining on paraffin-embedded tissues of the salivary glands were previously described . In brieSingle-cell suspensions of salivary glands were obtained as previously described . After aAnti-nuclear antibodies were detected in the sera of mice by using HEp-2 ANA kit . All procedures were performed according to the manufacturer's instructions. Mouse sera were diluted 1:80 and incubated on HEp-2-fixed substrate slides for one hour at room temperature in a humidified chamber. After three 5-min washes with PBS, the substrate slides were treated with a 1:100 dilution of AF 488 goat anti-mouse IgG (H+L) (Life Technologies) for 45 min at room temperature. After three washes, slides were treated with Vectashield DAPI-mounting medium (Vector Laboratories) and overlaid with glass coverslips. Fluorescence was detected by fluorescence microscopy at 400× magnification by using a Nikon microscope, and all images were obtained with exposure of 200 ms.To measure stimulated flow rates of saliva, individual mice were weighed and given an intraperitoneal (ip) injection of 100 μl of a mixture containing isopreterenol (0.2 mg/1 ml of PBS) and pilocarpine (0.05 mg/1 ml of PBS). Saliva was collected for 10 min from the oral cavity of individual mice using a micropipette starting one minute after injection of the secretagogue. The volume of each saliva sample was measured., La Jolla, CA, USA).Statistical evaluations for sialadenitis, B and T infiltrates in the glands, Th17 cells and Th17 cell proliferation were determined by using two-way ANOVA. A Chi squared test was used for autoantibody patterns and one-tailed Mann–Whitney test was used for salivary function. Analyses were generated by the GraphPad Prism 6 software (GraphPad Software


Suppressor of cytokine signaling (SOCS) proteins are key regulators of innate and adaptive immunity. There is no described biological role for SOCS4, despite broad expression in the hematopoietic system. We demonstrate that mice lacking functional SOCS4 protein rapidly succumb to infection with a pathogenic H1N1 influenza virus (PR8) and are hypersusceptible to infection with the less virulent H3N2 (X31) strain. In SOCS4-deficient animals, this led to substantially greater weight loss, dysregulated pro-inflammatory cytokine and chemokine production in the lungs and delayed viral clearance. This was associated with impaired trafficking of influenza-specific CD8 T cells to the site of infection and linked to defects in T cell receptor activation. These results demonstrate that SOCS4 is a critical regulator of anti-viral immunity. The suppressor of cytokine signaling proteins are key regulators of immunity. As yet there is no described biological role for SOCS4, despite its broad expression in cells of the immune system. Given the important role of other SOCS proteins in controlling the immune response, we have generated SOCS4-mutant mice and used a mouse influenza infection model to investigate the biological function of SOCS4. We demonstrate that mice lacking SOCS4 rapidly succumb to infection with a pathogenic H1N1 influenza virus and are hypersusceptible to infection with the less virulent H3N2 strain. This is the first demonstration of a functional phenotype in SOCS4-deficient mice. Our study reveals that in SOCS4-deficient animals, there is a dysregulated pro-inflammatory cytokine and chemokine production in the lungs and delayed viral clearance. This is associated with impaired trafficking of virus-specific CD8 T cells to the site of infection and linked to defects in T cell receptor activation. These results demonstrate that SOCS4 is a critical regulator of anti-viral immunity. Understanding the regulation of the inflammatory response to influenza is particularly relevant given the current climate concerning pandemic influenza outbreaks. Influenza is a highly infectious, acute respiratory disease that causes profound morbidity and mortality. Annual seasonal influenza epidemics result in ∼500,000 deaths worldwide and substantial losses to global economies The initial innate immune response to viral infection is characterized by an influx of neutrophils, monocytes and macrophages into the lung parenchyma and alveolar spaces, with the elevated levels of inflammatory cytokines/chemokines correlating strongly with pathogenesis and viral load The adaptive response subsequently results in the generation of strain-specific B cells and cross-strain protective CD4 and CD8 T cells. Influenza-specific CD8 T cells are largely responsible for host immunity to primary influenza infection and operate to promote the efficient elimination of virus, and host recovery, via the production of pro-inflammatory cytokines and direct killing of virus-infected cells Cytokine binding to the cognate receptor complexes triggers an intracellular signaling cascade, most often coupled to the JAK-STAT pathway, which orchestrates an intricate series of transcriptional changes leading to the appropriate cellular response. The suppressors of cytokine signaling (SOCS) proteins are key negative regulators of the JAK/STAT pathway and are thus involved in the fine-tuning of the cytokine networks responsible for an adequate and efficient innate and adaptive immune response c-cytokine-dependent T cell homeostasis Socs3 gene has identified a role for SOCS3 in regulating IL-6 and G-CSF signaling The generation of knockout mice has proven a powerful tool in defining the physiological role of the SOCS proteins. SOCS1 for instance, was revealed as a critical regulator of IFNγ signaling and γIn vitro studies have suggested that SOCS4 is involved in regulating epidermal growth factor (EGF) signaling Socs4, a point mutation identified in a library of ENU-mutagenised mice R108X/R108XSocs4 mice were highly susceptible to infection with influenza A, showing increased morbidity and a delay in viral clearance comparable to that observed for mice lacking CD8 T cells R108X/R108XSocs4 mice displayed impaired trafficking of virus-specific CD8 T cells to the lungs. The defect in trafficking appeared to be qualitatively linked to the activation status of these cytotoxic T lymphocytes and reveals a novel role for SOCS4 as a positive regulator of TCR signaling.In the current study, we have generated the first loss of function allele of murine R108X/R108XSocs4 mice were viable, fertile and showed no overt phenotype under steady-state conditions, including normal thymocyte development and composition of peripheral immune cells  with 20 pfu of the virulent H1N1 influenza strain A/Puerto Rico/8/34 (PR8) and monitored for weight loss. In accordance with ethical guidelines, mice were considered moribund upon losing greater than 20% of their initial body weight, and removed from the study. At this relatively low challenge dose, R108X/R108XSocs4 mice exhibited significantly enhanced disease progression and mortality compared to wild-type controls (ne cells . To invecontrols  and thiscontrols .R108X/R108XSocs4 mice and wild-type controls were subsequently infected i.n. with 103 pfu of the less virulent H3N2 A/Hong Kong x31 (X31) influenza virus. Similarly to infection with PR8, R108X/R108XSocs4 mice showed enhanced susceptibility to X31 infection with significantly greater weight loss  on day 3 and day 5 post-infection and this difference was greatly exacerbated by day 6 (2 log difference), when the majority of control mice had cleared the infection. Virus was undetectable in the lungs of wild-type controls on day 7, however some of the R108X/R108XSocs4 mice still retained low viral loads (R108X/R108XSocs4 mice on the C3H/He and C57BL/6 backgrounds (data not shown).To dissect the defective response in more detail, al loads . The augR108X/R108XSocs4 or Thy1.2 wild-type mice into lethally irradiated Thy1.1 wild-type recipients. The chimeric mice were subsequently infected i.n. with 103 pfu of X31 virus and disease progression monitored for 6 days. Although not significantly different, weight loss and particularly viral load were both increased in R108X/R108XSocs4 mice compared to controls . However, at day 3 post-infection, the R108X/R108XSocs4 mice showed significantly higher levels of cytokines and chemokines, such as IL-1β, IL-4, IL-5, IL-6, IL-12p40, IL-13, IFN-γ, and KC (CXCL1), MIP-2 (CXCL2) and MCP-1 (CCL2), respectively (Socs4 mutant mice. No difference was observed between R108X/R108XSocs4 and control mice in cytokine and chemokine production in either spleen or lungs on d3 following systemic administration of polyinosinic-polycytidylic acid (poly I:C), a non-dynamic, virus-like stimulus  and inflammatory monocytes (CD11c- Ly6Glow Ly6C+ F4/80+) (+) including higher levels of CD4 (CD44+) and CD8 (CD44+) T cells were detected in the lungs on day 3 post-infection . Socs4 mRNA expression increased over time, peaking at day 3 post-infection (due either to the changing cellular composition or to up-regulation within the infiltrating cells). In contrast, Socs4 mRNA was ∼15-fold lower in equivalent amounts of total lung RNA  accumulate in the lungs during the initial phase of infection in response to the elevated cytokine and chemokine levels. This may be a response to elevated chemoattractant levels or may potentially result from cytokine-driven proliferation (a bystander reaction rather than antigen-driven expansion). In summary, there is an increased net production of pro-inflammatory cytokines and chemokines, and while the initiating defect remains unclear, SOCS4 appears to have a classical role as a negative regulator of cytokine production and/or response in the innate immune reaction to influenza infection.Together, these data suggest that activated T cells  CD8 T cells in MLN, BAL and spleen, at days 5, 6 and 7 post-infection. Overall, the expansion of KdNP147-CD8 T cells did not differ between R108X/R108XSocs4 mice and wild-type controls (R108X/R108XSocs4 mice had significantly less virus-specific cells at the site of infection (BAL) when compared to controls and instead, these cells appeared to localise to the spleen. No difference was observed on day 7 post-infection  controls . Howevernfection , at whicn levels . No diffdNP147-CD8 T cells were analysed for granzyme B (GzmB) production as well as IFN-γ, TNF and IL-2. Alternatively, cells were stimulated ex vivo with KdNP147 peptide and stained with antibodies against lysosome-associated membrane protein 1 . No differences in GzmB production, cytokine production or CD107a mobilisation were observed between R108X/R108XSocs4 mice and wild-type controls . KdNP147-CD8 cells to the site of infection could result from reduced activation of the T cells following MHC-antigen presentation or alternatively, from altered homing signals. To investigate this further, we examined a panel of T cell activation markers  and homing receptors  at days 5, 6 and 7 post-infection. The majority of markers examined did not differ between wild-type and R108X/R108XSocs4 CD8 T cells (data not shown). However, at day 5 post-infection R108X/R108XSocs4 CD8 cells in the draining lymph nodes (MLN) showed comparatively higher levels of CD62L expression (R108X/R108XSocs4 CD8 cells in the lymph nodes (MLN), with no differences in the percentage of CD62Lhi versus CD62Llo expressing cells   were approximately equal  , comparely equal . Interes9 levels , whilst 9 levels .R108X/R108XSocs4 mice, results in their inability to migrate to the site of infection. In turn, this results in a reduced ability to clear the virus. To investigate this further, CD4 and CD8 T cells were isolated from R108X/R108XSocs4 and wild-type mice, and signaling responses analysed following CD3 engagement.Together, these data point to influenza-specific T cells receiving inadequate and/or incorrect signals in the lymph nodes, which in the Socs4 mRNA induced within 24 h of TCR stimulation with anti-CD3 antibodies, and peaking at 72 h. In addition, the magnitude of Socs4 expression was 4-fold higher in CD8 versus CD4 cells . To further investigate the consequences of reduced TCR responses, cells were labelled with Cell Trace Violet (CTV) dye, and proliferation measured in response to stimulation with anti-CD3 antibodies. As shown in R108X/R108XSocs4 CD8, but not in CD4, T cells. The defective proliferation seemed to result from a decrease in the number of cells that were mobilizing per division (R108X/R108XSocs4 and wild-type cells (data not shown).We initially examined SOCS4 expression in purified splenic CD4 and CD8 cells following activation with anti-CD3 antibodies. SOCS4 expression was compatible with a role in TCR signaling, with D4 cells . To invetivation . CD69, Cdivision  and resuR108X/R108XSocs4 T cells and further suggest that signaling through the TCR may be qualitatively different, thus accounting for the reduced activation observed in the lymph nodes during infection and in vitro following TCR engagement.These results indicate a defect that is intrinsic to the R108X/R108XSocs4 mice were highly susceptible to primary infection with the virulent PR8 H1N1 strain exhibiting increased mortality associated with weight loss and delayed viral clearance. Similar results were observed following infection with the less virulent X31 H3N2 strain, however the anorexia was somewhat ameliorated, enabling us to dissect the underlying pathology in greater detail. The transient weight loss induced by influenza infection is known to reflect viral pathogenicity and as in this study, correlates with increased cytokine/chemokine levels in the lungs in vivo, the 108-residue fragment could retain binding to its endogenous target and compete off other signaling intermediates, acting as a dominant negative. It might be speculated that residues 1–108 are involved in binding to a receptor subunit in a similar fashion to SOCS5 binding to the IL-4Rα Loss of functional SOCS4 protein led to a dramatic phenotype following influenza A infection. So far very little is known regarding the role of different SOCS proteins in influenza infection, outside of the ability of SOCS1 and SOCS3 to regulate specific cytokine receptor complexes. SOCS1 and SOCS3 have been implicated as negative regulators of innate immune responses (type I interferons) via a RIG-I dependent pathway R108X/R108XSocs4 mice, but it is unclear whether this increased cytokine production also contributes to the delayed viral clearance. Given that the delay in clearance is most pronounced during the adaptive phase of the immune response we postulate that the two phenomena  are independent of each other and result from multiple defects caused by the absence of a functional SOCS4.The pathogenicity of virulent influenza infections is not well understood, but it is accepted that pathogenic strains (such as PR8) cause dysregulation of innate immunity resulting in aberrant cytokine and chemokine production , which results in the lung tissue damage. Following infection with a viral strain of relatively moderate virulence, the absence of a functional SOCS4 protein led to dysregulated cytokine production akin to a cytokine storm. This uncontrolled inflammatory response is most likely responsible for the excessive weight loss in R108X/R108XSocs4 mice  and are confident that SOCS4 does not directly inhibit gp130 signaling. The elevated levels of MCP-1 and IL-12p40 are also of interest. IL-12p40 is produced by epithelial cells -/-Ifitm3 mice infected with influenza Ifitm3 gene Airway epithelial cells are the primary targets for influenza infection and are capable of producing cytokines as early as 3–6 hours post-infection 08X mice . IL-6 isR108X/R108XSocs4 and control mice. Numbers of neutrophils, NK cells, macrophages and inflammatory monocytes were comparable between the two groups. We do not understand the mechanism by which the lack of functional SOCS4 protein results in enhanced cytokine/chemokine production, but given the negligible expression of Socs4 in lung tissue and the relatively equal number of infiltrating cells, we propose that multiple immune cell types are producing greater amounts of cytokine per cell. This is also consistent with retention of the weight loss phenotype in wild-type mice reconstituted with R108X/R108XSocs4 bone marrow.Surprisingly, we did not detect major differences in the inflammatory cell infiltrates in the lungs of hi phenotype. This increased number of T cells in the lungs of R108X/R108XSocs4 mice is suggestive of proliferation due to bystander activation driven by cytokines rather than antigen-driven expansion of specific T cells or stimulation of cell division by cross-reactive antigens R108X/R108XSocs4 mice we observed significantly increased levels of IL-4 and IL-6 as well as elevated levels of IL-10, which combined with other cytokines in a complex lung milieu might lead to the activation and proliferation of T cells already present in the lungs However, our analysis of infected lungs did reveal significantly elevated numbers of T cells on day 3 post-infection, with both CD4 and CD8 showing an activated CD44R108X/R108XSocs4 mice, with tetramer positive cells appearing to accumulate in the spleen of R108X/R108XSocs4 mice instead of migrating to the site of infection, and this discrepancy was particularly apparent by day 5 post-infection  CD8 T cells migrating to the lungs had significantly lower levels of CD69 expression than wild-type (Thy1.1) CD8 cells. Although both subsets appear to be recruited to the lungs in similar ratios, their activation status differed significantly in an identical environment. Given that this result is in the context of a chimeric bone marrow reconstitution where up to 60% of the hematopoietic cells were wild-type, it strongly indicates an intrinsic CD8 T cell defect. CD69 expression declines in the absence of antigenic stimulation R108X/R108XSocs4 T cells showed a much weaker activation compared to the wild-type cells.In addition to the early excessive inflammation, we observed a clear difference in the distribution of virus-specific CD8 T cells in nfection . ActivatR108X/R108XSocs4 CD8 cells to the lungs is likely to be linked to the reduced activation of the virus-specific cells, which in turn suggests impaired TCR activation and signaling. The significantly lower numbers of influenza-specific effector cells available in the R108X/R108XSocs4 lungs no doubt result in the much higher viral loads observed .R108X/R108XSocs4) were generated through ENU mutagenesis by Ingenium Pharmaceuticals AG  on the C3HeB/FeJ background. The R108XSocs4 mutation was subsequently backcrossed onto both the Balb/c and C57BL/6 backgrounds for 10 generations.SOCS4 mutant mice (3-104 pfu of X31 (A/X31(H3N2)) influenza virus in 30 µL PBS. Virus stocks were grown in the allantoic cavity of 10 day old embryonated chicken eggs, from which the viral titre was determined by plaque assay on monolayers of Madin derby canine kidney (MDCK) cells. The weight of mice was monitored daily from day 3 post-infection. Mice were euthanised at various time points following infection and tissues collected for analysis.Mice were lightly anaesthetized by inhalation of methoxyflurane, and infected intranasally (i.n.) with 20 plaque forming units (pfu) of PR8 (A/Puerto Rico/8/1934 (H1N1)) or 10Lungs taken from mice after primary viral infection were homogenised and the virus-containing supernatant above the cell debris was harvested and stored at −80°C. Titres of infectious virus in the lung supernatants were determined by plaque assay on monolayers of MDCK cells.Spleen, mediastinal lymph node (MLN), and bronchoalveolar lavage (BAL) samples were recovered from mice at different stages during the acute phase of the primary infection. BAL samples were incubated on plastic petri-dishes for 1 h at 37°C to remove macrophages for tetramer experiments or used for phenotypic staining without adherent cell depletion. BAL fluid was collected and stored at −80°C for cytokine analysis. The spleens and MLN were disrupted, processed to single-cell suspensions and enriched for CD8 T cells by negative depletion using goat anti-mouse IgG and IgM antibodies to non-CD8 cell lineages . Lungs collected from infected mice were minced, incubated in 2 mg/mL collagenase for 30 min at 37°C, and processed to a single-cell suspension.dNP147) for 1 h at room temperature (RT). All batches of tetramer used in this study were titrated and the optimal dilution was based on both the percentage of epitope-specific CD8 T cells and the mean fluorescence intensity (MFI) of tetramer staining. Cells were washed twice in FACS buffer, and stained with antibodies to CD4, B220 and F4/80 , CD8-APC Cy7, CD3ε-PerCP Cy5.5, CD62L-APC, CD44-PE Cy7 (BD Biosciences or BioLegend) for 30 min on ice, washed twice and analysed by flow cytometry on the FACS Canto (BD Biosciences) and analysed by FlowJo software (Tree Star). For phenotypic staining, different combinations of antibodies were used as indicated in the text.Cells from BAL, MLN and spleen were stained with a tetramer conjugated to Strepavidin-PE  at an optimal staining dilution . Monensin  was then added and cells were incubated for a further 4 h. Cells were stained for surface CD8α expression, then fixed and permeabilized before intracellular staining with anti-IFN-γ FITC  and anti-GzmB APC. Negative controls incubated in the absence of peptide were used to control for spontaneous production of cytokine or expression of CD107a.Cells stained for tetramer and CD8α were fixed and permeabilized using a BD Cytofix/Cytoperm kit (BD Pharmingen), then stained for intracellular GzmB using anti-human GzmB-APC  as previously described 6 T cell-depleted bone marrow (BM) cells isolated from femurs and tibias of donor mice . Briefly, bone marrow was flushed from the femurs of 5–7 week old mice, washed once and incubated in complement-fixing antibodies anti-CD4 (RL172), anti-CD8 (3.186) and anti-Thy-1 (J1j) for 30 min on ice. After washing the cells once in HEPES Earle's medium containing 2.5% fetal calf serum (FCS) (HEM2.5), antibody-binding cells were incubated in rabbit complement at 37°C for 20 min. The mice were allowed to reconstitute for at least 8 weeks prior to use, blood samples were collected , and reconstitution of T cell compartment assessed by FACS analysis.To establish chimeras, recipient mice  were irradiated with two doses of 550R 3 h apart. At 3–5 h after the final irradiation, recipients were reconstituted with 3×102SO4. Absorbance values were read at 450 nm. TGF-β ELISA was performed according to the manufacturer's instructions .Cytokine levels were assessed by capture ELISA or BioPlex Pro Assay . Lung homogenates were stored at −80°C prior to analysis. BioPlex detection was performed according to the manufacturer's instructions. For ELISAs, U-bottom 96-well plates  were coated overnight at 4°C with capture antibodies against IL-4, IL-6, IL-10, IFN-γ, GM-CSF, KC, MIP-1α or MIP-2  and were then incubated with serial dilutions of samples for 2 h at RT, washed and incubated with the appropriate biotinylated detection antibody for 2 h at RT, followed by incubation with streptavidin-horseradish peroxidase. Plates were developed with 3,3′,5,5′-tetramethylbenzidine (TMB) in 0.1 M sodium acetate pH 6, and colour development terminated by the addition of 2N HIn vitro stimulation assays were performed by plating cells at 104 cells per well in RPMI 1640 medium containing 10% (v/v) heat-inactivated FCS , 5×10−5 M 2-ME (Sigma-Aldrich), 100 µg/mL streptomycin and 100 U/mL penicillin (Invitrogen Life Technologies) into anti-CD3-coated  96-well plates. Recombinant mouse IL-2 (20 ng/mL) was also added to wells. Cells were harvested at different timepoints, propidium iodide (2 µg/mL) and 5000 of Sphero Nile Red Fluorescent Particles (BD Biosciences) added per well and cell division analysed by flow cytometry. Each sample was analysed in duplicate.Single-cell suspensions were generated from spleens, and CD8 or CD4 T cells purified by negative selection using magnetic beads . Enrichment of cells was verified by flow cytometry. Purified T cells were labelled with Cell Trace Violet (CTV)  according to the manufacturer's instructions. Socs4 mRNA expression was performed essentially as described Q-PCR analysis of http://bioinf.wehi.edu.au/.Statistical analyses were performed using the unpaired t-test provided within GraphPad Prism 5 software and Compare Groups of Growth Curves software package available on the Walter and Eliza Hall Institute Bioinformatics Division's website Figure S1R108X/R108XSocs4 mice show normal composition of peripheral immune cells and normal thymic development. (A) Peripheral blood differential counts of R108X/R108XSocs4 and Balb/c mice. White Blood Cell differential count (left graph) and Complete Blood Counts for red and white blood cells (middle graph) and platelets (right graph) were performed using an ADVIA 2120 Haematology Analyser. Mean values from 18 R108X/R108XSocs4 mice and 10 Balb/c mice are plotted; error bars represent S.D. (B) Normal thymic development in R108X/R108XSocs4 mice. Thymocytes were stained with anti-CD4, CD8, CD44 and CD25 antibodies and DN (double negative CD4/CD8-) subdivided based on their CD44 and CD25 profile  as well as CD4 and CD8 expression. Mean values (n = 5) were plotted, error bars represent S.D. (C) Percentage of T and B cells in various naïve tissues of R108X/R108XSocs4 and Balb/c mice. Cells were gated as double positive T cells, either TCRβ+CD4+ or TCRβ+CD8+, or as B220+ B cells. Mean values are plotted (n = 5); error bars represent S.D.(TIF)Click here for additional data file.Figure S2Cytokine and chemokine production in spleen and lungs of R108X/R108XSocs4 following administration of polyinosinic-polycytidylic acid.R108X/R108XSocs4 and control mice were injected intraperitoneally with 2 µg of poly I:C per g of body weight. Lungs and spleens were harvested on day 3 post-injection and cytokine and chemokine levels in tissue homogenates were analysed by Bioplex. Mean ± S.E.M. are shown for biological replicates .(TIF)Click here for additional data file.Figure S3Phenotypic analysis of lung hematopoietic subsets in R108X/R108XSocs4 and Balb/c mice at day 2 (A) and day 6 (B) post-infection. Flow cytometry analysis was performed on homogenized lungs. * indicates p<0.05, **<0.005.(TIF)Click here for additional data file.Figure S4R108X/R108XSocs4 mice reconstituted with wild-type bone marrow show reduced engraftment and impaired activation of Thy1.2 T cells following H3N2 influenza A infection. (A) Irradiated R108X/R108XSocs4 mice were reconstituted with wild-type Thy1.1 bone marrow and expression of congenic markers  analysed in the T cell compartment in peripheral blood at 8 weeks post-reconstitution. (B–F) Lethally irradiated R108X/R108XSocs4 Thy1.2 mice reconstituted with wild-type Thy1.1 bone marrow were infected with H3N2 X31 influenza A virus. Spleen (SPL), mediastinal lymph node (LN) and BAL were collected on day 6 post-infection and single cell suspensions were analysed by flow cytometry to examine (B) percentage of Thy1.1 vs Thy1.2 cells, (C) CD69 and (D) CD44 expression on Thy1.1 and Th1.2 CD4 and CD8 T cells. Mean±S.D. are shown from biological replicates (n = 6), ** p = 0.001–0.01, ***p = 0.0001–0.001, ****p<0.0001. MFI, Mean Fluorescence Index. R108X/R108XSocs4 mice reconstituted with wild-type bone marrow (WT > R108X/R108XSocs4) show no difference in disease severity when compared to wild-type (Thy1.2) mice reconstituted with wild-type (Thy1.1) bone marrow (WT(Thy1.1)>WT(Thy1.2)). Mice were infected with H3N2 X31 influenza A virus and weight loss (E) was monitored for 6 days, at which timepoint lungs were harvested for viral titre estimation (F). No statistically significant differences were observed between the groups.(TIF)Click here for additional data file.


We have identified a quadruplex-binding protein, Lia3, that controls the boundaries of germline-limited, internal eliminated sequences (IESs) of Tetrahymena thermophila. Differentiation of this ciliate’s somatic genome requires excision of thousands of IESs, targeted for removal by small-RNA-directed heterochromatin formation. In cells lacking LIA3 (ΔLIA3), the excision of IESs bounded by specific G-rich polypurine tracts was impaired and imprecise, whereas the removal of IESs without such controlling sequences was unaffected. We found that oligonucleotides containing these polypurine tracts formed parallel G-quadruplex structures that are specifically bound by Lia3. The discovery that Lia3 binds G-quadruplex DNA and controls the accuracy of DNA elimination at loci with specific G-tracts uncovers an unrecognized potential of quadruplex structures to regulate chromosome organization.Guanine (G)-rich DNA readily forms four-stranded quadruplexes  in vitro, but their regulatory importance within cells has been particularly challenging to demonstrate conclusively. We have discovered a G4 DNA binding protein, Lia3, that specifically regulates programmed DNA elimination events in Tetrahymena thermophila. This ciliate deletes nearly one-third of its germline genome from each developing somatic nucleus. These genomic deletion events must be accurate as the thousands of DNA regions excised are located near genes and/or their promoters, thus aberrant excision may alter gene expression. When we knocked out the gene encoding Lia3, we found that the boundaries of the excised regions were heterogeneous for a subset of loci that are flanked by G-rich (5’-AAAAAGGGGG-3’) boundary controlling sequences. When we tested whether Lia3 bound this sequence, we discovered that the sequence itself formed G4 DNA and that Lia3 bound only when the sequence adopted this conformation. Our findings that Lia3 binds G4 DNA and that deletion of the gene encoding Lia3 perturbs the boundaries of the excised loci which are flanked by this quadruplex-forming DNA provides compelling evidence that this non-canonical DNA structure has a critical role during development of these cells.Non-canonical DNA structures, including four-stranded Guanine quadruplexes (G4 DNA), have been observed readily  Tetrahymena thermophila, more than 6,000 dispersed loci, comprising nearly one-third of the genome, are eliminated  ATP for 1 hr at 37°C and then purified using Roche oligo spin columns. Oligos were made double-stranded by mixing equal amounts of complementary oligonucleotides in 10 mM Tris pH 7.5, 5% glycerol, and 100mM LiCl and boiling for 5 min, followed by slowly allowing the oligonucleotides to cool to RT. Prior to gel shifts, oligonucleotides were boiled for 5 min in 10 mM Tris pH7.5, 5% glycerol, and either 100 mM KCl or 100 mM LiCl and slow cooled to RT to allow structures to form.Oligonucleotides  were lab32P-labeled oligonucleotide and incubation for another 15 min at RT before 4 μl was loaded onto a 4.5% polyacrylamide (75:1 acrylamide:bisacrylamide) gel. After pre-running gels at 140V for 30 min, samples were fractionated by electrophoresis at 140V for 1 hr 45 min. Gels were subsequently vacuum dried for 1 hr prior to exposure to X-ray film or to a Phosphorimager screen. For binding curve experiments, 4 nM 32P-labeled oligo was incubated with 0, 50, 75, 100, 150, 200, 250, 300, 400, 500, 700, 900, 1100, 1300, 1500, 1900, 2100, 2500, or 3000 nM His-Lia3 for 20 min at RT prior to loading on gel.For binding and competition experiments, 50–400 nM His-Lia3, MBP-Lia3, or MBP-MS2 was incubated with unlabeled competitor oligonucleotides  in bindiOligonucleotides were boiled for 5 min in 10 mM Tris pH 7.5, and either 100 mM KCl or 100 mM LiCl and slow cooled to 4°C. The CD spectra were recorded on a J-810 spectropolarimeter (Jasco). The measurements were carried out with 500 μL 3 μM ODN samples at 4°C under nitrogen. Spectra shown are the average of 3 scans in a range from 220 to 300 nm with a band width of 1 nm, response time of 0.5 s, data pitch of 0.2 nm, and scan speed of 50 nm/min. A blank sample of 10 mM Tris pH 7.5 with 100 mM KCl or 100 mM LiCl was used for baseline correction.Tetrahymena cells were fixed in 3% PHEMS-paraformaldehyde essentially as described [Strains CU428 and B2086 were starved in 10 mM Tris-HCl (PH 7.5) and mixed to induce mating. Between 9 and 10 hours post-mixing, mating escribed , incubatescribed , and detS1 FigΔLIA3 alleles are illustrated on the right. The LIA3 gene was replaced with the NEO3 selectable marker, which confers paromomycin resistance upon induction with CdCl2. The HindIII (HIII) restriction enzyme cut sites used to differentiate these two alleles, and the expected sizes of genomic DNA fragments are indicated above each diagram. The region of DNA that was radiolabeled and used as a probe is also shown. The first lane contains genomic DNA from parent strain CU428; the remaining lanes are DNA isolated from: initial germline-transformants after phenotypic assortment (1B2 and 1C2); germline homozygous lines resulting from crossing B*VI to 1C2; complete macronuclear (Mac) and micronuclear (Mic) ΔLIA3 lines resulting from crossing lines 5A and 5B. An additional high-molecular weight DNA fragment in lines 1A and 1B result from transgene deletion (tgd) occurring in the final cross to make the homozygous ΔLIA3 lines, which removes the HindIII within NEO3. The Mic and Mac genotype of each strain is indicated at the bottom of each lane. The migration of Lambda DNA (PstI digest) size markers of specific sizes is given on the left.Representations of wild-type and (EPS)Click here for additional data file.S2 FigLIA3 matings as indicated. (a) IES 54 is flanked by A5G5 sequences and shows aberrant rearrangement. Lanes 1–4 are from the wt and ΔLIA3 parent lines used for the matings in lanes 5–13. (b-f) PCR across rearrangement junction of IES that rearrange normally in ΔLIA3 matings. Larger black arrows, pcr primers; asterisks, polypurine tracts; grey boxes, flanking sequences; white boxes, IES; smaller black arrows; predominant wild-type products; grey arrowhead, non-specific PCR products. IES 92 has a sequence resembling a polypurine tract only on its right flank. Although some IES rearrangement is highly variable, those without flanking A5G5 sequences show similar variability in ΔLIA3 and wild-type progeny lines.PCR amplification of IES rearrangement junctions in progeny of wild-type and Δ(EPS)Click here for additional data file.S3 Fig(a) Western blot of the dialyzed final isolated protein sample detected using anti-6xhis antisera. Lane 1 is the size marker and lane 2 is the isolated His-Lia3 sample. (b) Coomassie from same gel as (a) confirming that His-Lia3 is the primary protein in the sample. For A and B, arrows point to His-Lia3.(EPS)Click here for additional data file.S4 Fig5G5 tract has been mutated.(A) M1 and M1-C3 oligos were boiled in 10mM Tris with or without 100mM KCl or 100mM LiCl, slowly cooled to room temperature and then run on a 10% native gel. Black arrows point to the quadruplex form, grey arrows to the ss linear form. (B) CD spectra for M1 and M2 oligos in different salt conditions. M1 and M2 display characteristic spectra for parallel G quadruplex in 100mM KCl but not in 100mM LiCl or when the A(EPS)Click here for additional data file.S5 FigDecreasing amounts  of MBP-Lia3 (top panel), MBP-MS2 (middle panel), or BSA (bottom panel), were loaded into four wells, fractionated by SDS-PAGE, and visualized by coomassie staining. A known amount (4.0–0.15 ug) of BSA was loaded and used to estimate the amount of purified Lia3 and MS2 protein.(EPS)Click here for additional data file.S6 FigTetrahymena telomere (d) sequence. Increasing concentrations of protein, from 50 to 3000 nM, were added to labeled probes (4 nM). (a) M1 probe DNA in 100 mM KCl allowing quadruplex formation (G4). (b) ssM1 probe DNA or (c) dsM1 annealled with its complementary sequence in 100 mM LiCl preventing quadruplex formation, binding reactions contained 100mM LiCl in place of KCl. (d) Tetrahymena telomeric DNA in 100 mM KCL allowing quadruplex formation. The position of migration unbound  probe and Lia3 bound probe are indicated by arrowheads.Purified Lia3 protein was incubated with 30 base oligonucleotides corresponding to the M1 boundary (a-c) or the (EPS)Click here for additional data file.S7 Fig32P-labeled G4-M1. Black arrows point to the bound M1 probe; grey arrows point to the unbound ss or G4 form of the probe. LiCl was substituted for KCl in binding reaction with non-G4 competitors.Competition gel shift in which different unlabeled competitor oligonucleotides  are incubated with His-Lia3 at either 5 fold(X) or 20X excess relative to the amount of (EPS)Click here for additional data file.S8 FigTetrahymena cells was performed using a 1:200 dilution of anti-G4 antisera  [Immunofluorescent detection of G4 DNA in unmated (A) and 9–10 hour mating (B) ica, MA)  on cellsica, MA)  and dete(EPS)Click here for additional data file.S9 FigLIA3 containing an N-terminal HA epitope was integrated into the endogenous locus. A copy of the neo3 paromomycin resistance selection cassette was incorporated downstream if the coding region to allow selection of biolistic transformants. Replacement of all copies of the untagged LIA3 with the HA-LIA3 allele in macronuclei (M) or both micro- and macronuclei (C) was confirmed by PCR-based tests. Protein was isolated from either a somatic (M) or complete (C) knock-in of Hemaglutanin (HA)-tagged LIA3 line matings and fractionated by SDS-PAGE. Arrow points to HA-tagged Lia3. Hours indicate times post-mixing that protein was extracted.Western blot using anti-HA. A tagged copy of (EPS)Click here for additional data file.S10 Fig32P-labeled G4-M1. Black arrows point to the bound M1 probe; grey arrows point to the unbound ss or G4 form of the probe.Competition gel shift in which different unlabeled RNA corresponding to the M1 flanking DNA was incubated with His-Lia3 at either 1, 5, 10, or 20 fold(X) excess relative to the amount of (EPS)Click here for additional data file.S11 FigLTL1, Ttherm_00499370; LTL2, Ttherm_00600360; LTL3, Ttherm_00787380. A multiple sequence alignment of the conserved region was generated with ClustlW2 . Open arrow, LIA3 coding region, shaded box, conserved region; the amino acids of Lia3 aligned are indicated above the diagram. B) Screenshots of microarray expression data from the Tetrahymena Functional Genomics Database [LIA3 and LTL genes are expressed exclusively in post-zygotic development. C) and D) Expression of yellow fluorescent protein-LTL C-terminal fusions show that both LTL1 and 3 are localized to developing macronuclei when DNA rearrangement occurs. The coding region of each was inserted into pIGF-gtw to generate N-terminal fusions with Green Fluorescent Proteins (GFP) and introduced into Tetrahymena cells by conjugative electroporation [A) Schematic diagram of the Lia3 protein denoting an ~100 amino acid region that shares similarity with three other Tetrahymena-encoded proteins: Database  reveals poration . GFP locporation .(EPS)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


In vitro RNA interference confirmed that c-Fos is one of the AP1 members driving the expression of viral oncogenes E6/E7. Mutation analysis and in vivo RNA interference identified an essential role for c-Fos/AP1 and also for the bromodomain protein Brd4 for papillomavirus-induced tumorigenesis. Lastly, chromatin immunoprecipitation analysis demonstrated that E2 binds together with Brd4 to a canonical E2 binding site (E2BS) in the promoter of c-Fos, thus activating c-Fos expression. Thus, we identified a novel way how E2 activates the viral oncogene promoter and show that E2 may act as a viral oncogene by direct activation of c-Fos involved in skin tumorigenesis.We investigated the mechanism of how the papillomavirus E2 transcription factor can activate promoters through activator protein (AP)1 binding sites. Using an unbiased approach with an inducible cell line expressing the viral transcription factor E2 and transcriptome analysis, we found that E2 induces the expression of the two AP1 components c-Fos and FosB in a Brd4-dependent manner.  Human Papillomaviruses (HPV) are the etiological agents of cervical cancer and of skin cancer in individuals with the inherited disease epidermodysplasia verruciformis (EV). While the role of the viral oncogenes E6/E7 as drivers of tumorigenesis in cervical cancer has been firmly established, the contribution of the early viral genes in skin cancer is less clear. For EV-associated HPV8 and for the skin cancer model system using cottontail rabbit PV, an important role of the viral E2 protein in tumorigenesis was suggested earlier and regulation of cellular genes by E2 through different mechanisms was demonstrated. We show now that the viral E2 and cellular Brd4 act together to induce the cellular gene c-Fos, which as a member of the AP-1 complex, is involved in the regulation of cellular genes and the viral promoter driving the expression of viral oncogenes. As c-Fos has also been shown to be essential for skin cancer, E2 contributes to tumorigenesis via expression of E6/E7 as well as by increasing c-Fos. Expressn cancer . Those an cancer . Dysregun cancer . Papillon cancer ,6 and bin cancer –9. Both n cancer . Transcrn cancer . In addin cancer . The mecn cancer  and cancn cancer ,12, whiln cancer . Interesn cancer ,15. AP1 n cancer . Interesn cancer ,18 and cn cancer ,20. As tn cancer  we now iTo study the ability of CRPV E2 to activate AP1-dependent reporters, we performed luciferase experiments with artificial promoter constructs consisting of multimers of four different TPA- (TREs) and as control one cAMP-responsive element (CRE). Seven copies of each sequence element originating from the human MMP9 promoter (AP-MMP9), from the URR of HPV18 (AP18), from the URR of CRPV  and from the CRE element were cloned upstream of the luciferase gene  into theOur previous studies demonstrated that the AP1-mediated activation of the MMP9 promoter by CRPV E2 does not require E2BS in the promoter region . TherefoSince Brd4 interacting with PV E2 proteins is required for stability of E2 and its transactivating capability ,8, we teE.coli [To overcome limitations using artificial reporters with multimerized AP1BS, we next investigated the natural CRPV URR containing nine E2BS and two putative AP1BS. The entire URR encompassing the L1 stop codon and the E7 ATG driving the luciferase reporter was cloned  and the double-mutation (dpAP) into the URR of the whole CRPV genome . The skin of New Zealand White (NZW) rabbits was infected with these CRPV genomes and tumor growth was assessed 6 months post infection. We observed only 33.3% papilloma induction with pLAII-CRPV-mdAP as compared to pLAII-CRPV-mpAP and the wt CRPV positive control  that mediate Brd4 binding severely impaired the ability of CRPV to induce tumors in rabbits , which aTo identify the mechanism of E2-mediated activation of AP1-responsive promoters, we used an unbiased approach to search for cellular genes with altered expression levels in the presence of CRPV E2. For this we established an inducible E2-expressing cell line using the pRTS1-vector . The sysin vitro activity of the CRPV URR either in the presence or absence of CRPV E2. For this, c-Fos was silenced in C33A cells using a pool of three siRNAs  experiments and investigated the binding of different AP1 components to FAP1 and the potential AP1BS at -4961 in the endogenous c-Fos promoter. C33A cells were serum-starved for 18h and after addition of serum ChIP experiments were performed using antibodies targeting respective AP1components c-Fos, FosB, c-Jun, JunB and JunD and primers flanking the AP1BS at positions -454 and -4961. All AP1 members, except JunB, were enriched at the FAP1 site and at the AP1 site at position -4961 .To investigate how E2 activates c-Fos expression, binding of E2 and Brd4 to the consensus E2BS in the c-Fos promoter at position -2411 as well as to both AP1 sites was investigated by ChIP. A distinct enrichment compared to the empty vector of both CRPV E2-HA (4.1 fold) and Brd4 (2-fold) at the canonical E2BS was observed, indicating that CRPV E2 indeed binds together with Brd4 to this sequence. In contrast, no enrichment of E2 or Brd4 was observed at the AP1BS at -4961 while Brd4, but not E2, was enriched approximately 6-fold at the FAP1 . This mi7 C33A cells were transiently transfected with the respective expression vectors for ChIP experiments. Again a specific enrichment of both CRPV E2-HA and Brd4 at the canonical E2BS was observed  in up to 26% of treated melanoma patients, while the combination of vemurafenib with the MEK-inhibitor cobimetinib reduced the appearance of SCC to 5% . The ideConstruction of plasmids, cell culture conditions, transient luciferase assays, siRNA transfections, quantitative real-time PCR, lentiviral infection, microarray processing and data analysis, immunoblot analyses and detailed chromatin immunoprecipitation (ChIP) are listed in supplemental experimental procedures .Animal experiments were reviewed and approved (Permit Number: H1/03 and H1/08) by the responsible authority  according to the German Animal Welfare Act (TierSchG §8 Abs. 1) and were performed according to national regulations (TierSchVersV).New Zealand White rabbits were obtained from Charles River Laboratories. Rabbits were infected with different recombinant CRPV genomes using the “helios gene gun” (Bio-Rad) as described previously . Tumor gC33A cells were transfected with pRTS1-CRPVE2-HA or empty vector pRTS1 and selected with 250 μg/ml of hygromycin for 5 days. CRPV E2-HA expression was induced in stable pooled cell lines by the addition of 1 μg/ml of doxycycline for 48h. The cells were then harvested and total cellular RNA was isolated for microarray analysis.32P  using Polynucleotide Kinase (Fermentas) and purified with illustra ProbeQuant G-50 Micro columns . 5–10 fmol/μl were used in binding reactions with 50 ng of recombinant protein in Bclw/BSA buffer . Binding reactions were performed for 30–40 min at 30°C and separated on a 4% polyacrylamide gel with 0.25x TBE  as running buffer for 1h. The gel was dried, exposed and then visualized by using Typhoon 9200 PhosphorImager . For oligo competitions, a 100-fold molar excess of unlabeled DNA fragments containing either wild-type or mutated distal AP1 sequences was included at the beginning of the reaction.Double-stranded oligos  were seeded and treated with 1 μM MG132 16 h before harvest to prevent protein degradation. Cells were harvested in cold PBS, lysed in high salt NP-40 buffer  and incubated for 30 min on ice. The lysate was centrifuged, and the supernatant was diluted to a final NaCl concentration of 150 mM. After that, the supernatant was incubated with 30 μl of anti HA-magnetic microbeads (Miltenyi biotec) for 1h. The supernatant was discarded and the beads were washed 2–3 times with NP-40 buffer containing 150 mM NaCl. Bound protein complexes were eluted using SDS sample buffer (Carl Roth).For transfection for ChIP assays, one 100-mm plate per construct of C33A cells was transfected with 10 μg DNA using Fugene HD (Promega) according to the manufacturers’ instructions. ChIP was carried out as previously described . See supGraphPad (version 5) was used to calculate unpaired and paired p-values.All transcriptional profiles have been submitted to the GEO database at NCBI (Accession no. GSE67345).S1 Fig(A) E2 K320M/C321R is unable to bind to DNA. Transient replication assay in C33A cells using the CRPV URR, CRPV E1, HA-tagged and untagged E2 and Brd4-deficient binding mutants of E2 ) as well with the DBD-deficient mutant E2 K320M/C321R . Wt E1 and E2 together with the empty vector were used as a control. (B) Brd4-CTD blocks activation of the AP18 reporter devoid of E2BS by E2 in a dose-dependent manner. Luciferase activity of C33A cells cotransfected with constant amounts of wt CRPV E2 and AP18 and increasing amounts of the dominant negative variant Brd4-CTD.(TIF)Click here for additional data file.S2 Fig4) were transiently transfected with 0.5 μg of empty vector (pSG) and wt E2 proteins. 48h later the cells were harvested and analyzed for c-Fos mRNA induction compared to the control (empty vector).c-Fos mRNA induction by E2 proteins from different HPVs. HeLa cells (8x10(TIF)Click here for additional data file.S3 Fig4) were transfected with a pool of three c-Fos siRNAs (siFos) or a control siRNA (siControl). 24h later the cells were transiently transfected with 10ng of the reporter (pC18-SP1-luc) and 10ng of the E2 expression vector (CRPVE2) or the empty vector. Measurement was carried out 48h after DNA transfection. The induction of the pC18-SP1-luc is shown in relative light units (RLUs). The values indicate mean ± standard deviation of four independent experiments.Activation of the pC18-SP1-luc by CRPV E2 after c-Fos silencing. C33A cells (~ 7x10(TIF)Click here for additional data file.S4 FigImmunohistochemistry of normal skin and of CRPV induced papillomas using an antibody against c-Fos. Control: Incubation with secondary antibody only. The dashed line indicates the basal lamina of the skin.(TIF)Click here for additional data file.S5 FigThe skin of 3 different rabbits was shaved and irritated using ultrafine sandpaper. Punch biopsies were taken after the indicated timepoints. C-Fos expression was analyzed by qRT-PCR and normalized to alpha-tubulin. Relative induction was compared to timepoint 0 min (before irritation). Standard deviation is indicated by error bars.(TIF)Click here for additional data file.S6 FigUpper panel: Schematic description of the Δ c-Fos promoter reporters used in the luciferase assays. Canonical and non-canonical E2BS are shown as black and grey boxes, respectively. The FAP1 site is shown as hatched box, the AP1BS (AP1) as white box. (lower panel) Luciferase assay with truncated c-Fos-promoter constructs and wt E2 (grey bars) and mutant E2 constructs impaired in DNA- and Brd4-binding ) and the empty vector as control (dark grey bars). The induction of each construct is shown in relative light units (RLUs). The values indicate mean ±SD of five independent experiments.(TIF)Click here for additional data file.S7 Fig(A) Protein expression levels of C33A cells transfected with HA-tagged CRPV E2 (overexpression) or empty vector (control). Alpha-tubulin was used as a loading control. Marker in kDa is indicated on the left. (B) cp-values of the mRNAs encoding different AP1 monomers in C33A cells transfected with CRPV E2 (wt E2) or empty vector (control).(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


Hepatitis C virus (HCV) poses a global threat to public health. HCV envelop protein E2 is the major component on the virus envelope, which plays an important role in virus entry and morphogenesis. Here, for the first time, we affinity purified E2 complex formed in HCV-infected human hepatoma cells and conducted comparative mass spectrometric analyses. 85 cellular proteins and three viral proteins were successfully identified in three independent trials, among which alphafetoprotein (AFP), UDP-glucose: glycoprotein glucosyltransferase 1 (UGT1) and HCV NS4B were further validated as novel E2 binding partners. Subsequent functional characterization demonstrated that gene silencing of UGT1 in human hepatoma cell line Huh7.5.1 markedly decreased the production of infectious HCV, indicating a regulatory role of UGT1 in viral lifecycle. Domain mapping experiments showed that HCV E2-NS4B interaction requires the transmembrane domains of the two proteins. Altogether, our proteomics study has uncovered key viral and cellular factors that interact with E2 and provided new insights into our understanding of HCV infection. HCV is an important human pathogen that primarily infects human hepatocytes and causes chronic liver diseases . This deTo fill this knowledge gap, we developed a strategy to purify intact E2 complex formed in HCV infected human hepatoma cells and reproducibly identified 85 HCV E2 binding proteins. Our comparative proteomics and functional analyses revealed an important interaction between HCV E2 and the endoplasmic reticulum (ER) protein UGT1, which regulates the production of infectious HCV. Interestingly, another viral protein, NS4B, was also found to interact with E2. Multiple domains of HCV NS4B coprecipitated with HCV E2 and this interaction was abolished when E2 transmembrane domain was removed. Characterizing these interactions in detail would provide a deeper understanding of HCV infection and also potentially present targets for antivirals to disrupt virus biology.The human kidney epithelial cell line Lenti-X 293T was purchased from Clontech. The human liver cell line Huh7.5.1 was provided by Dr. Francis Chisari (Scripps Research Institute) . All celJFH1-Flag-E2, which expresses Flag-tagged JFH1 E2, has been described previously. pLVX-Fl8) were infected by JFH1-AM2 and JFH1-Flag-E2-AM2 viruses. On day 3 postinfection, cells were trypsinized and washed with ice-cold phosphate-buffered saline and then Dounce homogenized in 10 ml of immunoprecipitation (IP) buffer  supplemented with a protease inhibitor cocktail. Centrifugation-cleared lysates were then subjected to IP with 50 μl anti-Flag M2 affinity resin and rotated 4 hours at 4°C. After four washes with the IP buffer, bound proteins were eluted with Flag peptide  in 100 μl Tris-buffered saline and resolved on SDS/PAGE, and the proteins were visualized by SYRO Ruby staining. Roughly 20 gel slices were excised from either JFH1-AM2 or JFH1-Flag-E2-AM2 lane and subjected for LC-MS/MS.Huh-7.5.1 cells  was added and samples were allowed to incubate on ice for 45 minutes. The supernatant was removed and the gel bands were covered with 25 mM NH4HCO3, pH 8.4, and incubated at 37°C overnight. Tryptic peptides were extracted from the gel pieces with 70% acetonitrile, 5% formic acid, lyophilized to dryness and resuspended in 10 μL of 0.1% formic acid prior to MS analysis.Gel bands of interest were subjected to in-gel digestion according to established protocols . BrieflyNanoflow reversed-phase liquid chromatography (RPLC) was performed using a Dionex Ultimate 3000 LC system  coupled online to an LTQ-Orbitrap XL mass spectrometer . Separations were performed using 75 μm i.d. x 360 o.d. x 20 cm long fused silica capillary columns  that were slurry packed in house with 5 μm, 300 Å pore size C-18 silica-bonded stationary phase . Following sample injection onto a C-18 trap column (Dionex), the column was washed for 3 min with mobile phase A  at a flow rate of 0.3 μL/min. Peptides were eluted using a linear gradient of 0.34% mobile phase B (0.1% formic acid in acetonitrile) / min for 117 minutes, then to 95% B in an additional 10 min, all at a constant flow rate of 0.2 μL/min. Column washing was performed at 95% B for 20 minutes, after which the column was re-equilibrated in mobile phase A prior to subsequent injections.http://www.ebi.ac.uk/integr8) and the Hepatitis C virus genotype 2a (isolate JFH-1) protein sequence (UniProt Accession Q99IB8) using SEQUEST (ThermoFisher Scientific). For a fully tryptic peptide to be considered legitimately identified, it had to achieve stringent charge state and proteolytic cleavage-dependent cross correlation (Xcorr) scores of 1.9 for [M+H]1+, 2.2 for [M+2H]2+ and 3.5 for [M+3H]3+, and a minimum delta correlation (ΔCn) of 0.08. The false discovery rate cutoff was set as < 1%. Additionally, peptides were searched for methionine oxidation and cysteine carboxyamidomethylation with a mass addition of 15.99492 and 57.02416, respectively. The obtained proteins were further filtered by removing proteins identified by less than two unique peptides. Finally, those proteins identified from the JFH1-AM2 lane were subtracted from the total proteins that were identified in the JFH1-Flag-E2-AM2 lane (comparative or subtractive approach) in order to obtain the real E2 interacting partners. The purification, LC-MS/MS and subtractive analyses were done three times independently.The LIT-MS was operated in a data dependent MS/MS mode in which each full MS scan was followed by seven MS/MS scans where the seven most abundant peptide molecular ions are selected for collision-induced dissociation (CID), using a normalized collision energy of 35%. Data were collected over a broad mass to charge (m/z) precursor ion selection scan range of 300–1800, utilizing dynamic exclusion to minimize redundant selection of peptides previously selected for CID. Tandem mass spectra were searched against a combined UniProt human protein database (03/2011) from the European Bioinformatics Institute .Four shRNA clones targeting human UGT1 and the pLKO.1 control plasmid were purchased through TRC consortium from Sigma. To generate lentivirus, 3 μg of shRNA clone, 3 μg of pCMV8.2ΔR, and 1.5 μg VSV-G expression plasmid were transfected into 293T cells in 6-cm plates by lipofectamine 2000 (Invitrogen). Viruses were collected at 48 hours post-transfection and cleared through filtration. 500 μl viruses were added to Huh7.5.1 cells in a 12-well plate and selected by puromycin (0.6 μg/ml). To verify the knockdown efficiency, cell lysates were prepared and analyzed by Western blotting.Briefly, 30 μg of proteins were run on 4–20% precast polyacrylamide gel  and transferred to nitrocellulose membranes. Membranes were blocked with Odyssey Blocking Buffer  followed by incubation with primary antibodies at a 1:1000 dilutions. Membranes were washed three times with 1X TBS, incubated with IRDye secondary antibodies  for 1 h and washed again to remove unbound antibody. Western blotting images were taken using the ODYSSEY CLx .Bar graphs were plotted to show mean ± standard deviation (SD) of at least two independent experiments. Statistical analyses were performed using Graphpad Prism 5. A p value of <0.05 in the Student's test was considered statistically significant.The urgent need of prophylactic vaccines and alternative therapies demands for better understanding of the virus life cycle –23. HoweWe have previously engineered a Japanese fulminant hepatitis 1 (JFH-1) clone that expresses a FLAG tag in fusion with its glycoprotein E2 . This clTo depict the networks to which the 85 factors are clustered, we used Ingenuity Pathway Analysis (IPA) software to curate information on protein-protein interactions (PPIs) and molecular pathways. Indeed, many of the identified proteins interact with each other according to the database and can be linked to the same protein network, which further validates the success of our affinity purification. Well-represented networks include molecular transport and cell signaling; carbohydrate metabolism, lipid metabolism; cell-to-cell signaling and interaction . PHB1 anAmong the 85 cellular proteins, endoplasmic reticulum chaperones such as calnexin  and calrHCV E2 is synthesized at rough endoplasmic reticulum (ER) and then glycosylated at eleven sites before secretion. It is known that the eleven N-glycans of HCV E2 greatly influence its function in E2 ER-localization, dimerization, maturation, binding to CD81, protein folding, virus entry, assembly, and protection against neutralization, etc –34. One To investigate the functional role of UGT1 in HCV life cycle, we tested four short-hairpin interfering RNA (shRNA) clones that target human UGT1. Western blotting showed that #2 and #4 shRNA markedly reduced the endogenous level of UGT1 in Huh7.5.1 cells . ConcomiThe main function of E2 is to bind cellular cell surface molecules, namely the tetraspannin CD81 and scavenger receptor SR-BI , and triEmerging evidence has indicated that p7, NS2, and NS3-NS4A participate in virus particle assembly. In particular, NS2 has been shown to interact with both E1 and E2 during viral assembly and plays an important role in the assembly process , 41. TheTo characterize the molecular determinants of E2-NS4B interaction, we created deletion constructs containing the N-, C-, TM, TMC domain of NS4B to express Flag tagged proteins. Domain mapping study revealed that the N-terminus and C-terminus regions of NS4B efficiently precipitated with E2 . The traIdentifying which host proteins and complexes come into physical contact with the viral proteins is crucial for a comprehensive understanding of how HCV usurps the host's cellular machinery during the course of infection. To our knowledge, this is the first detailed interactome analysis of HCV E2 in the context of infection, which reduces identifying false interactions that only occur in the artificial yeast two-hybrid assay or when a singly overexpressed viral protein is used as a bait in immunoprecipitation. Three independent trials plus stringent criteria for protein identification yielded highly reproducible data that warrant future mechanistic investigations. In a recent study , Ramage The 85 cellular factors identified in our experiment may not be binding to E2 directly, because we were using a fully infectious molecule clone. HCV E2 interacts with E1 and NS2 as other have published –60, it iThe immediate application of use of the identified interactions is to develop better understanding of the molecular biology of HCV envelope protein E2. Heterodimers between E2 and E1 viral glycoprotein together make up the virus envelop spikes that mediate viral attachment and entry into host cells, and the assembly of infectious virus particles . The funOur E2 interaction map now provides an excellent opportunity to look for druggable targets. In a separate publication, we described a detailed validation and functional characterization of E2-prohibitin interaction . In the S1 Fig(TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.