Tumour necrosis factor (TNF) is produced by primary human macrophages in response to stimulation by exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) via Toll-like receptor (TLR) signalling. However, uncontrolled TNF production can be deleterious and hence it is tightly controlled at multiple stages. We have previously shown that Bruton's tyrosine kinase (Btk) regulates TLR4-induced TNF production via p38 MAP Kinase by stabilising TNF messenger RNA. Using both gene over-expression and siRNA-mediated knockdown we have examined the role of Btk in TLR7/8 mediated TNF production. Our data shows that Btk acts in the TLR7/8 pathway and mediates Ser-536 phosphorylation of p65 RelA and subsequent nuclear entry in primary human macrophages. These data show an important role for Btk in TLR7/8 mediated TNF production and reveal distinct differences for Btk in TLR4 versus TLR7/8 signalling. •Btk is required for TLR7/8 signalling in primary human macrophages.•R848-induced TNF mRNA is more Btk dependent than LPS-induced TNF mRNA.•Btk transcriptional control of TNF following R848 requires the promoter and 3′UTR.•Btk knockdown reduces p65RelA translocation to the nucleus upon TLR7/8 stimulation. Toll-liNon-receptor tyrosine kinases play a major role in TLR signalling , and in In HEK293 cells Btk physically interacts with the cytoplasmic Toll/IL-1 receptor (TIR) domains of TLRs 4, 6, 8 and 9 as well as the adaptor molecules Myd88 and Myd88-adapter-like  . FollowiHere we provide evidence for Btk in TLR7/8 signalling in human primary macrophages. Btk regulates TLR7/8-induced TNF production at early time points via the 3'enhancer region of the TNF gene. Moreover, we show that Btk controls the initiation of TNF transcription through NF-κB recruitment. Interestingly, TNF transcription in response to LPS was less affected, revealing a previously unreported distinction between TLR4 and TLR7/8-mediated TNF gene transcription.2Reagents and Antibodies. R848 and LPS were from Alexis Biochemicals and macrophage colony-stimulating factor (M-CSF) was from Peprotech. Polyclonal rabbit anti-Btk antibody for immunoprecipitation was a gift from M. Tomlinson , mouse anti-Btk antibody (clone 10D11) for Western blotting was from BD Bioscience and anti-GAPDH (ab9484) and rabbit isotope control antibodies were from Abcam. The phosphotyrosine clone 4G10 was from Millipore. Cell Signaling provided anti-IκBα (#9242), anti-phospho-p65RelA (Ser536) , and anti-p65RelA . Anti-p65RelA (sc-372) for Western blotting was from Santa Cruz Biotechnology.Monocyte isolation and adenoviral infection. Following ficoll-hypaque centrifugation, monocytes were elutriated from PBMC as previously described [escribed . Monocytescribed . For douescribed ,18.Gene knockdown by siRNA. 5 × 106 primary human monocytes were transfected with targeting siRNA or control oligunucleotides  at concentrations ranging from 100 to 300 nM using Human Monocyte Nucleofector Kit  according to manufacturer's instructions. After 24 h, monocytes were cultured in 5% HIFCS phenol red free RPMI with 100 ng/ml M-CSF for a further 72h. STAT1 phosphorylation by Western blot following siRNA nucleofection was assessed after a further 24h in the absence of M-CSF and stimulation ± IFN (1 ng/ml) for 5 min.Immunoprecipitation and Western Blotting. M-CSF-differentiated macrophages were plated on 10 cm2 petri dishes and serum starved for 2h prior to stimulation. Cells were lysed in ice-cold lysis buffer , containing freshly added 10 mM DTT, 100 μM Na2VO3, 5 mM NaF, 1x Protein Inhibitor Cocktail (Sigma). Debris was removed by centrifugation, and supernatants were pre-cleared with protein G-sepharose. Btk was precipitated with polyclonal rabbit anti-Btk anti-sera and protein G-sepharose for 1.5 h. Immunoprecipitated complexes were washed in lysis buffer before resolving on 10% SDS-PAGE gel and transferring to nitrocellulose membrane (Millipore). The membrane was blocked for 1h in TBS-Tween (0.1%) with 2% BSA for the detection of phosphorylated proteins or in 5% skimmed milk for other proteins.Immunocytochemistry. After siRNA transfection, monocytes were differentiated in M-CSF for 72h. Macrophages were plated on glass coverslips  and stimulated with R848 (1 μg/ml). Cells were fixed with 4% (w/v) paraformaldehyde in PBS for 15 min at 37 °C, quenched with 50 mM NH4Cl/PBS for 10 min, and permeabilised with 0.1% (w/v) Triton X-100 in PBS for 5 min. Samples were blocked with 3% (w/v) BSA in PBS for 30 min at room temperature followed by incubation with anti-phospho-p65RelA (Ser536) diluted in 3% (w/v) BSA in PBS for 1 h at room temperature. After washing, secondary antibody was added; phosphorylated p65RelA (Ser536) with Alexa Fluor 488 , actin cytoskeleton with Alexa Fluor 546 Phalloidin  and the nucleus with DAPI . Samples were mounted with ProGold antifade mounting media . Confocal ‘z’ stack were used to quantify the intensity of staining by measuring the respective brightness of the pixels for each of detection channels using Fiji image analysis software.Cytokine measurements by ELISA. TNF concentration in supernatants was determined by ELISA (BD Biosciences), according to the manufacturer's instructions. Absorbance was read and analysed at 450 nm using a Fluostar Omega  plate reader and analysed using MARS data analysis software.Real-time RT-PCR. RNA was extracted from macrophages using Blood RNA extraction kit (QIAGEN), and genomic DNA removed using TURBO DNA-free kit (Applied Biosystems). cDNA was subjected to real-time PCR analysis using SYBR Premix Ex Taq (Lonza) on a Corbett Rotor-Gene 6000 (Qiagen). Primers for measuring primary human TNF transcripts were 5′-GCAGTCAGATCATCTTCTCG-3′ and 5′-GGTACAGGCCCTCTGATGGCAC-3’. Mature human TNF transcripts were 5′-CCTGCTGCACTTTGGAGTGATCGG-3’ & 5′-GTACAGGCCCTCTGATGGCACCACC-3′, respectively. Primers for actin-related protein transcripts (ARP) were 5′- CGACCTGGAAGTCCAACTAC-3′ and 5′- ATCTGCTGCATCTGCTTG-3’. Relative quantification of gene expression was expressed as fold mRNA/ARP as determined using the comparative ΔΔCT method.Statistical analysis. Values correspond to mean ± SEM or SD. In experiments with multiple groups, differences were first evaluated using repeated-measures ANOVA and then Dunnett's test to compare group means. Unpaired Student's t-test was used when comparing differences between two groups.33.1Enzymatic activation of Btk is marked by tyrosine phosphorylation. Primary human macrophages were stimulated with either LPS (10 ng/ml) or R848 (1 μg/ml) for 5, 10, 15 or 20min and tyrosine phosphorylation of Btk determined by Western blot analysis. As previously observed  an increTo determine whether Btk modulates TLR7/8 driven TNF, Btk was over-expressed as previously described  as confi3.2TNF is controlled at a number of different stages in its production. Our previous work showed that message stability is an important control point for TNF production . TNF is To establish whether this is also true of primary human cells we examined mature TNF transcripts in macrophages following siBtk. Real-Time PCR with a primer set spanning an exon-exon TNF sequence A were usIn response to Btk knockdown, primary TNF transcripts  within the promoter region and a fourth cluster  in the 3′UTR. Site 4 plays an essential role in LPS-mediated TNF transcription in macrophages and DCs [Transcription of TNF is mediated by a number of transcription factors including AP-1, Oct-1 and NF-κB . 4 disti and DCs ,21 and o and DCs .To investigate the NF-κB sites, and other regions in the TNF locus, controlling Btk-mediated TLR7/8-induced transcription we used a series of adenoviral constructs containing the TNF promoter, 3′UTR and other downstream regions, linked to a luciferase reporter A. Over-eIn contrast, the 5'only construct, the 4X NF-κB construct , and the 5′785 construct, which lacks a 252bp region in the 3′UTR, were not able to increase luciferase production after Btk over-expression B. Thus d3.4Studies to determine the effect of Btk on TLR7/8 mediated IκBα activity showed that knockdown of Btk had no effect TLR7/8-mediated phosphorylation and degradation of IκBα A. Howeve4Understanding how TNF expression is controlled is crucial for the next generation of anti-inflammatory therapies . TLR7 anBtk is a critical signalling component of a wide variety of immune receptors, including Fc receptors, gp130 containing cytokine receptors, and the B cell receptor where it is required for the activation of NF-κB ,27; the Phosphorylation of p65RelA occurs after release from the inhibitory IκBα complex and the importance of Ser536 phosphorylation of p65RelA is demonstrated by the mutation of Ser536 to alanine which abolishes the interaction of p65RelA with the transcriptional coactivators p300 and CREB-binding protein (CBP) thereby decreasing transcriptional activation ,30. Btk Non-receptor tyrosine kinases are attractive targets for therapeutic intervention as small molecule inhibitors are readily synthesised . Btk inhWe have used loss of function and overexpression manipulation technologies to demonstrate the importance of Btk in response to different TLR ligands in primary human macrophages. Btk plays an important role in the TLR7/8 mediated induction of TNF and there are clear differences between TLR4 and TLR7/8-mediated TNF production. These data add important insight into the control of TNF production in primary human cells and may suggest molecular targets for the development of more efficient anti-inflammatory therapeutics.


CAPOW, is presented along with enhanced visualization tools for analysing statistical distributions of data.A new standalone program to calculate optimized values for weighting parameters,  CAPOW , a new program for the calculation of optimal weighting parameters for a SHELXL weighting scheme, is presented and an example of its application in a multipole refinement is given.The rigorous analysis of crystallographic models, refined through the use of least-squares minimization, is founded on the expectation that the data provided have a normal distribution of residuals. Processed single-crystal diffraction data rarely exhibit this feature without a weighting scheme being applied. These schemes are designed to reflect the precision and accuracy of the measurement of observed reflection intensities. While many programs have the ability to calculate optimal parameters for applied weighting schemes, there are still programs that do not contain this functionality, particularly when moving beyond the spherical atom model. For this purpose,  For a refinement on F2, q varies depending on the sign of parameter c:Advanced weighting schemes can be applied to give an improved distribution of residuals if the refined structural model is understood to be complete and correct. The potential weighting schemes that can be applied, unless direct manipulation of the standard uncertainties occurs, are dependent upon what is available within the refinement packages being used. One popular weighting scheme for small-molecule crystallography is employed in the c = 0,c > 0, andc > 0.Here, λ is the wavelength of the X-ray radiation.a and b parameters are routinely calculated for data refined in native SHELXL refinements, with the other parameters remaining fixed  has been created to allow straightforward operation of the program and provide informative output. The current version of CAPOW features a window with two different tabs: one for the calculation of the optimal weighting scheme and another for the creation of a normal probability plot  of each of the ten bins is calculated for a 9 × 9 grid of incrementally increasing a and b values. The combination of a and b that gives the minimum variance within the grid is used to give a new starting point for the next grid. The step size, i.e. how much a or b increases between subsequent grid points, is reduced, provided that the minimum variance did not occur at the edge of the grid. The process is repeated until a stopping condition, usually based on the size of the a or b step, is fulfilled. The values of a and b that give the minimum variance from the final grid are taken as the optimal parameters to be used in the weighting scheme. The user interface has been customized to allow a choice of both stopping condition and initial starting values for a and b.The optimal weighting is determined using a grid-search method. After the initial starting values for CAPOW were compared with those calculated in Olex2 . Selected weights can then be transferred to the normal probability plot tab using the ‘Send Weights to Tab 1’ button.The interface to the weighting scheme calculation is displayed in Fig. 12.2.matplotlib package  or .fco [XD2016  and .ins/.mas file . The program outputs optimized values for both a and b to be applied in further refinement.The The source code has been tested using Scientific Linux 7.0 and Windows 8, and has been written with no operating system dependencies.3.XD2016, and prior versions, is a widely used crystallographic refinement package for aspherical atom refinement. Atom-centred spherical harmonic (multipolar) functions are used to describe aspherical electron density distributions  it was important to examine the spread of the variance of the wGooF of binned data from a charge density refinement: the value to be minimized in the weighting scheme optimization. The variance was calculated for SHELXL weighted data with different combinations of a and b and displayed as a contour plot  did not result in convergence. The calculated a and b starting values are used to determine initial grid step sizes, giving values that were smaller than the stopping conditions (thus preventing the grid search from running). To counteract this, we recommend using a and b stopping points of a_stop > 0.00001 and b_stop > 0.0005. The problem could also be overcome by changing the initial starting points for a and b. The weighting applied in this refinement was calculated with stopping points a_stop = 1 × 10−7 and b_stop = 1 × 10−6.When using data from a multipole refinement, the stopping points originally applied in the a) shows the normal probability plot created using statistically weighted data. The values for a normal distribution of residuals are shown as a dashed red line that can be compared with the experimental values. The deviation of the residuals from a normal distribution can be assessed by computing a line of regression using data between the lower and upper quartiles to remove bias from any large outliers. For the statistically weighted data, this gives an equation of y = 1.3985x − 0.02406. As previously stated, if the residuals were normally distributed a gradient of 1 and an intercept of 0 would be expected. The result shown, alongside visual inspection of the graph, highlights that these data do not demonstrate a normal distribution.Fig. 4SHELXL weighting scheme, optimal parameters determined by CAPOW are input into the refinement master file. Several iterations of the least-squares refinement were undertaken until convergence was achieved. Optimal a and b parameters for the newly converged model are calculated and then applied. The process of calculation and refinement is repeated until convergence of the a and b parameters occurs.To apply the SHELXL weighted refinement  is much closer to that which would be expected from a normal distribution of residuals. The line of regression of data between the lower and upper quartiles has an equation of y = 0.9707x − 0.00006, i.e. a gradient much closer to the ideal value of 1, with an intercept of close to zero.The normal probability plot from the converged nt Fig. 4b is mucSHELXL weighting are shown in Table 1SHELXL weighting is found, giving a value much closer to 1, along with an increase in the value of the weighted residual. For the data set used in the above refinements, there was no significant statistical difference observed for atom positions, bond lengths, bond angles or multipolar populations between the statistical and SHELXL weighted refinements. However, the multipole parameters calculated from the SHELXL weighted refinement have larger standard uncertainties than the statistically weighted refinement. This causes some populations to become less statistically significant.Key refinement statistics from both the statistical and the 4.CAPOW, embedded within a user-friendly graphical interface, enabling the calculation of the optimal a and b values of a SHELXL weighting scheme. We have shown that, by applying a correctly determined weighting scheme, a more normal distribution of residuals can be produced. However, caution must always be exercised when applying a weighting scheme that is based upon a model. This situation relies on the assumption that the model is correct. Therefore, as a non-normal distribution of residuals can indicate the presence of systematic errors, it is always advisable to use statistical weights initially and eliminate as far as possible any systematic errors before applying a more complex weighting scheme.We have demonstrated the use of a standalone weighting program, SHELXL refinement has three other parameters that could also be optimized . Further work is being conducted to analyse the impact of and to expand the optimization routines to evaluate these additional parameters.The CAPOW and its source code are distributed via the nu-xtal-tools repository which can be found at http://github.com/nu-xtal-tools.


NFκB activation and regulated cell death are important in tissue homeostasis, inflammation and pathogenesis. Here we show the role of the p55TNFR–IKK2l–Ripk3 axis in the regulation of synovial fibroblast homeostasis and pathogenesis in TNF-mediated mouse models of arthritis. Mesenchymal-specific p55TNFR triggering is indispensable for arthritis in acute and chronic TNF-dependent models. IKK2 in joint mesenchymal cells is necessary for the development of cartilage destruction and bone erosion; however, in its absence synovitis still develops. IKK2 deletion affects arthritic and antiapoptotic gene expression leading to hypersensitization of synovial fibroblasts to TNF/Ripk1-mediated death via district mechanisms, depending on acute or chronic TNF signals. Moreover, Ripk3 is dispensable for TNF-mediated arthritis, yet it is required for synovitis in mice with mesenchymal-specific IKK2 deletion. These results demonstrate that p55TNFR–IKK2–Ripk3 signalling orchestrates arthritogenic and death responses in synovial fibroblasts, suggesting that therapeutic manipulation of this pathway in arthritis may require combinatorial blockade of both IKK2 and Ripk3 signals. TNF is a major therapeutic target for rheumatoid arthritis (RA) and synovial fibroblasts are central to the pathogenesis of RA. Here the authors dissect TNF-induced death and activation signalling in RA synovial fibroblasts and TNF-driven arthritis and indicate that a successful therapeutic strategy might be to target both IKK2 and RIPK3 at the same time. The socioeconomic burden of rheumatic diseases is thus amplified more than once thought. Regardless of the therapeutic advances provided by effective new drugs, absolute remission of disease has not been achieved. Further investigation of the aetiopathogenesis of RA is required.Rheumatoid arthritis (RA) is an inflammatory disease that primarily affects the synovial joints, especially the fingers, wrists, feet, and ankles, resulting in painful deformities and immobility. RA has now been associated with multi-disease syndromes and comorbidities that affect bone and lipid metabolism, the cardiovascular, gastrointestinal and respiratory systems, and increased cancer susceptibility3. Functional genetic analyses in TNF-overexpressing mice (hTNFtg4 and TNFΔARE45) highlight the fundamental role of synovial fibroblasts (SF) and p55TNFR in mediating arthritogenesis, demonstrating that TNF/p55TNFR signalling in SFs is sufficient to orchestrate proliferation, inflammation, and tissue destruction in these models6. In principle, p55TNFR signalling is driven by complex I and II, which consist of Tradd/Ripk1/Traf2/cIAP1 (I) and FADD/caspase-8/Ripk1 (IIa) or Ripk1/Ripk3/Mlkl (IIb). These complexes lead to survival and inflammatory signalling (such as nuclear factor κB (NFκB), as well as death pathways that are reliant on the availability of NFκB and caspases (reviewed in ref. 7).Current understanding of RA pathogenesis is focused mainly on inflammation in the joints, which is maintained by the deregulated production of inflammatory mediators by immune and mesenchymal cell types that are fueled mainly by tumour necrosis factor (TNF)8). However, there are instances of TNF-induced necroptotic death happening in the presence of caspase machinery such as the case of L929 fibrosarcoma cell line9. Some other studies challenge the idea that NFκB/caspase inhibition is required for activation of Ripk1/Ripk3/Mlkl signalling and suggest that necroptosis induction is dependent on both the stimuli and the cell context. For example, Toll-like receptors (TLR) are equally effective in driving Ripk1/3-dependent and Mlkl-dependent necroptosis with different molecular prerequisites13; the kinase activity of Ripk1 is required for both p55TNFR-induced or TLR-induced necroptosis in macrophages15, but this activity is dispensable for TLR-induced necroptosis in endothelial cells or fibroblasts12.Aside from apoptosis, necroptosis is another form of programmed cell death controlled by the p55TNFR pathway upon the suppression of both NFκB and caspase signalling and is dependent on the kinase activity of Ripk1 and the interaction of Ripk1/Ripk3/Mlkl  mice29 with Col6a1-Cre mice6 in order to generate animals which lack mesenchymal p55TNFR in their SFs (p55TNFRMs-KO)  cocktail. None of the p55TNFRMs-KO mice developed any joint inflammation either when monitored in the course of the disease or in the histopathological analysis, in sharp contrast to p55TNFR-sufficient littermate controls, which exhibited the reported clinical and histopathological manifestations at the peak of disease  demonstrated the essential role of p55TNFR in the development of this induced arthritic pathologyase Fig. . Thus p57, so we targeted IKK231 (encoded by the Ikbkb gene and from now on referred as Ikk2) in a mesenchymal-specific manner. We generated Ikk2Ms-KO animals, which either were crossbred into hTNFtg background or subjected to CAIA. Interestingly, the clinical manifestations in both cases were ameliorated -positive cells in the joint area, detection of some cleaved-caspase-3-positive (CC3+) cells, mainly in articular cartilage, was evident in hTNFtg mice. The Ikk2Ms-KO mice in the arthritic models and, interestingly, also in non-arthritic conditions exhibited a readily detectable signal of TUNEL-positive cells in the all compartments of the synovium . To clarify the potential efficacy problems, we employed Col6a1-Cre Ikk2f/f in combination with RosamT/mG mice35, in order to observe Cre-mediated recombination events through the expression of GFP reporter gene. The limited expansion of SFs derived from the double mutant Col6a1-Cre Ikk2f/fRosamT/mG mice, as this was detected by the flow cytometric detection of Cre+ (GFP+) cells in culture, compared to Col6a1-Cre RosamT/mG SFs suggested defective survival and/or expansion of Ikk2-null SFs ex vivo  displayed altered TNF- and interleukin (IL)-1-induced responses, as indicated by failure of IkB degradation, whereas phosphorylation of c-Jun N-terminal kinase1/2 (JNK1/2) was prolonged 37, is required for the prevention of death in hTNFtg Ikk2Δ/Δ SF cultures. Collectively, our results show that IKK2 signals operating downstream of the p55TNFR in SFs, either acutely or chronically experienced to TNF, are strongly required for their survival in vivo and ex vivo.Owing to aforementioned limitations in ex vivo culturing, we first applied an IKK2 inhibitor (ML120B)say Fig. . In all say Fig.  being evsay Fig. , none ofivo Fig. . The ex ged Fig.  in agreeres Fig. . Additiores Fig. . The hTNell Fig. , yet theIkk2-deficient SFs in vivo and ex vivo by examining proliferation capacity and the expression of TNF/NFκB-induced genes implicated in cell survival and activation. Synchronized, TAT-Cre-treated SFs of Ikk2f/f and hTNFtg Ikk2f/f cultures  as well as the antioxidant enzyme Sod2 failed to upregulate their expression in Ikk2-null SFs, whereas this was not evident in the case of hTNFtg Ikk2-null SFs , Mmp-3, -13 and -14 in all Ikk2-null SF extracts; however, the Timp1 expression remained unaltered  gene expression, which is merely changed upon further TNF stimulation  exhibited less profound changes in pro-inflammatory activation and no overt differences in antiapoptotic responses.Quantitative PCR analysis revealed the defective RNA expression of NFκB-regulated molecules, such as red Fig. . The hTNion Fig. . InteresSFs Fig. . CollectIkk2-deficient SFs and the sensitivity of the hTNFtg Ikk2-deficient SFs to a caspase3/8-independent death, we subjected the Ikk2-null SF cultures to TNF treatment in the presence of pan-caspase and/or Ripk1/Mlkl inhibitors . Interestingly, TNF-induced death was partially rescued in Ikk2-null SFs by either treatment, indicating that besides the caspase-mediated death, a caspase-independent and Ripk1/Mlkl-mediated death is also at play. The hTNFtg Ikk2-null SFs, even in the absence of additional TNF treatment, were extremely sensitive to caspase inhibition that led to a high percentage of dead cells . The absence of any effect of Ripk3 deficiency in the development of arthritis in hTNFtg mice  expression remained elevated compared to Ikk2Δ/ΔRipk3−/− cells -SFs with no significant differences between the two types 6, innate (as in TNFΔARE)6 or adaptive (as in CAIA)43 stimuli are instigating pathology. These findings may translate into respective TNF-driven mechanisms operating in several subclasses of human arthritides and explain the comparatively broad success of anti-TNF therapies in RA patients.We have previously demonstrated the sufficiency of TNF/p55TNFR-signalling in SFs for the orchestration of murine TNF-dependent arthritis34. Cell-specific KO approaches revealed the essential role of IKK2 in TNF- and TLR-mediated activation of NFκB and its importance in regulating inflammatory and proliferative as well as death responses (reviewed in ref. 45). Owing to such pro-inflammatory and pro-survival functions, NFκB appeared as an appealing pharmacological target. Indeed, several IKK2 inhibitors have been developed; however, almost none achieved the criteria to pass into clinical applications. This was mainly due to the lack of specificity and multiple side effects that were poorly reported in the preclinical studies most probably due to the short term application of the protocols (reviewed in ref. 46). Interestingly, longer IKK2 inhibition led to leukocyte death and increased neutrophilia, rendering the mice severely immunocompromised47, susceptible to endotoxemia48 and autoimmune diseases49, indicating that inactive IKK2 leads to miscellaneous responses depending on the cell target. Our observations revealed that targeted mesenchymal IKK2 deficiency did not lead to overt phenotypes. By employing the hTNFtg model of chronic arthritis, we observed that, regardless of the ameliorated arthritic phenotype and the preserved joint architecture, mice with joint mesenchymal cells deficient in IKK2 still developed a sustained synovitis throughout the course of chronic disease, indicating that IKK2 acts as a checkpoint regulator of the progression from synovitis to pannus formation.In light of this evidence, we then examined the mesenchymal-specific function of IKK2, a natural mediator of TNF/p55TNFR signals. IKK2 deficiency in mice is lethal due to a massive apoptotic response in the embryonic liver caused by defective NFκB activationIkk2-null SFs suggest that the naive Ikk2-null SFs exhibit an inflammatory and antiapoptotic transcriptional insufficiency caused by the defective NFκB and caspase-3 activation in response to TNF, in line with previous reports50. Surprisingly, the hTNFtg Ikk2-null SF population was selectively expressing some inflammatory profiles despite their chronic TNF exposure , a caspase-independent, yet Ripk1/Mlkl-dependent death is taking place in the absence of IKK2. Even though the GW806742X inhibitor (targeting Mlkl) had been recently reported to affect the human RIPK1/3 activity too52, the TNF-induced phosphorylation of Mlkl in Ikk2-null SFs and the spontaneous phospho-Mlkl expression in hTNFtg Ikk2Δ/Δ SFs  indicate a possible Mlkl involvement in our experimental system. Moreover, we show that IKK2 regulates hTNF, Ripk3 and Mlkl protein expression under chronic inflammatory conditions . Notably, the hTNFtg Ikk2-deficient synovium is surprisingly less prone to CC3-mediated apoptotic events marked with a few CC3+ cells, located mainly in sublining area regardless of uniform staining of TUNEL throughout the synovium. This and our ex vivo findings suggest that either Mlkl phosphorylation and caspase activation is happening within the same Ikk2-deficient cell or that distinct cells within the Ikk2-deficient SF population die by different modes of death. In this regard, it is possible that distinct SF types exist within the synovium and differentially regulate local responses that affect the physiology of the joints leading to pathologic situations. Moreover, numerous reports have analysed the resistance of arthritic SFs to apoptosis (reviewed in ref. 51) rendering the re-activation of their death machinery as a potential therapeutic approach. However, the segregation of programmed cell death into apoptosis, necroptosis, pyroptosis, etc, depending on the molecular players and the processes involved, has started clarifying their functional significance in physiology and disease. The inflammatory nature of necroptotic death due to the release of damage-associated molecular patterns compared to the immunological quiescent apoptotic death40 may explain the abrogation of the residual inflammation seen in the synovium in the absence of both IKK2 and Ripk3 signalling in SFs, whereas Ripk3 were dispensable for the development and progression of the chronic TNF-mediated arthritis. Interestingly, this latter finding contrasts the previously reported acceleration of resolution phase of arthritis caused by the Ripk3 deficiency in the KRN-serum transfer arthritis model13. Of note, the KRN-serum transfer model of arthritis is minimally TNF dependent and more TLR4 and IL-1β dependent55, and the diminished TLR4/TRIF/Ripk3-dependent IL-1β expression by macrophages, and not macrophage necroptosis, could explain the observed differences13. In our study, we clearly show that the mesenchymal compartment of the hTNFtg joints, namely SFs, is the major responder to TNF arthritogenic signals that could act independently to induce synovitis even in defective IL-1 conditions57, as this could happen, e.g., in a Ripk3-null background. Moreover, IKK2/Ripk3 deficiency is rather relevant to the downregulation of hTNF release by the double IKK2/Ripk3-deficient hTNFtg SFs compared to hTNFtg Ikk2-null SFs  depending on the context and independently of NFκB activation; however, single Ikk2 deficiency was not sufficient to provide the same result in that study58. Interestingly, another recent report suggested non-necroptotic functions of either Mlkl and/or Ripk3 in controlling, at least partially, lymphoid tissue homeostasis via production of chemokines and cytokines under caspase-8 or FADD deficiency59. Whether Ripk3 could further regulate altered gene expression in the arthritic Ikk2-null SFs, as it has been reported for other cell types deficient in IAPs63, and/or death is yet to be explored. Further studies employing several mutant mice for the molecular players of necroptotic and apoptotic pathway will shed light on the contribution of death pathways under mesenchymal IKK2 deficiency in TNF-mediated arthritis.Another possible explanation for the differential death susceptibility of 19, MG132, a potent NFκB modulator acting through proteosomal inhibition and suggested for the treatment of arthritis reviewed in64, provoked both RIPK1-mediated caspase-dependent and -independent death in SFs from some patients, providing translational evidence to our murine-based observations.Interestingly, even though IKK2 inhibition in OA- or RA-SFs is not sufficient to induce similar effectsIkk2-null SFs to switch from a caspase-dependent to a Ripk3-mediated caspase-independent mode of death following chronic TNF stimulation suggests that combination of mesenchymal IKK2 targeting along with Ripk3 blockade may be considered as an optimal therapeutic strategy for the treatment of chronic arthritis.In conclusion, our study provides novel mechanistic insights into the mesenchymal-specific role of the IKK2 and Ripk3 axes during TNF-dependent arthritogenesis, offering ex vivo and in vivo evidence on their complementary functions in regulating differential modes of pro-inflammatory synovial cell activation and death with significant impact on the physiological outcome in modelled arthritis. Moreover, the distinct property of hTNFtg), TNFΔARE, Col6a1-Cre6, p55TNFRf/f29, Ikk2f/f33, p55TNFR−/−65, RosamT/mG37 and Ripk3−/−66 mice have been previously described. Col6a1-Cre Ikk2f/f and Col6a1-Cre p55TNFRf/f mice are referred to as Ikk2Ms-KO and p55TNFRMs-KO, respectively. All mice were maintained on a C57BL/6J except from hTNFtg Ikk2Ms-KO and littermate mice, which were maintained in a CBA;C57BL/6J genetic background. Mice were maintained under specific pathogen-free conditions in conventional, temperature-controlled, air-conditioned animal house facilities of BSRC Alexander Fleming with 14–10 h light/dark cycle and received food and water ad libitum.Human TNF transgenic . Briefly, arthritis was induced in 6–8-week-old littermate male mice of the indicated genotypes by intravenous injection of ArthritoMabTM (4 mg) on day 0. On day 3, mice also received an intrapritoneal injection of 75 μg lipopolysaccharide diluted in 150 μl phosphate-buffered saline .CAIA was induced with a cocktail of four monoclonal IgG Abs, specific for four well-defined major epitopes on triple helical Collagen II, the C1 (CIIC1 clone), J1 (M2139 clone), D3 (CIIC2 clone) and U1 epitopes (UL1 clone)hTNFtg animals was evaluated in ankle joints in a blinded manner using a semiquantitative arthritis score ranging from 0 to 4: 0: no arthritis ; 1: mild arthritis (joint swelling); 2: moderate joint swelling and digit deformation, reduced grip strength; 3: severe joint swelling and digit deformation, impaired movement, no grip strength; 4: severe joint swelling and digit deformation, impaired movement, no grip strength and obvious weight loss cachexia.Arthritis in 68. Briefly, arthritis was evaluated in a blinded manner for each of hindpaw and forepaw, according to the following scale: 0 for normal appearance, 1 for swelling and/or redness in one joint, 2 for swelling and/or redness in more than one joint, 3 for swelling and/or redness in the entire paw, and 4 for maximal swelling. The final score value is the sum of the scores obtained from the four limbs of each mouse.CAIA clinical evaluation was performed as previously described69 , Toluidine Blue or Tartrate-Resistance Acid Phosphatase (TRAP) Kit [Sigma-Aldrich]. H&E-, TB- and TRAP-stained joint sections were semiquantitatively blindly evaluated for the following parameters: synovial inflammation/hyperplasia , cartilage erosion , and bone loss  based on an adjusted, previously described methodTUNEL was performed in tissue sections according to manufacturer's instructions . The slides were deparaffinized, rehydrated and permeabilized with 20 μg/ml proteinase K for 25 min at room temperature. The TdT-mediated labelling reaction was performed for 60 min at 37 °C using fluorescent-labeled nucleotides. DAPI  was used to stain the nuclei. Quantitation of TUNEL+ cells were performed with the ImageJ software.Cleaved caspase-3 detection was performed according to Ab manufacturer's instructions . Briefly, the slides were deparaffinized and rehydrated. Antigen unmasking was performed in 1× Citrate Buffer, 45 min, at 90 °C. Upon endogenous peroxidase quenching and blocking, the incubation with the Ab was performed overnight, and visualization was performed using the ABC and DAB Kit .70. Briefly, scanning was conducted at 50 kV, 100 mA using a 0.5-mm aluminum filter, at a resolution of 6 mm/pixel. Reconstruction of sections was achieved using the NRECON software (Bruker) with beam hardening correction set to 40%. The analysis was performed on a spherical volume of interest (diameter 0.53 mm) within 62 slides of the trabecular region of calcaneus. Morphometric quantification of trabecular bone indices such as trabecular bone volume fraction (BV/TV%), bone surface density (BS/TV%), trabecular number  and trabecular separation  were performed using the CT analyser program (Bruker).Microcomputed tomography (mCT) of excised joints was carried out by a SkyScan 1172 CT scanner  following the general guidelines used for assessment of bone microarchitecture in rodents using mCTIkk2−/− MEFs were previously described71. Primary mouse SFs were isolated from mice with the indicated genotypes and cultured for four passages as previously described72. For Cre transduction experiments, 5 × 105 cells/well (from passage 2) were plated on a 6-well plate, washed with PBS and incubated for 16 h in 1:1 filtered mixture of protein-free Dulbecco’s modified Eagle’s medium (DMEM) and PBS containing 3 μM TAT-Cre . After transduction, cells were washed and cultured at least 2 days in normal DMEM/10% foetal bovine serum, and transduced cultures were further expanded for 3 more passages.WT and 19 was performed for 3 h before addition of other reagents. zVAD , Nec-1 , Nec1s  and GW806742X  were used in concentrations of 20, 30, 30 and 0.5 μM, respectively. TNF cytotoxicity assays were performed in 96-well plates, terminated 18 h post TNF addition and evaluated upon crystal violet staining, solubilization in 33% acetic acid solution and determination of optical density at 570 nm as a test filter and 630 nm as a reference filter.In experiments with the IKK2 inhibitor, pretreatment of cells with ML120B (10μM)4 cells/well (5–7 wells/genotype), plated in 96-well plates, were incubated for 24 h and then they were treated with MTT . Plates were incubated for 5 h in the CO2 incubator and the MTT-induced formed formazan is then solubilized in DMSO, and the concentration was determined by optical density at 540 nm.For MTT assay, briefly, 10Flow cytometric analysis was performed in cells stained with the following Abs: CD90.2 , VCAM-1 , CD45 , ICAM-1 , and CD11b  using the FlowJo analysis software.Synovial tissues were obtained from patients diagnosed with OA or RA undergoing joint replacement surgery at the Schulthess Clinic Zurich. RA patients fulfilled the American Rheumatism Association 1987 revised criteria for the classification of RA. Patient’s characteristics are summarized in Supplementary Table SFs were isolated by digestion with dispase  and cultured in supplemented DMEM. All reagents were purchased from Life Technologies.4 in 96-well plates. After 24 h, cells were treated for 24 h with 10 ng/ml recombinant human TNF-alpha , 50 µM InSolution™ MG-132 , 20 µM zVAD, 30 µM Nec-1 and/or 30 µM Nec-1s in duplicates. The evaluation of viability was performed as described for murine SFs.SFs were seeded at a density of 1.5 × 10Samples were collected in RIPA buffer, containing 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 150 mM NaCl, 10 mM Tris HCl, pH 7.4, 1 mM EDTA, protease inhibitors [Roche] and phosphatase inhibitors [Sigma-Aldrich], separated by SDS/polyacrylamide gel electrophoresis (8–12.5%), transferred to nitrocellulose membranes (Millipore) and probed with the following Abs: IKK2 , pJNK1/2 , cleaved caspase-3 , PARP-1 , SOD2 , FLIP , XIAP  [Cell Signaling]; ikB , tubulin , actin  ; XIAP , cIAP1 , Ripk3  [ENZO]; Mlkl ; and pMlkl . Relative quantitation of blots was performed either with the ImageLab (BioRad) or Gene Sys (SYNGENE) software.For human samples, probing were performed with the following Abs: tubulin , MLKL , and RIP3 . Relative quantitation has been performed with the ImageJ software.t-test  was used for evaluation of statistical significance (SigmaPlot 11 and GraphPad 6 software). P-values < 0.05 were considered significant.Data are presented as mean ± SE. Student’s Experiments with mice were performed in the conventional unit of the animal facilities in Biomedical Sciences Research Center (BSRC) “Alexander Fleming” under specific pathogen–free conditions, in accordance with the guidance of the Institutional Animal Care and Use Committee of BSRC “Alexander Fleming” and in conjunction with the Veterinary Service Management of the Hellenic Republic Prefecture of Attika/Greece. Experiments were monitored and reviewed throughout its duration by the respective Animal Welfare Body for compliance with the permission granted. Experiments with human SFs were approved by the Swiss Ethical Commission and patients signed an informed consent form.The data that support the findings of this study are available from the corresponding authors upon reasonable request.Supplementary InformationPeer Review File


Heart Failure with preserved Ejection Fraction (HFpEF) represents a major public health problem. The causative mechanisms are multifactorial and there are no effective treatments for HFpEF, partially attributable to the lack of well-established HFpEF animal models. We established a feline HFpEF model induced by slow-progressive pressure overload. Male domestic short hair cats (n = 20), underwent either sham procedures (n = 8) or aortic constriction (n = 12) with a customized pre-shaped band. Pulmonary function, gas exchange, and invasive hemodynamics were measured at 4-months post-banding. In banded cats, echocardiography at 4-months revealed concentric left ventricular (LV) hypertrophy, left atrial (LA) enlargement and dysfunction, and LV diastolic dysfunction with preserved systolic function, which subsequently led to elevated LV end-diastolic pressures and pulmonary hypertension. Furthermore, LV diastolic dysfunction was associated with increased LV fibrosis, cardiomyocyte hypertrophy, elevated NT-proBNP plasma levels, fluid and protein loss in pulmonary interstitium, impaired lung expansion, and alveolar-capillary membrane thickening. We report for the first time in HFpEF perivascular fluid cuff formation around extra-alveolar vessels with decreased respiratory compliance. Ultimately, these cardiopulmonary abnormalities resulted in impaired oxygenation. Our findings support the idea that this model can be used for testing novel therapeutic strategies to treat the ever growing HFpEF population. Heart Failure (HF) is the consequence of many forms of CVD and is a major public health problem. Corresponding total direct costs for HF are predicted to increase from $21 billion in 2012 to $53 billion by 20303. Novel therapies to reduce the morbidity and mortality of CVD and HF are clearly needed.Cardiovascular disease (CVD) is the leading cause of death worldwide, with almost 20% of deaths being cardiac related. By 2030, almost 41% of the US population will be affected by some form of cardiovascular disease4. HFpEF accounts for about 50% of all cases of HF6 and its prevalence relative to HFrEF is growing by 10% per decade3. The underlying pathophysiological initiators as well as factors that drive the progression of HFrEF are well-characterized7, in part because there are well-established animal models that adequately mimic the human condition and have reliably predicted the effects of therapeutics that are now known to improve outcomes of HFrEF patients.HF is classified into three main categories, HF with reduced ejection fraction (HFrEF), HF with mid-range ejection fraction (HFmrEF), or HF with preserved ejection fraction (HFpEF)8. However, the effects of neurohumoral inhibition in HFpEF have consistently failed to reach positive primary outcomes15. The fact that therapies for HFrEF are not effective in HFpEF patients likely reflects the distinct differences in the pathophysiological mechanisms of the two diseases16. What we do know is that the fundamental cardiac pathophysiology of HFpEF is complex and not well established, and truly representative experimental HFpEF models that capture the complexity of this disease have not yet been adequately established17. Our view is that the lack of well-established HFpEF animal models has limited the determination of HFpEF therapeutic targets and the preclinical testing of novel therapeutics18. There is clearly a need for new HFpEF therapeutics. HFpEF related mortality has not changed over the past 3 decades, with 5-year survival rates as poor as 50%20. While rodent HFpEF models have contributed to our understanding of underlying mechanisms of LV diastolic dysfunction, rodents have inherent limitations due to their size, cardiac structure and function that preclude comprehensive hemodynamic assessments that can be performed in large mammalians and humans. In addition, most of these models develop dilated phenotypes which are more representative of HFrEF than HFpEF. This includes most mouse models of transverse aortic constriction (TAC)21. Canine23 and swine25 models have revealed important pathophysiological aspects of HFpEF and might be useful, but the majority of preclinical HFpEF models have focused on cardiac function and structure, without characterizing cardiopulmonary interaction, which is a critical component of the HFpEF phenotype.Large clinical outcome trials in HFrEF patients have proven the efficacy of neurohumoral inhibition by significantly decreasing mortality29. Furthermore, PH in the early stages of HF has recently become a target of novel therapeutic strategies30. Although PH in HF patients is a common and life-threatening complication associated with a poor prognosis, it remains widely underestimated in clinical cardiology31. The understanding of PH in HFpEF is still in its early stages. For example, changes in lung parenchyma and lung mechanics that could limit functional capacity and quality of life have not been systematically studied. Many underlying concepts in this field come from observational studies and in vivo human data characterizing pulmonary hemodynamics33. Hoeper et al. have suggested that deep phenotyping of animal models with HFpEF cardiopulmonary features will advance the field34.Dyspnea, particularly during exercise, is one of the cardinal symptoms in HFpEF patients and significantly limits their functional capacity and quality of life. Pulmonary hypertension (PH) is found in 36–83% of HFpEF patients and is strongly associated with morbidity and mortalityTherefore, the aim of this study was to characterize a feline model of slow-progressive pressure overload to determine if it has critical structural and functional cardiac phenotypic features of HFpEF, and for the first time determine if this model has a HFpEF pulmonary phenotype that includes changes in lung mechanics, gas exchange and hemodynamics. These studies should reveal new mechanistic insights into the development of pulmonary impairment due to elevated left-sided filling pressures and establish this as a suitable model for studying HFpEF mechanisms and testing new therapeutics.The aortic banding/sham procedure was performed in 2-month-old male kittens following baseline (BL) echocardiography (ECHO), electrocardiogram (ECG) and collection of blood samples. Animals were studied for 4-months post-banding with serial ECHOs and ECGs performed at 1, 2, 3 and 4-months   hypertrophy, starting at 1-month post-banding and continued to progress throughout the study Fig. . Global 01) Fig.  and left01) Fig. . Global 01) Fig. . Represe01) Fig.  and LA e01) Fig.  at 4-mon01) Fig. .Figure 135, revealed an increase in banded cats compared to sham cats  Fig.  and tissats Fig. . E/e′ plats Fig. . All banmax) was significantly higher in banded cats compared to sham cats  versus sham cats  , cardiac output (CO), pulmonary and cardiac pressures were recorded. Then, functional stress testing was performed by intravenous infusion of dobutamine (5 µg/kg/min). First, we utilized fractional flow reserve (FFR) techniques to measure pressure gradients across the aortic band. The mean systolic aortic pressure gradient was 52 ± 12 mmHg  Fig. . Banded 05) Fig.  and mean05) Fig.  compared05) Fig.  and dP/dced Fig.  in bandeced Fig. .Figure 22 vs. 278 ± 9µm2; p < 0.001)  Fig. , and ske01) Fig. . Represe01) Fig. . LateralES)  . At 4-months banded cats had significantly elevated NT-proBNP levels compared to sham cats  Fig. . NT-proB01) Fig. .2/FIO2 ratio   Fig.  and intr 2) Fig.  comparedinsp) was then adjusted to support a tidal volume of 6–8 mL/kg and target PaCO2 within 35–45 mmHg. Banded cats required slightly higher Pinsp (12.1 ± 0.9 cmH2O) compared to sham cats  to ensure sufficient ventilation. Under these conditions there was an inverse linear correlation between LVEDP and PaO2 , as well as between mPAP and PaO2    guidelines, this model meets all criteria necessary for a HFpEF diagnosis37, which causes an immediate and severe pressure overload, our approach allows the heart to slowly adjust to the increased afterload and does not result in LV dilation and systolic dysfunction. The slow-progressive overload model takes longer to develop but produces a phenotype that more closely mimics the human conditions that can lead to HPpEF. Rodent HFpEF models generally progress to HFrEF over time21, which means that HFpEF is just a transient stage in the development of HFrEF. In addition, myocyte properties of rodents, such as regulation of [Ca2+] are fundamentally different than in large mammals , making translation of results from preclinical HFpEF studies in rodents to humans unreliable43. In the model developed in the present study, functional (diastolic dysfunction) and structural  cardiac changes post-aortic banding progressed slowly over time without developing LV dilation.We performed transverse aortic constriction in kittens using a customized pre-shaped band that caused slow-progressive pressure overload as the animals matured. In contrast to most available transverse aortic constriction (TAC) models44. Recently, Zakeri et al. reported that diminished LA compliance and reduced contractile function compromise overall cardiac performance in a hypertensive canine model of HFpEF45. The same group showed that in human patients with HFpEF, reduced global LA EF and increased LA volume were associated with an increased risk of death46. Consistent with these findings, we observed remarkable LA enlargement and reduction in LA EF in banded cats. LA EF was positively correlated with respiratory compliance and PaO2. ECHO revealed impaired diastolic function after aortic banding, reflected in abnormal transmitral inflow pattern (E/A ratio) and increased E/e′ ratio, a surrogate for elevated LV filling pressures47.Echocardiographic and hemodynamic features are the most important components for a HFpEF diagnosis, both of which can be performed in a comprehensive and highly translational fashion in cats. Banded cats developed concentric LV hypertrophy without LV dilation, LA enlargement and dysfunction and LV diastolic impairment, all of which resemble key echocardiographic findings seen in human HFpEF patientsmax) were comparable between groups at baseline. Interestingly, after dobutamine infusion the increase in dP/dtmax was attenuated in banded cats, supporting characteristics of an impaired systolic reserve. Several studies have reported that patients with HFpEF, despite having preserved ejection fraction, display subtle but significant abnormalities in systolic function at rest when utilizing more sensitive and load-independent techniques50. The subtle impairments in resting systolic function become dramatic during physiological stress52. Limitation in systolic reserve also affects diastolic function, because recoil and suction forces during early diastole are attenuated53. Even though dP/dtmax varies directly with preload, it is independent of afterload54, thus the blunted increase in dP/dtmax after dobutamine cannot be explained by a higher afterload in banded cats.Indices of LV systolic function  at rest, which is defined as the inability to fill the LV to an adequate volume at acceptable low pressures55. Elevated LV filling pressures are the ultimate downstream expression of LV diastolic dysfunction and provide sufficient evidence to secure a HFpEF diagnosis in symptomatic patients56. Additionally, prolongation of τ is a common finding in human HFpEF patients58. The observed delay in LV isovolumic relaxation in banded cats  will limit LV filling, impair diastolic suction, and increase end-diastolic pressures because relaxation remains incomplete (t-dia/ τ < 3.5)59. In the present study, T-dia/ τ - ratio in banded cats was 3.8, arguing against incomplete relaxation. However, Schwarzl et al. showed in a very elegant study using healthy pigs that incomplete relaxation occurred at ratios clearly higher than 3.560. Isovolumic active relaxation is related to myofilament dissociation and ATP-dependent calcium reuptake53 and is a sensitive marker for myocardial ischemia61. Therefore, coronary microvascular dysfunction could have caused prolongation of τ in banded cats with significant LV hypertrophy.Banded cats showed significant diastolic dysfunction  and higher respiratory rates, particularly after physical exertion. As an alternative to controlled exercise, we used pharmacologic stress testing. Dobutamine infusion did not cause the hemodynamic profiles in banded cats to decline. LVEDP and mPAP did not change and active relaxation improved in response to dobutamine, which is not surprising since ß-adrenoreceptor signaling accelerates relaxation and therefore counteracts acquired diastolic dysfunction63. Our findings are also consistent with the results of a hemodynamic study performed in human HFpEF patients. Penicka et al. reported a decrease in both LVEDP and τ, and an increase in dP/dtmin after dobutamine infusion, while performing hand-grip exercises increased LVEDP and caused a prolongation of τ64.Exercise intolerance is one of the hallmark clinical features of HFpEF and is attributed to impairments in systolic and diastolic reserve, chronotropic incompetence and peripheral autonomic and vascular dysfunction65 and a result of fibroblast activity and extracellular matrix remodeling66. We consistently found that both lateral and septal LV walls of the banded group had a higher percentage of fibrosis compared to the sham group. Interestingly, the fibrosis was diffuse, and more pronounced in the lateral free wall compared to septal wall in banded cats. This difference in fibrosis between the LV walls in HFpEF has not to our knowledge been reported elsewhere. Furthermore, the LV fibrosis was more pronounced in the subendocardium vs the subepicardium. A similar pattern was observed in dogs with LV hypertrophy and failure induced by chronic pressure-overload and related to a limited subendocardial coronary reserve67 and latent ischemia during adrenergic stimulation68. In line with that, isovolumic relaxation is prolonged in banded cats, which could relate to less potential recoil energy stored in the damaged matrix of the subendocardium during systole69. It is likely that latent myocardial ischemia during pressure-overload is a major trigger for primarily subendocardial fibrosis also in our HFPEF model. We speculate that LV fibrosis in HFpEF might evolve with a subendo- to subepicardial gradient, similar to the wavefront of myocardial infarction during acute coronary occlusion70. NT-proBNP, a surrogate marker for increases in atrial and ventricular size as well as cardiomyocyte stretch, has been validated in cats and is released in proportion to the degree of stretch and stress on the myocardium71. The ESC defined elevated natriuretic peptides  as a criterion to secure a HFpEF diagnosis4. Due to the high LV filling pressures and significant LA enlargement in banded cats, it was not surprising that we found elevated NT-proBNP levels at 4-months post-banding, which further correlated positively with LA size. Oyama et al. reported that in cats with respiratory symptoms, plasma NT-proBNP levels > 270 pmol/L are suggestive of HF as the likely cause with high sensitivity and specificity71. Hypertensive heart disease (HDD) without heart failure and HFpEF share common features, such as LVH and diastolic dysfunction, but there are also distinct differences between these two entities. Zile et al. reported that HFpEF patients had increased LVEDP, LA volume, NT-proBNP levels, and myocardial passive stiffness compared to both control and HDD without HF patients. In comparison to control patients, HDD without HF patients had no change in myocardial passive stiffness or LVEDP. The authors also showed that NT-proBNP levels were elevated in HDD compared to controls and these levels were further increased in HFpEF patients72. A recent study73 in hypertensive cats reported NT-proBNP levels of 236 pmol/L, which were measured by the same commercial laboratory that we used. Our HFpEF cats had higher NT-proBNP levels, which is consistent with the findings reported by Zile et al.72. We did not perform passive stiffness experiments, but all our banded cats had increased LV filling pressures. Speckle-tracking based longitudinal strain has been shown to discriminate between HFpEF and hypertensive patients with diastolic dysfunction but no HF. In comparison with hypertensive patients, HFpEF patients had significantly lower longitudinal strain values74. Unfortunately, STE analyses in cats are not well established and poorly validated.HFpEF is associated with interstitial fibrosis, which is mostly reactivein vivo human pulmonary hemodynamic studies. Furthermore, histological data supporting vascular remodeling in HFpEF is lacking33. In this study, we present a pre-clinical model of HFpEF with PH that allowed us to assess the structural and functional pulmonary changes as a result of high left-sided filling pressures and provides new mechanistic insights about PH in HFpEF. Our concept of pathobiological and functional abnormalities is displayed in Fig. 30. The observed increase in wet/dry lung weight ratio and the elevated protein content following aortic banding are suggestive of capillary endothelial disruption and impaired permeability due to increased hydrostatic pressure with subsequent interstitial edema and protein loss from the vasculature into the lung tissue proper. These reversible changes have been described in Ipc-PH and are summarized under the term ‘alveolar-capillary stress failure’31. In addition, we found extra-alveolar vessel abnormalities including perivascular fluid cuff formation, impaired expansion of the lung (decreased expansion index), smaller alveolar areas, and thickening of alveolar-capillary membrane in banded cats. To the best of our knowledge, this is the first time perivascular cuff formation has been described in HFpEF PH. Lowe et al. reported perivascular fluid cuff formation along extra-alveolar vessels without alveolar flooding or blood gas abnormalities in a lung injury model with thapsigargin. The authors showed that perivascular fluid cuffs reduced lung compliance by increasing tissue resistance and impairing mechanical coupling between bronchovascular bundle and the lung parenchyma75. Consistent with these findings, we also observed cuff formation and stiffening of the lungs. Finally, these changes led to an increase in intrapulmonary shunt fraction and impaired oxygenation.Largely due to limitations of current available HFpEF animal models, current knowledge of pulmonary hypertension (PH) in HFpEF is derived from et al. there is an imbalance between the urgent need to treat PH-HFpEF safely and effectively and a lack of conclusive scientific evidence supporting its presence. Collectively, our findings support the translational efficacy of this animal model and support its use as a platform to test novel therapeutic strategies to treat the ever growing HFpEF population.The present experiments showed that slow-progressive pressure overload of the LV caused LV concentric hypertrophy, diastolic dysfunction with elevated LV filling pressures, and LA dilation with reduced LA systolic function. These are critical features of HFpEF. In addition, our experiments document that the HFpEF phenotype included early stages of PH (Ipc-PH) with endothelial stress failure affecting not only the alveolar-capillary membrane but also extra-alveolar vessels, which subsequently causes significant pulmonary functional impairments with impaired oxygenation and respiratory symptoms. Since most of the pulmonary changes may likely be reversible at this stage, effective therapeutics could yield very promising outcomes. As highlighted by Hoeper 16. However, the cats in our study are still young (age 6 months) at the time they undergo hemodynamic testing, and except for hypertension, do not share other comorbidities and risk factors of human HFpEF patients. It is likely that in contrast to young cats, aged cats would have impaired cardiovascular reserve. Another limitation of the study is that once the aortic constriction surgery is performed, the band is in place for the entire duration of the study since it cannot easily be removed. Furthermore, the pericardial sack is dissected during both the sham and aortic constriction procedures, which could have potentially resulted in an underestimation of the left-sided filling pressures76.We did not collect data on pressure volume (PV) loops due to technical difficulties. PV catheters that are used for animals this size are 3 F and bigger. A 3 F catheter would very likely compromise aortic blood flow through the band and make comparison between sham and banded cats difficult. Therefore, we used a 2 F micro-tip pressure catheter  which is used for hemodynamic studies in rodents in order to minimize the effects on the hemodynamic study. RV function is known to be abnormal in HFpEF, but was not assessed is this study. Echo based imaging parameters, such as TAPSE, MAPSE, and speckle-tracking based strain analyses are used to assess RV and LV function, but are not well established and validated in cats. Invasive hemodynamics were performed in anesthetized cats in the supine position, which is an unusual posture for quadrupeds. Dobutamine stress testing does not perfectly replicate physical exercise, therefore results need to be interpreted with caution. Furthermore, it is well established that with normal aging LV stiffness increases, diastolic relaxation becomes compromised and diastolic suction is impaired, which may further compromise exercise tolerance in human HFpEF patientsAll animal procedures were approved by the Temple University Lewis Katz School of Medicine Institutional Animal Care and Use Committee and all experiments were performed in accordance with relevant guidelines and regulations. We utilized a total of 20 male short hair kittens, aged 2 months (1.3 kg), that underwent either aortic constriction (n = 12), with customized pre-shaped bands, or a sham procedure (n = 8). The pre-shaped band was placed around the ascending aorta and gently tied down without causing significant constriction of the aorta.Echocardiography (ECHO) was performed with a Vivid q Vet Premium BT'12 using a 12S-RS sector probe at baseline and 1, 2, 3, and 4-months post-surgery. Echocardiographic measurements were subsequently performed offline in a blinded fashion with EchoPAC SW v201.2O, PEEP = 3 cmH2O, respiratory rate = 16 breaths/min, FIO2 = 100%, I:E = 1/4.5) of time-cycled, volume-controlled, pressure-limited ventilation, independent of study group, and data generated from the integrated pulmonary mechanics module utilizing airway manometry and pneumotachography were recorded. The peak inspiratory pressure was then adjusted to support a tidal volume of 6–8 mL/kg. Arterial blood samples were analyzed and respiratory phase timing was adjusted to maintain PaCO2 within 35–45 mmHG, with all other ventilator settings remaining constant, and the animals were then fully instrumented . Trans-aortic band pressure gradients were measured invasively using the Fractional Flow Reserve (FFR) module . Pulmonary pressures were measured through the lumen of a thermodilution catheter (5 Fr). LV pressure was measured with a 2 F micro-tip pressure catheter  and cardiac output (CO) was determined using thermodilution methodology  Measurements were recorded at end-expiration with a steady PEEP of 3cmH2O. Afterwards, dobutamine was infused at 5 µg/kg/min and all measurements outlined above were performed again. Data were acquired and analyzed using Powerlab and LabChart Pro 8.1.5 . The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.At 4-months post-surgery, comprehensive hemodynamic studies were performed. In order to perform cardiopulmonary measurements under comparable intrathoracic pressure profiles and free of spontaneous respiratory efforts, anesthesia and paralysis were maintained with sodium pentobarbital (10 mg/kg/hr) and pancuronium bromide (0.1 mg/kg/hr), respectively. Animals were initially supported (Dräger Babylog 3000) with the same settings  with repeated measures on time. Treatment group means were compared vertically using the Dunn-Bonferroni procedure and horizontally with their respective baseline. Group comparisons for all other parameters were performed using the Mann-Whitney test. Statistical significance was accepted at p < 0.05.The data that support the images and plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.Supplementary Information


The antibodies also inhibited the binding of V2 peptide to the HIV-1 α4β7 integrin receptor. Antibody titers to MorHap, even with the presence of injected cyclic peptide, were very high, and resulted in inhibition of the hyper-locomotion and antinociception effects of injected heroin. From these initial experiments, we conclude that with a potent adjuvant and mostly synthetic constituents, a vaccine directed to heroin and HIV-1 (H2 vaccine) could be a feasible objective.A synthetic heroin analog (MorHap) and a synthetic 42 amino acid V2 loop peptide from A/E strain of HIV-1 gp120 envelope protein that was previously used in a successful phase III vaccine trial were constructed as antigens together with liposomes containing monophosphoryl lipid A as an adjuvant, to explore the feasibility of producing a dual use vaccine both for treatment of heroin addiction and prevention of HIV-1 infection among injection drug users. The V2 peptide was tethered by a palmitoyl fatty acyl tail embedded in the liposomal lipid bilayer, and the heroin analog was conjugated to tetanus toxoid as a carrier protein that was mixed with the adjuvant. Upon comparison of a linear V2 peptide with a cyclic peptide, differences were found in the secondary configurations by circular dichroism, with the tethered cyclic peptide  entirely in a random coil, and the tethered linear V2 peptide  entirely in a beta-sheet. Upon immunization of mice, palm-cyclic peptide induced anti-cyclic peptide endpoint titers >10 A vaccine designed to treat heroin addiction while at the same time preventing HIV infection elicited strong immune responses in mice. Scientists from the US government led by Carl Alving from the Walter Reed Army Institute of Research in Bethesda, Maryland, created a dual vaccine formulated with three main components: a segment of a protein expressed on the surface of HIV; synthetic molecules that resemble heroin and its degradation products; and a potent adjuvant to stimulate the immune system. Mice immunized with this vaccine had high antibody titers against the HIV surface protein as well as heroin and its derivatives. These mice also showed dulled responses to injected heroin. The findings suggest this vaccine strategy could help fight heroin abuse and the high risk of HIV infection among intravenous drug users. The rationale behind this approach mirrors the strong association between injection drug use and the risk for HIV-1 infection, and it highlights the possibility of additive societal and health benefits of creating a single combination heroin-HIV-1 (H2) vaccine that has twin goals for alleviation of the two diseases.Addiction to opioid drugs is a major source of morbidity and mortality worldwide, including the US, where it has become a national epidemic with an estimated 2.5 million adults afflicted in 2014.6 However, because of the extremely rapid degradation of heroin after injection, the bulk of euphoria is caused by 6-acetyl morphine and morphine, so although binding to heroin itself would be useful, it may not be absolutely necessary for achieving at least a high degree of vaccine efficacy.Among the challenges for such a heroin-HIV vaccine are included individual antigen selection for the heroin and HIV-1 arms. In the case of heroin, rapid degradation to 6-acetyl morphine and morphine occurs after injection of heroin, and antibodies ideally are desired that would exhibit type 2 cross-reactivity both with heroin itself and with the major degradation products.7 To date, the only phase III clinical efficacy trial to demonstrate efficacy remains the RV144 Thai trial.8 The RV144 trial resulted in 31.2% efficacy for preventing HIV-1 infection, which, although modest, was significant at the primary endpoint of 3.5 years. However, post hoc analysis revealed apparent efficacies of 60 and 44%, respectively, at 12 and 18 months.9 Subsequent immune correlate analysis of RV144 revealed that non-neutralizing antibodies that were induced to the V1V2 loop of the HIV-1 gp120 envelope protein were inversely correlated with the risk of HIV-1 infection.10 In the gp120 protein, the V2 loop is located at the apex of the protein and has at least two α4β7 integrin-binding sites that play an important role in viral entry into susceptible cells, and could be important for vaccine design.14 The V1V2 region of gp120 has multiple N-linked glycan sites and the α4β7 integrin-binding site presumably could be obscured and protected by a “glycan shield”.13 As V2 is likely a relatively protected site, antibodies to gp120 injected as a vaccine antigen might be predominantly directed to sites, such as the V3 region, that are more immunodominant.15For HIV-1, the exact immune response needed for efficacy and the optimal antigen required to achieve it are still unclear.17 For the HIV-1 arm, we examined two different antigens, consisting of either a synthetic linear (LV2) or a cyclic (CV2) 42 amino acid-free peptide, both of which were based on the V2 loop of an HIV-1 subtype AE (CRF01_AE) gp120 protein.18 The synthetic LV2 and CV2 peptides each contained palmitic acid at the N-terminus, resulting in palm-LV2 or palm-CV2. Antibodies to V2 from CRF01_AE HIV-1 in plasma samples obtained from the RV144 phase III Thai trial were previously detected by enzyme linked immunosorbent assay (ELISA) using linear and cyclic V2 peptides as capture antigens.18Based on the above considerations, in this study for the heroin arm, we included a synthetic hapten (MorHap) conjugated to tetanus toxoid that was predicted to display epitopes that would induce antibodies to heroin and particularly to its degradation products and inhibit the antinociception effects of heroin Fig. .5, 6, 1622 In this study, we incorporated either palm-LV2 or palm-CV2 as an immunogen into the surfaces of the ALF liposomal vaccine adjuvant particles, and we mixed the resultant particles together with the antigen consisting of MorHap conjugated to tetanus toxoid to make a multi-specific vaccine formulation  adjuvant (liposomes containing monophosphoryl lipid A (MPLA)) was used as a potent adjuvant that would be expected to induce high levels of specific antibodies. The ALF family of adjuvants has been used in numerous phase I and II human trials since 1992 with various types of antigens, and has exhibited considerable safety and effectiveness. In previous studies, both in the development of a vaccine to heroin, and for induction of antibodies to peptides and proteins, ALF has shown a high level of potency as an adjuvant when compared with aluminum salts or many other adjuvants. green) fluorescence outside the lipid bilayers (red).To visualize association of the V2 peptides with the ALF liposomal bilayer, palm-CV2 Fig.  or palm-In order to determine the secondary structural configurations of the peptides, circular dichroism (CD) analysis was performed with liposomal palm-CV2 Fig.  or liposImmunization with liposomal palm-CV2 induced antibodies to CV2 with an endpoint titer of 3,959,467 Fig. . The ant18 In RV144, antibodies to V2 bound more strongly to Scr flanks peptides than to Scr crown peptide.18 In the present study, antibodies to palm-CV2 bound both to the Scr flank and Scr crown peptide. In contrast, antibodies to palm-LV2 bound predominantly to peptide containing Scr crown  rather than to peptide containing Scr flank . Based on these data, it was concluded both that palm-CV2 induced antibodies that bound to both the crown and flank regions of intact V2, and that palm-CV2 was a stronger immunogen than palm-LV2. Because of this, further experiments used liposomal palm-CV2 as an immunogen to determine the effect, if any, of simultaneous immunization with non-liposomal TT-MorHap as a second antigen.In order to localize the specificities of induced anti-V2 peptide antibodies to regions of the intact V2 peptide, separate peptides were synthesized in which either the flank or the crown regions of intact V2 were scrambled .4β7 integrin receptor. Antibodies induced either by palm-CV2 or palm-CV2 plus TT-MorHap also strongly inhibited the α4β7 receptor  , or with TT-MorHap together with palm-CV2 (H2 vaccine), high titers of antibodies were obtained to MorHap, but the titers of H and H2 vaccine to MorHap were not statistically different from each other  Fig. . Althougly) Fig. .Fig. 5Inmiddle and right bottom panels are compared to the control left bottom panel). Inhibition of hyper-locomotion by each type of immunization was highly significant when compared to the controls . Immunization with TT-MorHap either alone (H vaccine) or TT-MorHap together with palm-CV2 (H2 vaccine) caused significant reduction of heroin-associated hyper-locomotion . This type of adjuvant has been successfully used previously for immunization with both heroin haptens and synthetic peptides.23 and hyper-locomotion after heroin injection.25 In each of the two types of assay, highly significant inhibition was observed with mice immunized either with TT-MorHap alone or with TT-MorHap+CV2, when compared with non-immunized animals ,als Fig. . Howeverals Fig. . In contals Fig. . The tai4β7 receptor that is present at the apex of the V2 loop of the HIV-1 gp120 envelope protein. The α4β7 receptor, which comprises at least two α4β7 integrin-binding sites,14 is closely associated with the CD4 HIV-1 binding site and is thought to play a role in HIV-1 infection.27 As noted in the Introduction, non-neutralizing antibodies to V1V2 were correlated with decreased risk of HIV-1 infection among vaccine recipients in the RV144 phase III trial,10 and this has brought attention to the potential importance of induction of antibodies to this region for vaccine design.13 Weakly neutralizing and non-neutralizing human monoclonal antibodies (mAbs) to the V2 loop were used as probes to study the interactions of anti-V2 antibodies with HIV-1, gp120 envelope protein and with the highly conserved V2 region, and its neighboring regions on gp120.28 Human anti-V2 mAbs derived from a volunteer vaccinee in RV144 bound both the free LV2 and CV2 peptides, including the conserved region of the α4β7 receptor, and inhibited the binding of HIV-1 to the receptor.29 Anti-V2 mAbs derived from RV144 also captured a fraction, but not all, infectious HIV-1 virions from multiple HIV-1 strains in an in vitro capture assay.30 Interestingly, combinations of different mAbs that bound to different regions of the HIV-1 gp160 envelope protein, such as mAbs binding both to V1/V2 on gp120 and to gp41, resulted in higher capture of infectious virions.30 This suggests that although antibodies to V2 might provide a useful degree of viral capture, future addition of multiple antigenic specificities to an H2 vaccine, such as addition of both V2 and gp41 epitopes, may result in the induction of antibodies that would provide better capture of infectious virions.For HIV-1, the CV2 peptide with a palmitoyl anchor in the surface of the ALF membrane induced antibodies that, both in the presence and in the absence of the heroin hapten-protein conjugate, resulted in strong inhibition of binding of HIV-1 to the α4β7 integrin receptor on CD4+T cells, as shown in this study, has been highlighted recently by the important observation that a monoclonal antibody to the α4β7 integrin receptor on SIV-infected cells, when combined with antiretroviral treatment, prevented rebound of suppressed SIV infection over a prolonged period, with no further treatment over the length of the study.31 The mechanism of this fascinating observation is currently unknown, but it would seem likely that inhibition or prevention by anti-α4β7 of initial homing of SIV to α4β7 on CD4+T cells in the mucosa was a major effect. Interestingly, the combined treatment of the infected macaques also resulted in increased titers of circulating antibodies to V2.31 Although the mechanism(s) for the increase in anti-V2 titers is unknown, it seems reasonable to assume that preservation of CD4+T cells from infection by SIV must have played a role. One could even speculate that blocking by anti-α4β7 antibodies of the binding of V2 of the SIV gp120 to α4β7 might have led somehow to increased exposure of the V2 binding site, thus allowing greater induction of anti-V2 antibody levels.The potential importance of high titers of antibodies to V2, particularly with respect to the inhibition of binding of V2 to the αβ-sheet  were greater than any of the inhibitions of binding of CV2 previously observed by us with human mAbs CH58, CH59, or 2158, or with individual plasmas obtained from infected patients in the RV144 study.14In the present study, distinctive secondary conformational differences based on CD analysis were observed between palm-CV2 and palm-LV2 upon association with liposomes, in that palm-CV2 was entirely in a random coil and the palm-LV2 was entirely in a eet Fig. . The relrin Fig. . It shou4β7 on CD4+T cells in the mucosa, ADCC induction by NK cells, or even through induction of neutralizing antibodies. In the present study, we have demonstrated that extremely high levels of antibodies to V2 that inhibit binding of HIV-1 to the α4β7 receptor can be achieved by direct attachment of the CV2 peptide to the highly potent ALF adjuvant. Thus, the feasibility and potential practicability has been demonstrated of combining a CV2 epitope together with a candidate heroin hapten along with a potent adjuvant formulation to achieve high levels of antibodies with small epitopes in a mostly synthetic H2 vaccine formulation. The resultant antibodies mitigated the effects of heroin injection and also exhibited high levels of functional antibodies to the V2 loop.The route to developing a maximal protective vaccine to HIV-1 is still not fully understood. The studies with mAbs suggest that anti-V2 loop antibodies may contribute at least partially to protective efficacy through a variety of mechanisms, possibly including inhibition of viral binding to αFrom a larger HIV-1 vaccine perspective, it might seem useful to include immunization with a V2 peptide as a complementary modality to induce high antibody titers to V2 that might contribute to protection, together with a protein antigen that might enlist additional protective mechanisms. Further studies will examine the addition of other haptens and peptides, and an HIV-1 protein to determine whether mixture with other antigenic epitopes could prove useful for additional additive or synergistic effects. However, in the present context, the H2 combination of a heroin vaccine with a simultaneous vaccine to the V2 loop of HIV-1 would treat the detrimental effects of injection drug use and might reduce the possibility of HIV-1 infection.18 The resin was treated with either palmitic acid N-hydroxysuccinimide ester  or biotin N-hydroxysuccinimide ester (for ELISA reagents) after the completion of the peptide synthesis. Palm-CV2, palm-LV2 and other peptides were cleaved from the resin using Reagent K and purified by reversed-phase high-performance liquid chromatography and characterized by MALDI-TOF MS. Palm-V2-OG conjugates were prepared using Oregon Green® 488 (OG) carboxylic acid succinimidyl ester .Peptides were synthesized on an Applied Biosystems 433 A peptide synthesizer using Fmoc chemistry. The two terminal cysteines were replaced with alanines to make the LV2 sequence.16 TT-MorHap conjugates with 33–37 haptens were obtained by treating TT with 1600-fold excess, on a molar basis, of the heterobifunctional cross-linker.TT-MorHap conjugate was synthesized, purified and characterized as previously described.®; Avanti Polar Lipids, Alabaster, Alabama, USA) (400 μg/mL) were dissolved in lipid solvents as described earlier.5 To this were added either palm-CV2 or palm-LV2 peptide (400 μg/mL) in chloroform/methanol. The formulation for the HIV leg alone (50 μL dose) contained 50 mM phospholipid, 20 μg palm-V2 peptide and 20 μg MPLA in Dulbecco's phosphate buffered saline (DPBS), pH 7.4. For the heroin/HIV combination, preformed ALF was mixed 1:1 with the TT-MorHap conjugate. The heroin/HIV vaccine (50 μL dose) contained 10 μg of TT-MorHap, 20 μg palm-CV2 peptide, and the same amounts of phospholipids and MPLA as the HIV formulation.ALF, consisting of dimyristoyl phosphatidylcholine: cholesterol:dimyristoyl phosphatidyglycerol (9:7.5:1 molar ratio), and synthetic MPLA  were purchased from Jackson Laboratories . Two sequential independent animal studies were conducted to assess the immunogenicity of the vaccine formulations: (1) comparison of palm-LV2 and palm-CV2 (6 mice/group), and (2) comparison of palm-CV2, TT-MorHap and palm-CV2+TT-MorHap (10 mice/group). Mice were immunized IM with 50 μL doses of the vaccines at weeks 0, 3, and 6 in alternating rear thighs. For the first animal study, six mice/group were terminally bled at week 9. For the second animal study, ten mice/group were bled at week 8, with a terminal bleed at week 15.16 The estimated lethal dose of heroin in humans is 50 mg (range 12–180 mg).33 Assuming a 70 kg person, this corresponds to 0.7 mg/kg. The dose of injected heroin used in the current mouse studies is thus in-line with the toxic dose of heroin in humans.The TT-MorHap and palm-CV2+TT-MorHap groups of mice were challenged with heroin  at week 10.Liposomes were labeled with Texas Red-DHPE  (Thermo Fisher Scientific) and the Palm-V2-OR peptides were added as 0.16% (8.0 μM). For confocal fluorescence microscopy, the total phospholipid concentration was 5 mM. Images were taken with a Zeiss LSM 710 confocal microscope  equipped with excitation laser lines at 405, 458, 488, 514, 561, and 633 nm. Images were taken using a 63×/1.4 Oil DIC Plan Apo. The liposome-containing peptide formulations were placed in a CoverWell™ perfusion chamber gasket (Thermo Fisher Scientific), and palm-CV2 and palm-LV2 peptides and Texas Red-DHPE were observed with fluorescein and rhodamine filters, respectively. All images were stored and processed with the Zeiss Zen 2012 Lite software. Green fluorescent, red fluorescent, and gray images are Oregon Green, Texas Red and DIC channels, respectively. Superimposition of the Oregon Green and Texas Red channels yield the overlay images.34CD experiments were performed under three conditions: liposomes ; 1% SDS in DPBS; and 50% TFE in DPBS (1%). The experiments were performed using 400 μg/mL of liposomal peptide, and 50 μM peptide in 1% SDS or 50% TFE, at 25 °C in a Hellma quartz cell (1 mm path length). Data points were recorded between 195 and 250 nm in 0.5 nm increments on a JASCO J-815 CD Spectrometer . Spectra were collected using scan speed of 10 nm/min and a band-pass of 1 nm. For each peptide condition assayed, three scans were recorded, averaged, and corrected for appropriate blanks. For CD of palm-V2 peptides in liposomes, the CD data of control empty liposomes was analyzed at 2 mM liposomal phospholipid concentration and subtracted from the sample data. The % secondary structure of the peptide was calculated by fitting the CD data through CDFIT Program.17 V2, A244 gp70-V1V2, and gp120 ELISAs were performed as described previously,18 with modifications, as summarized in Table SMorHap ELISA was performed with 100 uL (1 μg/mL) of coating antigen.14 Sera from animals immunized with palm-CV2 and palm-LV2 were evaluated at week 9, while sera from animals immunized with palm-CV2 together with TT-MorHap were evaluated at week 15, for inhibition of binding of CV2 peptide to α4β7 on RPMI 8866 cells.Serum samples pre-immunization (pooled) or post-immunization  at a dilution of 1:50 in sample buffer was examined as previously described.16 For the locomotion test, the same mice from the tail flick test were placed in a black chamber (11 inches × 11 inches) with an EverFocus® Polestar II camera  mounted above (56 inches from the chamber), for 5 min. The locomotion of the mice was processed by EthoVision® XT software . The baseline responses prior to heroin injection were measured on the tail flick followed by the locomotion test, after which the animals were injected SC with 1 mg/kg of heroin HCl in saline (400 μg/mL) between the front shoulders. Thirty minutes after injection, the tail flick responses were measured and the motions of the mice inside the box were tracked for 5 min. The distance of the tracks were recorded at 0.5 min interval for 5 min and the distance travelled at a given time was calculated using the formula:Mice were challenged with heroin at week 10. Tail flick test was performed as previously described.In each case, the distance travelled prior to heroin injection was subtracted as a baseline from the distance travelled after heroin injection.Supplementary information


Rousettus and Eonycteris spp. bats in China exhibited 61%–99% nt identity with reported filoviruses, based on partial replicase sequences, and they demonstrated lung tropism. Co-infection with 4 different filoviruses was found in 1 bat. These results demonstrate that fruit bats are key reservoirs of filoviruses.Genetically divergent filoviruses detected in  Filoviridae) are nonsegmented, negative-strand RNA viruses belonging to 3 genera: Marburgvirus, Ebolavirus, and Cuevavirus. Marburgvirus comprises 1 species, Marburg marburgvirus, which includes Marburg virus (MARV) and Ravn virus. Ebolavirus comprises 5 species, Zaire ebolavirus (ZEBOV), Sudan ebolavirus, Bundibugyo ebolavirus, Taï Forest ebolavirus, and Reston virus (RESTV). Cuevavirus comprises 1 species, Lloviu cuevavirus  in China ; the specimens comprised 10 (23.3%) of 43 E. spelaea and Rousettus sp. collected in 2009 and 5 (11.9%) of 42 collected in 2015. Using qPCR, we detected filovirus RNA in 20 specimens from E. spelaea (n = 4) and Rousettus sp. (n = 16) bats: 10 (23.3%) of the bats were collected in 2009 and 10 (23.8%) in 2015. No filovirus was detected in other bat species studied . Results showed that filoviruses were mainly located in the lung and that genome copy numbers ranged from 29 to 523,582/mg of tissue . Only 2 Escherichia coli and used them as antigens . In this experiment, we used 25 bat samples from 2015 that had enough serum volume for testing; 14 samples showed a strong cross-reaction with the ZEBOV nucleoprotein, and among them, 7 showed a weak cross-reaction with RESTV nucleoprotein. We used Western blotting to confirm these results; 11 of the 25 samples were positive for ZEBOV nucleoprotein and 4 for RESTV nucleoprotein  cross-reactive with ZEBOV nucleoprotein or that nucleoproteins of the novel filoviruses were cross-reactive with ZEBOV and RESTV nucleoproteins.We detected novel filovirus sequences with high divergence in The bat samples in this study were collected from 2 caves in 2009 and 2015, respectively; the caves are ≈200 km metric apart. Across the 2 different years and locations, we detected closely related viruses and found 1 bat that was acutely co-infected by 4 different filoviruses; this finding suggests that these viruses have been circulating in the 2 bat species and that densely populated bat caves provide opportunity for cross-infection with different viruses. However, considering the migration ability of the fruit bat, we cannot exclude the possibility that there are exchanges of virus between the bats in these two caves. Longitudinal surveillance with tracking tags may help to better understand the spatial–temporal distribution of these viruses in bat populations.,In previous reports, filoviruses were primarily detected in liver and spleen tissues (These results will be helpful in providing a better understanding of the distribution and diversity of filoviruses, which may have implications for public health. Considering their feeding habitats, fruit bats are often in close contact with domestic animals and human populations. It is therefore necessary to establish long-term and proactive surveillance of these viruses and related diseases.Rousettus and Eonycteris spp. bats, China, 2009 and 2015.Detailed methods and primer sequences used in a study of genetically diverse filoviruses in