gingivalis Mitomycin C solubility dmso [64]. Notably, P. gingivalis does not rely on immunological mechanisms for C5aR activation, since it can activate this complement receptor through C5a generated locally by its Arg-specific gingipains (HRgpA, RgpB) that have C5 convertase-like activity [64, 65]. Porphyromonas gingivalis also expresses a number of potent TLR2 ligands including serine lipids and lipoproteins [66, 67]. At the molecular level, the P. gingivalis-induced C5aR-TLR2 cross-talk in macrophages leads to synergistic activation of cAMP-dependent protein kinase A for inhibition of glycogen synthase kinase-3β and of iNOS-dependent

intracellular bacterial killing [64] (Fig. 3). In the murine periodontal tissue, C5aR signaling synergizes with TLR2 to induce secretion of cytokines that promote periodontal inflammation and bone loss (TNF, IL-1β, IL-6, and IL-17A). This is likely to enhance the fitness of P. gingivalis and other periodontitis-associated bacteria that require an inflammatory environment to secure critical nutrients, i.e. tissue breakdown products including peptides and hemin-derived iron. In stark contrast to the upregulation of bone-resorptive inflammatory cytokines, P. gingivalis-induced C5aR signaling in macrophages downregulates TLR2-induced MG-132 molecular weight IL-12 and hence inhibits IFN-γ production and cell-mediated immunity against the bacteria [63, 65]. The selective inhibition

of

bioactive IL-12 (IL-12p35/IL-12p40) associated with C5aR-TLR2 cross-talk involves ERK1/2 signaling-dependent suppression of the IFN regulatory factor-1 (IRF-1), a transcription Nintedanib (BIBF 1120) factor that is crucial for the regulation of IL-12 p35 and p40 mRNA expression [65, 68]. Importantly, genetic ablation of C5aR or TLR2 promotes the killing of P. gingivalis in vivo [64, 69]. The inhibitory ERK1/2 pathway that regulates TLR2-induced IL-12 is also activated when P. gingivalis binds complement receptor 3 (CR3) on macrophages [70, 71] (Fig. 3). CR3 is a β2 integrin (CD11b/CD18) that can bind ligands when its high-affinity conformation is transactivated via inside-out signaling by other receptors such as chemokine receptors. Porphyromonas gingivalis induces TLR2-mediated transactivation of CR3 through an inside-out pathway that involves RAC1, PI3K, and cytohesin-1 [72, 73] (see Fig. 3). Upon binding CR3, P. gingivalis not only downregulates IL-12 but also enters macrophages in a relatively safe way [74], perhaps because CR3 is not linked to strong microbicidal mechanisms such as those activated by FcγR-mediated phagocytosis [75]. Indeed, P. gingivalis can persist intracellularly in WT macrophages for longer times than in CR3-deficient macrophages [74]. As alluded to above, P. gingivalis can activate C5aR signaling independently of the canonical activation of complement [64, 65]. In fact, P.

[4-9] Hessell et al.[10] showed that an HIV-specific neutralizing R788 clinical trial antibody mutated in the Fc position was no longer able to elicit Fc-mediated functions, such as ADCC, and that the efficacy in preventing simian/human immunodeficiency virus (SHIV) infection of macaques was significantly decreased, suggesting that the ADCC function is important for the protection afforded by neutralizing antibodies. There is a more limited understanding of the role of ADCC in the small subset of HIV-infected subjects who naturally control chronic infection, although the role

of cytotoxic T lymphocytes and neutralizing antibodies has been extensively studied.[11-24] We previously detected ADCC-mediated NK-cell activation in a small cohort of six subjects with slow HIV progression, but found no clear correlation with the magnitude of the ADCC response and control of buy PD0325901 HIV. A recent study of 22 subjects indicated that elite controllers of HIV infection (subjects with consistent plasma HIV levels of < 50 copies/ml) have higher levels of ADCC antibodies than viraemic subjects, with an absence of correlation between cytotoxic T lymphocytes and neutralizing antibodies.[6] Whether these results are generalized across larger numbers of long-term slow-progressors

(LTSP) subjects is not clear. In addition, the HIV epitopes targeted by efficient ADCC are unknown but would logically be interesting vaccine targets. We analysed ADCC responses using an assay studying antibody-mediated interferon- γ (IFN-γ) and CD107a expression of NK cells. We

studied serum samples from 139 HIV-infected subjects not on anti-retroviral therapy; 65 subjects were LTSP who maintained a CD4 T-cell count of > 500/μl for at least 8 years after infection and the remaining 74 subjects were non-LTSP. We found that ADCC responses in LTSP subjects were broadly reactive against multiple HIV proteins and that LTSP subjects disproportionally targeted three specific ADCC epitopes within Vpu (viral protein U). The characteristics of the 139 subjects are shown in Table 1. All subjects were HIV-infected Atazanavir and not on anti-retroviral therapy at the time of sampling. Subjects enrolled in both cohorts provided written informed consent and the relevant human research ethics committees approved all studies. Subjects were recruited both through the Long-term non-progressor network co-ordinated by the Kirby Institute, Sydney, Australia and through the Melbourne Sexual Health Centre, Australia. Sixty-five of the subjects met the pre-defined criteria as LTSPs, being HIV-positive for more than 8 years without anti-retroviral therapy and maintaining a peripheral CD4+ T-cell count above 500 cells/μl. There were no viral load entry criteria. The remaining 74 subjects did not meet the criteria for LTSP (i.e. had not maintained CD4 T-cell counts > 500 cells/μl for 8 years). For both cohorts, serum for ADCC testing was derived from the earliest time-point available.

100 Three proteins (SP-2, SP-3 and SP-4) were found in higher concentrations in stallions with low fertility scores, while SP-1 was positively correlated with

fertility and was suggested to be homologous to OPN.95 The spermadhesin PSP-I, common in pigs, seems negatively related to fertility58, while other molecules, such as TGF-β, appear unrelated to overall fertility in relation to levels in semen.89 However, as the SP of a boar differs somehow from that of another boar, maybe it is not the amount of the cytokine that Seliciclib mouse play the major role, but its capacity to differentially induce degrees of maternal tolerance by the female and thus attain differences in embryo survival, leading to variation in fertility. It is hoped that this line of research is followed. Proteins of the seminal plasma are relevant for sperm function particularly

for their interactions with the various environments of the tubular genital tract and the oocyte and its vestments. Moreover, specific peptides and proteins act as signals for the immune system of the female, ultimately modulating sperm rejection https://www.selleckchem.com/products/LBH-589.html or tolerance, perhaps even influencing the relative intrinsic fertility of the male and/or couple. Funding has been provided by The Swedish Research Councils Vetenskapsrådet (VR) and FORMAS, Stockholm, Sweden; and BFU2010-17373, Valencia, Spain. “
“Recent progress achieved by an impressive number of studies focusing upon the ontogenesis and immunobiology of epidermal Langerhans cells (LCs) and other cutaneous dendritic cell (DC) populations as well as DCs at oral mucosal tissue has profoundly revised Mephenoxalone our understanding of the role of DCs in different tissues and microenvironments. By sensing their environment for microbial

signals or allergens and bridging innate and adaptive immunity in a sophisticated manner, subtypes of DCs play a critical role in the maintenance of the immunological homeostasis in the periphery. Thereby, DCs, located directly at the interface to the environment, fulfil opposing tasks as they are key players in both the control and the generation of allergic inflammation. Furthermore, it is under ongoing debate whether DCs attenuate or aggravate allergic inflammation. As a consequence, accumulated knowledge gained in this field within the last few years has provided an excellent basis for innovative therapeutic opportunities which tend to target specifically the multi-faceted properties of DCs at distinct anatomical sites. Since the discovery of the classical epidermal dendritic cells (DCs) by Paul Langerhans in 1863 [1], DCs have fascinated researchers all over the world, but still remained enigmatic due to their complex characteristics and roles in our immune system. However, all DC subtypes display a few common features, such as their localization at the border zones to the environment, which is associated directly with their pivotal role as sentinels of the immune system.

Laboratory data revealed that our patient did not express donor-specific antibody and the peritubular capillaries did not exhibit C4d immunoreactivity. Upon consideration of both histological and laboratory findings, we diagnosed acute vascular rejection of Banff 2007 class ACR IIA. We commenced 3-day sessions of intravenous steroid

pulse therapy twice weekly and adjusted the trough TAC level to 5–8 ng/mL by varying the TAC dose. We next performed an allograft biopsy and found no evidence of rejection (the S-Cr level was 2.7 mg/dL on April 1 2013). The present case report demonstrates the difficulties associated with management of TAC-based regimens in kidney transplant patients undergoing antituberculosis therapy. We also review the relevant literature. The proportion of HSP activation kidney allografts that is not rejected has improved dramatically in the era of the calcineurin inhibitor (CNI), but the use of such a strong immunosuppressant increases the risk of infection. Of the various possible infections, tuberculosis is particularly problematic because infection of transplant patients is associated with a higher incidence of mortality than noted this website in the general population. The same antituberculosis agents are recommended for use in both transplant patients and the general population.[1] Rifampicin (RFP) plays a key role in antituberculosis therapy, but the

trough CNI level requires close attention because it is frequently decreased by RFP use. A 29-year-old man was admitted to our hospital in June 2013 for a scheduled biopsy 1 year after primary kidney transplantation. He had been diagnosed with IgA nephropathy at the age of 17 years. He underwent peritoneal dialysis in June 2011. In June 2012, he received a live-donor kidney transplant from his father. The ABO blood types of donor and recipient were compatible, and the HLA alleles were haplo-identical. The standard complement-dependent cytotoxicity cross-match test was negative. Immunosuppressive therapy consisted of tacrolimus (TAC), mycophenolate

mofetil, methylprednisolone and basiliximab. The allograft exhibited excellent early function, associated with an S-Cr Bcl-w level of 1.2 mg/dL. The 1 year protocol biopsy revealed no evidence of rejection. However, our patient was diagnosed with lung tuberculosis. The QFT was positive and the chest CT findings typical of tuberculosis. Standard therapy with antituberculosis agents, consisting of isoniazid (INH) 300 mg, rifampin (RFP) 450 mg, ethambutol (EB) 500 mg and pyrazinamide (PZA) 1500 mg daily, commenced on 9 June 2012. Despite increasing the TAC dose (512 mg, daily) and frequent monitoring of the serum TAC trough level, the serum TAC level decreased gradually from 3.1 ng/dL on 7 July 2012 to 1.6 ng/dL on 1 October 2012.

[1] Microvesicles have protein content similar to the plasma membrane of activated platelets and have procoagulant and inflammatory functions.[79, 80] In contrast, platelet exosomes only interact poorly with annexin-V and do not bind prothrombin and factor X. Platelet-derived exosomes are enriched in CD63, a tetraspanin protein also found on exosomes from other cell types.[81] Tetraspanin proteins have been implicated in adhesive as well as co-stimulatory and signalling functions. Platelet-derived exosomes may be released at

sites of vascular injury and could well Rapamycin function in promotion of platelet and neutrophil adhesion.[1, 82] Endothelial dysfunction and vascular calcification is a significant risk factor for cardiovascular morbidity and mortality in patients with renal disease. In vitro, vesicles appear to be important in mediating vascular smooth muscle cell calcification.[83] In a recent study, it was found that phosphorylated fetuin-A is present in the calciprotein particles in serum of predialysis chronic kidney disease (CKD) patients. Increased calciprotein particle fetuin-A levels reflect an increasingly procalcific milieu and are associated with increased aortic stiffness.[84] Increased levels of circulating microparticles

(MP) or microvesicles check details have been detected in patients with CKD. Circulating levels of MP and microvesicles derived from endothelial cells correlate with arterial stiffness in haemodialysis

patients.[85-87] It is unclear whether exosomes and/or other circulating MP may play an important role in transporting or promoting vascular calcification in CKD or in other calcification-associated Selleckchem Ixazomib diseases. Nephrolithiasis is associated with the formation of calcium oxalate, calcium phosphate, cystine, struvite or urate crystals in the kidneys. In vitro studies have demonstrated that renal brush border-derived exosomes/microvesicles of ∼100 nm in diameter can induce and promote calcium oxalate crystallization in nephrolithiasis.[88] In transplantation, it has been shown that the exchange of exosomes between dendritic cells may constitute a potential mechanism by which passenger leukocytes transfer alloantigens to recipient antigen-presenting cells, leading to an increased generation of donor-reactive T cells.[89] On the other hand, other studies have found that dendritic cell-derived exosomes may induce tolerance rather than immune stimulation.[90] Engineering of dendritic cells to release tolerogenic exosomes could be useful to prevent/ameliorate transplant rejection. Urine is the ideal biological sample for discovery of new biomarkers for kidney diseases because of the ease of non-invasive collection.

Neither short ZnT8R nor ZnT8W peptides displaced the labelled ZnT8R (268–369) in binding to ZnT8RAb in patient P1-R (Fig. 4, panels A and B) or P2-R (Fig. 4, panels C and D). At 50–100 μg/ml, the short ZnT8W peptide reduced the binding by 10–20% in patient P1-R (Fig. 4, panel B). Neither of the short ZnT8 (318–331) peptide variants were able to compete with the labelled ZnT8W (268–369) in binding to ZnT8WAb in patient P3-W (Fig. 5, panels A and B) or P4-W (Fig. 5, panels C and Cabozantinib research buy D). The reactivity against

the ZnT8 325-epitope was also tested with various dilutions of the non-radioactive long ZnT8 (268–369) proteins (Figs. 6 and 7). The long ZnT8R protein showed a displacement of the labelled ZnT8R (268–369) protein in patients P1-R (Fig. 6, panel A) and P2-R (Fig. 6, panel C) that amounted to a half-maximal displacement (Kd) at 3.0 and 4.1 pmol/l, respectively. In patients, P1-R and P2-R (Fig. 6, panels B and D, respectively) the long ZnT8W protein displaced

MK-2206 datasheet the labelled ZnT8R protein (Kd 26.1 and 11.1 pmol/l). In both ZnT8RAb-specific patients, the Kd of the long R protein was different from the W protein (P = 0.0003 and P < 0.0223, respectively; Table 2). Maximal displacement (Vmax) of the ZnT8RAb-positive patient sera with the long R protein was 90% and 87% (10% and 13% binding) (Fig. 6, panels A and C) compared to 67% and 78% (33% and 22% binding) with the long W protein (Fig. 6, panels B and D). Due to lack of serum from the ZnT8WAb-positive patients, P3-W and P4-W, two additional patients,

P5-W and P6-W, were selected for displacement with the long ZnT8 (268–369) protein. These patients were also tested with the short ZnT8 (318–331) peptide variants, which did not displace the labelled long ZnT8 protein in binding to ZnT8WAb (data not shown). In the P5-W and P6-W ZnT8WAb-positive sera, the long ZnT8W protein displaced the labelled ZnT8W (268–369) protein at Kd 10.4 pmol/l and Kd 15.5 pmol/l (Fig. 7, panels A and C, respectively). In the reciprocal permutation experiments, ID-8 a half-maximal displacement in patient P5-W (Kd > 108.6 pmol/l) was never achieved with the long ZnT8R protein as it was in patient P6-W (Kd 27.2 pmol/l) (Fig. 7, panels B and D). The Kd of the long W protein was markedly different from the R proteins in patient P5-W (P = 0.0016; Table 2), but not in patient P6-W (P = 0.2193; Table 2). In the ZnT8WAb-positive patient sera, Vmax with the long W protein was achieved at 89% and 75% (11% and 25% binding) (Fig. 7, panels A and C) compared to the Vmax for the long R protein at 44% and 68% (56% and 32% binding) (Fig. 7, panels B and D). It has been proposed that the specificity of autoantibodies for certain epitopes may be important to the prediction of the beta-cell destruction in T1D [23].

DEGs specifically modulated by MSU in WT and Nlrp3−/− DCs were further analyzed by MetaCore™ software to identify putative biological pathways and cellular processes they might participate in. Three major biological processes were statistically modulated by MSU in both WT and Nlrp3−/−

DCs compared with untreated controls: the DDR, cell cycle, and apoptosis/survival pathways (Fig. 1A). A significant increase in the expression selleck compound of several genes involved in double-strand and base-excision DNA repair (Xrcc1, Rad51, Ogg1, Brca1, Polb, and Tyms), cell cycle progression and proliferation (cyclin B and D, Ttk protein kinase, Prim1 and 2, and Rfc3 and 4), and repression of apoptosis (Xiap and Birc3) was observed only in Nlrp3−/− cells (Fig. 1B and Supporting Information Table 1). These data indicate that cells lacking NLRP3-mediated signaling exhibit a differential response to MSU compared with WT cells. Neratinib nmr To confirm the

physiological relevance of the MSU-induced pathways identified by gene expression array, we next assessed the extent to which MSU stimulation causes DNA damage in DCs. DCs generated from bone marrow (BM) of WT and Nlrp3−/− mice were therefore stimulated with MSU for 24 h and DNA fragmentation in individual cells was assessed by comet assay. This assay exploits a single-cell gel electrophoresis to progressively separate fragmented DNA from intact DNA from lysed cells. The resulting comet-like tail formation is then visualized Lumacaftor and quantitatively analyzed; tail length reflects the degree of DNA fragmentation (Tail DNA%), while the Olive Tail Moment is an index of DNA damage that considers both the migration of DNA as well as the relative amount of DNA in the tail. No tail was observed in untreated DCs (Fig. 2). Bright comets of fragmented DNA were detected in the majority of MSU-treated DCs, with mean% of total

DNA in the tail and olive moment significantly higher than in untreated controls (Fig. 2). Interestingly, DNA breaks were significantly diminished in Nlrp3−/− DCs compared with WT DCs after stimulation with MSU alone or in the presence of LPS, indicating that LPS priming was not required for DNA damage induced by MSU. Moreover, in the absence of Nlrp3, DNA damage in DCs treated with oxidative H2O2 was also significantly reduced (Fig. 2). We then tested H2AX histone phosphorylation on serine 139 (γH2AX), a primary marker of DNA damage required for triggering DDR in eukaryotic cells [9]. We found that H2AX was readily phosphorylated in WT DCs during MSU stimulation and that γH2AX levels were sustained for up to 24 h (Fig. 3A). Similarly to MSU, stimulation of WT DCs with silica robustly induced γH2AX, indicating that the same pathway is induced by other particulates (Supporting Information Fig. 1).

Upon recognition of pathogen-associated molecular patterns (PAMPs), i.e. danger signals and

sensing of the inflammatory cytokine environment, DCs undergo rapid maturation. The extent of their activation depends on the initial triggering stimuli 5 that can directly impact the fate of CD8+ T cells differentiation 1. In mice infected by Listeria monocytogenes (Lm), inadequate cDC activation correlates with impaired development of protective CD8+ T-cell memory 6–8. Evidence accumulated over the past years suggested that CD8α+ cDCs selleck screening library play a unique role in priming CD8+ T cells, in particular because of intrinsic features of their MHC class I processing machinery 9. CD8α+ cDCs have also been shown to be endowed with optimized functional characteristics to induce pathogen- and tumor-specific CD8+ T cells to differentiate into primary effector cells 10–13. However, whether these cells or even CD8α− cDCs, independently of their respective capacity to process MHC class I-associated antigens, are capable of integrating all pathogen-derived signals this website and conveying them to naïve CD8+ T cells to become long-lasting pathogen-specific

protective memory cells in vivo is not known. While both cytosolic and/or extracellular-derived signals likely contribute to such cDC licensing, the relative impact of these signals has not been extensively investigated. Lack of such knowledge is mostly due to technical limitations. In fact, adoptive transfer of DC subsets from immunized animals has been difficult to interpret since these cells contain virulent pathogens that can directly infect recipient hosts and activate long-term immunity. Selective in vivo depletion of APC subsets also suffered from the specificity of the depletion 4, 14. To circumvent these issues, we designed Methamphetamine an experimental system in which APC subsets could be purified from mice immunized with the intracellular bacterium Lm lacking the SecA2 auxiliary secretion system (secA2− or ΔSecA2 Lm) 15, 16 which induce protective immunity only upon infection with high numbers of bacteria (107). SecA2−Lm also exhibit impaired spreading from cell to cell and do not efficiently infect APCs from recipient mice. Thus, taking advantage

of this experimental set-up, we could ask whether a subset of cDC is indeed more efficient at inducing protective CD8+ T-cell memory in vivo. We previously demonstrated that mice immunized with low numbers (106) of secA2−Lm develop memory CD8+ T cells that do not protect against a secondary infection with wt bacteria 16, 17. Since SecA2 partially controls the secretion of a subset of bacterial proteins, we hypothesized that induction of protective memory CD8+ T cells may require the secretion of a sufficient amount of at least one SecA2 substrate protein inside the cytosol of infected host cells to generate the appropriate priming environment. Therefore, we reasoned that the cytosolic signaling defect should be restored by immunizing mice with an increased dose of secA2−Lm.

We confirmed that thymus NKT cells in humans were predominantly CD4+, but found that they were capable of significant cytokine production, including BGJ398 nmr IFN-γ, TNF and IL-4. Strong cytokine staining was also observed using NKT cells from cord blood,

illustrating that many CD4+ NKT cells in thymus and cord blood are functionally competent, although the pattern of cytokine expression was distinct from CD4+ NKT cells isolated from peripheral blood (Fig. 8). It also raises the question of whether or not there is a similar resident mature NKT cell population in the human thymus to that identified recently in mice [28]. We also performed the first analysis of NKT cells from human spleen. Fewer surface antigens were analysed for spleen NKT cells, but these appeared to be similarly heterogeneous in expression of cell surface antigens to blood-derived NKT cells, and were similar in their overall frequency and cytokine profile (IFN-γ, TNF and IL-4). This supports the analysis of blood NKT cells as a representative source of systemic NKT cells, at least relative to spleen, although more work is needed to confirm this, including comparative functional analysis of NKT cells from peripheral blood and from other peripheral tissues, such selleck inhibitor as liver and lymph nodes. Our data

clearly support the concept that heterogeneity within the NKT cell pool extends well beyond the CD4+ and CD4− subsets. More investigations are needed to define the functional diversity that exists within the human

NKT cell compartment and to correlate this with patterns of antigen expression and tissue residency, but it appears likely that that the diverse activities attributed to human NKT cells relies on an equally diverse array of subsets. The authors acknowledge the kind donation of tissue for research purposes by donors and their families. This research was supported by an NHMRC Project Grant (no. 454363) and an NHMRC Program Grant (no. 454569). S.P.B. was supported by an NHMRC Career Development Fellowship (no. 454731) and by the Australian Government Collaborative Research Network (CRN). S.P.B. is currently Methocarbamol supported as a Dorevitch Senior Research Fellow (at FECRI) and as a Robert H. T. Smith Fellow (Uni of Ballarat). D.I.G. is supported by an NHMRC Senior Principal Research Fellowship (no. 1020770). The authors declare no conflicts of interest. “
“The serine/threonine kinase LKB1 has a conserved role in Drosophila and nematodes to co-ordinate cell metabolism. During T lymphocyte development in the thymus, progenitors need to synchronize increased metabolism with the onset of proliferation and differentiation to ensure that they can meet the energy requirements for development.

The most important type I IFNs involved in an antiviral response are IFN-α and IFN-β, and it is well known that stimulation of TLR3 and RIG-I with viral RNA results in type I IFN production [[10, 20]]. As our data suggest that TLR3 and RIG-I play a role in the observed synergistic response to RSV and

MDP, we investigated the induction of IFN-β. As type I IFNs are generally regarded as early responders SAHA HDAC in vivo [[21]], we determined the kinetics of IFN-β induction at an early (4 h) and at a late (24 h) time point following stimulation with RSV, MDP, LPS, and both types of Poly(I:C). Infection with RSV, with and without MDP, showed a small increase of IFN-β mRNA compared with unstimulated KU-57788 in vivo cells after 4 h (Fig. 4A) and a strong increase of IFN-β mRNA after 24 h (Fig. 4B). Stimulation with MDP did not induce upregulation of IFN-β at either time point. Although LPS stimulation resulted in a temporary upregulation of IFN-β mRNA after 4 h, IFN-β was downregulated after 24 h, similar to previous observations [[21]]. Both Poly(I:C) HMW and Poly(I:C)-LyoVec LMW induced an upregulation of IFN-β after 4 h and 24 h (Fig. 4B). These data suggest that stimulation with dsRNA results in IFN-β transcription and stimulation with live RSV results in a delayed IFN-β response. Previous studies have shown

that TLR3, RIG-I, and NOD2 are upregulated by type I IFNs in response to Poly(I:C) and viral infection [[22, 23]]. To investigate whether IFN-β induces a transcriptional upregulation of these receptors, quantitative PCR was used to determine the expression levels of TLR3, RIG-I, and NOD2. Human PBMCs were stimulated with RSV, MDP, IFN-β, and both types of Poly(I:C). Stimulation of human PBMCs with RSV, with and without MDP, showed an upregulation of both TLR3 and RIG-I, although the most pronounced C59 cost effect was observed with RIG-I (Fig. 5A). Similar to RSV stimulation, IFN-β, Poly(I:C) HMW, and Poly(I:C)-LyoVec LMW also induced an increase in TLR3 expression

and a stronger increase in RIG-I transcription. In addition to TLR3 and RIG-I, NOD2 was also found to be upregulated after stimulation with all stimuli except MDP (Fig. 5B), suggesting that in this model RSV, Poly(I:C), and IFN-β affect the transcription of TLR3, RIG-I, and NOD2. Our results suggest that RSV infection induces upregulation of NOD2 in an IFN-β dependent manner. Subsequent stimulation of NOD2 with MDP then results in an elevated response in proinflammatory cytokines. As this implies that the order of events is important, we performed an experiment in which we sequentially stimulated PBMCs. Cells were first stimulated with RSV or MDP for 24 h and then subsequently stimulated with either RSV or MDP for another 24 h. The amount of cytokine release after these stimulations can be found in Supporting Information Fig. 3.