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International Journal of Microbiology and Current Research
TLR3 Deficiency Leads to a Dysregulation in the Global Gene-Expression Profile in Murine Oviduct Epithelial Cells Infected with Chlamydia muridarum
Ramesh Kumar and Wilbert A Derbigny*
Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana-46202, USA
*Corresponding author: Wilbert A Derbigny, Assistant Research Professor, Department of Microbiology and immunology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, Indiana, USA, Tel: +1 (317) 274-0697, Fax: (317) 274-4090, E-mail: wderbign@iupui.edu
Received: November 14, 2018 Accepted: November 28, 2018 Published: December 5, 2018
Citation: Kumar R, Derbigny WA. TLR3 Deficiency Leads to a Dysregulation in the Global Gene-Expression Profile in Murine Oviduct Epithelial Cells Infected with Chlamydia muridarum. Int J Microbiol Curr Res. 2018; 1(1): 1-13. doi: 10.18689/ijmr-1000101
Chlamydia trachomatis replicates primarily in the epithelial cells lining the genital tract and induces the innate immune response by triggering cellular pathogen recognition receptors (PRRs). Our previous studies showed that Toll-like receptor 3 (TLR3) is expressed in murine oviduct epithelial (OE) cells, is the primary PRR triggered by C. muridarum (Cm) early during infection to induce IFN-β synthesis, and that TLR3 signaling regulates the chlamydial induced synthesis of a plethora of other innate inflammatory modulators including IL-6, CXCL10, CXCL16 and CCL5. We also showed that the expression of these cytokines induced by Chlamydia was severely diminished during TLR3 deficiency; however, the replication of Chlamydiain TLR3 deficient OE cells was more robust than in WT cells. These data suggested that TLR3 had a biological impact on the inflammatory response to Chlamydia infection; however, the global effects of TLR3 signaling in the cellular response to Chlamydia infection in murine OE cells has not yet been investigated. To determine the impact of TLR3 signaling on Chlamydia infection in OE cell at the transcriptome level, we infected wild-type (OE-WT) and TLR3-deficient (OE-TLR3KO) cells with Cm, and performed transcriptome analyses using microarray. Genome-wide expression and ingenuity pathway analysis (IPA) identified enhanced expression of host genes encoding for components found in multiple cellular processes encompassing: (1) pro-inflammatory, (2) cell adhesion, (3) chemoattraction, (4) cellular matrix and small molecule transport, (5) apoptosis, and (6) antigen-processing and presentation. These results support a role for TLR3 in modulating the host cellular responses to Cm infection that extend beyond inflammation and fibrosis, and shows that TLR3 could serve a potential therapeutic target for drug and/or vaccine development.
Keywords: TLR3; Chlamydia; RNA; Micro-array; Epithelial; RANTES; Pathogenesis; Transcriptome; Genital tract.
Chlamydia trachomatis is a gram-negative, obligate intracellular bacterial pathogen causing the most common sexually transmitted infections (STI) worldwide, particularly among young women [1]. In women, chronic infection with the urogenital serovars (D-K) can cause pelvic inflammatory diseases (PID) and chronic pelvic pain, which can culminate into scarring and fibrosis of the Fallopian tubes leading to infertility or ectopic pregnancy [2-4]. According to the Centers for Disease Control and Prevention's (CDC's) report in 2016, a total of 1,598,354 Chlamydia infections were reported in USA alone, which was 4.7% higher than the reported number of cases in 2015 [5]. As per CDC's estimate, nearly 20 million healthy individuals are infected every year, accounting for almost $16 billion in health care costs annually (CDC 2016) [5].The asymptomatic nature of C. trachomatis infection means that the pathogen persists for a long time in some individuals, which suggests the effective evasion of host immune systems [6,7].
The innate immune system is known for recognizing a vast variety of pathogens, including viruses, bacteria and fungi via sensing the specific pathogen associated molecular patterns (PAMPs). The innate immune response induced by PAMPs includes the cellular production of a wide range of antimicrobial and inflammatory mediators. The recognition of PAMPs by pattern recognition receptors (PRRs) expressed by innate immune cells is crucial for maintenance of homeostatic immunity as well as an effective induction of an adaptive immune response [8-10]. However, an overly activated innate immune response can cause the overabundant production of inflammatory mediators which can result in tissue damage [11,12]. Among the PRRs, Toll-like receptors (TLRs) play a major role in innate immunity by recognizing structurally conserved microbial components [13]. TLRs are membrane bound PRRs that have been shown to be triggered by PAMPs from various bacterial, viral and fungal pathogens [14]. Engagement of the TLRs by the PAMPs can lead to the activation of phagocytosis and the production of inflammatory cytokines including TNF-α, IL-6, and GMCSF, as an important step prior to the switch from an innate immunity and the onset of an adaptive immune response [15-18].
Epithelial cells lining the mucosal surfaces of the female genital tract serve as the sentinels to the invading C. trachomatis by expressing TLRs that trigger the innate immune response by inducing a multitude of pro-inflammatory cytokines and chemokines during infection [19]. These cytokines and chemokines mediate resolution of Chlamydia infection and are responsible for polarizing the innate and adaptive immune responses [18-23]. We previously showed that Chlamydia muridarum (Cm), an orthologue of human C. trachomatis, induces TLR2 dependent secretion of acute-phase inflammatory cytokines including IL-6, GM-CSF, and TNF-α in murine oviduct epithelial (OE) cells [8]. Further, we showed that Chlamydia infection mediates IFN-β secretion in a mostly TLR3-dependent manner, and demonstrated a role for TLR3 in regulating the expression of a plethora of other innate-inflammatory modulators including IL-6, CXCL10, CXCL16 and CCL5 [24-26]. The Chlamydia-induced expression of these cytokines was severely diminished in TLR3 deficient OE cells whereas; replication of Cm in TLR3 deficient OE cells was more robust than in WT OE cells. These findings suggest that triggering the TLR3 pathway in OE cells during Cm infection invokes cellular mechanisms that inhibit the Chlamydial developmental cycle, and thereby implicate TLR3 in regulating cellular processes that extend beyond the syntheses of chemo tactic and inflammatory mediators.
In order to help identify other cellular pathways that are regulated by TLR3 signaling during Chlamydia infection, we conducted transcriptome analyses and comparative gene expression profiling on WT and TLR3-deficient murine OE cells that were infected with Cm. In the present study, we aim to identify target genes that are affected by Chlamydia infection of OE cells and to ascertain a role for TLR3 in regulating those pathways by using genome-wide microarray analysis followed by ingenuity pathway analysis (IPA). Our results indicate that TLR3 plays a significant role in modulating the host cellular responses that encompass a diverse subset of biological functions during Cm infection.
Oviduct epithelial cells and culture conditions
The murine oviduct epithelial cell lines OE-129TLR3-/- (C19) and OE-129WT [25] were grown at 37°C in a 5% CO2 humidified incubator in epithelial-cell media {Dulbecco's modified Eagle medium and F12K (Sigma-Aldrich) in 1:1 ratio}, supplemented with 10% fetal bovine serum (HyClone), 2mM L-alanyl-L-glutamine (Glutamax I; Gibco/ Invitrogen, Carlsbad, CA), 5µg bovine insulin/ml, and 12.5 ηg/ml recombinant human KGF (keratinocyte growth factor; Sigma-Aldrich) as previously described [18,24].
Chlamydia stocks
Mycoplasma-free C. muridarum Nigg, previously known as C. trachomatis strain (MoPn), was grown and titrated in McCoy cells (ATCC) as described [18, 27, 28]. The elementary bodies were harvested from infected McCoy cells, were re-suspended in SPG buffer (250mM sucrose, 10 mM sodium phosphate, and 5mM L-glutamic acid, pH 7.2), and quantified on McCoy cells using methodology described previously [18,26,29].
In vitro infection of oviduct epithelial cells
OE-129WT and OE-129TLR3-/- (C19) cells were seeded in 6-well tissue culture plates and grown until confluence. The cells were infected with 5 inclusion forming-units (IFU) of C. muridarum/cell in 900ul of culture medium as described previously [24, 25]. Mock-treated controls were incubated with an inoculum containing equivalent volume of SPG buffer, but were not infected with Chlamydia. Each cell type was seeded in duplicate wells of the 6-well plate and were infected when confluent. The experiments were repeated 3 times on different days to provide the appropriate number of biological replicates for proper statistical analyses.
Total RNA purification and Microarray analysis
Total RNA was purified from OE-129WT and OE-129TLR3-/-(C19) cells infected with C. muridarum(5 IFU/cell) at 24h post-infection using the Norgen Total RNA Purification Kit(Norgen Biotek; Thorold, Ontario). During purification, all RNA samples were treated with RNase-free DNase I (Qiagen) to remove all traces of genomic-DNA contamination. The RNA quantity and quality were measured using the Nano Drop 2000c spectrophotometer (Thermo Scientific; Pittsburgh, PA). The RNA purified from mock-infected cells was used as control sample. Purified total RNA was submitted to the gene-expression profiling services of Phalanx Biotech Group, Inc. (Belmont, CA) for microarray analysis. Genes that were up or down regulated ≥ 2.5-fold with p-values <0.05 were considered in the final analysis (Tables 1 and 2; and Supplementary Tables 1 and 2).
Note: OE-WT-Cm/OE-WT-Mockrefers to the gene-expression levels in Chlamydia muridarum (Cm) infected OE-WT cells derived versus the basal gene-expression levels in the Mock-infected OE-WT cells at the 24h time-point. OE-TLR3KO-Cm/OE-WT-Cm refers to the gene-expression levels in Cm-infected OE-WT cells versus the gene-expression levels in the Cm-infected OE-WT cells at the 24h time-point. For all genes, only fold changes above 2.5-fold with a p-value of<0.05 for changes in expression between above groups are shown, - (negative) sign with fold-change indicates down-regulated genes whereas; fold-change without - (negative) sign indicates up-regulated genes. (a) Table 1: represents differentially expressed genes up-regulated in OE-WT-Cm vs OE-WT-Mock and down-regulated in OE-TLR3KO-Cm vs OE-WT-Cm. (b) Table 2: represents differentially expressed genes down-regulated in OE-WT-Cm vs OE-WT-Mock and up regulated in OE-TLR3KO-Cm vs OE-WT-Cm.
Functional and canonical pathway analyses
The microarray gene expression data were analyzed by Qiagen's ingenuity pathway analysis (IPA; Ingenuity Systems, https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis; Qiagen Inc.) to determine whether genes are associated with particular diseases, exhibit prominent biological function, or if canonical signaling pathways were preferentially up- or down-regulated in murine OE cells [30]. Genes were selected for analyses if they had a p-value <0.05 and absolute fold change ≥2.5. The data are presented as the comparison of: (1) C. muridarum-infected wild-type (OE-WT-Cm)vs Mock-infected wild-type (OE-WT-Mock) and (2)C. muridarum-infected wild-type (OE-WT-Cm)vs C. muridarum-infected TLR3-deficient OE cells (OE-TLR3KO-Cm) (Table 3, Supplementary Table 3, and Table 4).
Identification of differentially-regulated genes in murine oviduct epithelial cells
Our previous studies showed that TLR3 is the primary PRR in OE cells triggered by Cm infection in the early synthesis of IFN-β, and in the syntheses of a multitude of other innate inflammatory modulators such as IL-6, CXCL10, CXCL16 and CCL5. Our data show that TLR3 has a biological impact on the innate immune response to Chlamydia infection; however, the comprehensive impact of TLR3 deficiency during Chlamydia infection of murine OE cells remains unclear. To determine the global significance of TLR3 signaling on Cm infection in OE cell lines, we infected wild-type and TLR3-deficient OE cells (here forth referred to as OE-WT and OE-TLR3KO cells, respectively) with 5 IFU/cell Cm for 24h post-infection (PI) and performed transcriptome analysis using microarray. We used a minimum significance criterium of p<0.05 and an absolute fold change ≥2.5 for comparative gene analyses (See Materials and Methods). Out of the several hundred genes that were differentially affected by Cm infection, candidate gene selection based on this criterion yielded 152 genes up-regulated and 56 genes down-regulated when comparing OE-WT-Cm vs the OE-WT-Mock control (Supplementary Tables 1 and 2). Interestingly, there were 600 genes up-regulated and 616 genes down-regulated when comparing OE-TLR3KO-Cm vs OE-WT-Cm cells, which shows that TLR3 deficiency results in the differential regulation of a multitude of cellular processes during Chlamydia infection of the OE cells.
We selected genes that were up-/or down-regulated > 2.5-fold in both comparative groups: [OE-WT-Cm vs OE-WT-Mock] and [OE-TLR3KO-Cm vs OE-WT-Cm] for our analyses (Tables 1 and 2).The most prominent genes up-regulated in OE-WT-Cm vs OE-WT-Mock are listed in Table 1. As indicated, many of the most highly up-regulated genes in the OE-WT-Cm cells such as Ccl5 (80.050-fold), Lamc2 (71.848-fold), Cxcl2 (70.603-flod), Lcn2 (67.864-fold), Cmpk2 (31.845 fold) and Csf3 (27.161-fold) were substantially and significantly down regulated in OE-TLR3KO-Cm cells. Not surprisingly, pro-inflammatory cytokines and chemokines showed the highest level of up regulation during Cm infection in the OE-WT cells. Amongst the most highly up-regulated pro-inflammatory mediators were Ccl5 (RANTES), Ccl4 (MIP-1b), and the monocyte chemo attractant proteins Ccl2 and Ccl7 (or MCP 1 and 3, respectively) belonging to C-C chemokine family. The C-X-C motif chemokines Cxcl2 (also called macrophage inflammatory protein2-alpha) and Cxcl5 (or epithelial neutrophil activating protein-78), and the acute inflammatory cytokines Csf3 and Il6 were also included in the most highly upregulated inflammatory mediators. These highly up-regulated cytokine and chemokines were severely down-regulated in the OE-TLR3KO-Cm cells relative to OE-WT-Cm cells (Table1).
The extracellular matrix (ECM) proteins play a critical role in the cell invasion, adhesion, cell patterning and architectural changes, and "outside-in" signal transduction [31]. Our microarray data showed that many host ECM moieties were differentially expressed, suggesting a rapid and dramatic remodeling of the extracellular milieu in response to Chlamydia infection (Tables 1 and 2; Supplementary Tables 1 and 2). The genes involved in remodeling included glycol proteins, metalloproteinases, numerous collagens, and several fibrosis-associated moieties that were highly upregulated in our data set (Table 1). The Cm induced ECM genes included Lamc2 (LAMC2), Fst (Follistatin), Mmp10 (metalloproteinase MMP10), Ereg (epiregulin EREG), Enpp2 (ATX or autotaxin, ENPP2), Fgf23 (fibroblast growth factor FGF23), Steap4 (metalloreductase STEAP4), Madcam-1(mucosal vascular address in cell adhesion molecule-1, MAdCam-1) and Icam-1 (intracellular adhesion molecule-1, ICAM-1). However, as we have seen with many of the highly up regulated pro-inflammatory mediators, transcription of these ECM genes in the Cm-infected OE-TLR3KO cells resulted in a substantial down regulation or inhibition when compared to the Cm-infected OE-WT cells.
Other genes that were highly up regulated in response to Cm infection in WT-OE cells but attenuated during TLR3 deficiency included components of various cellular metabolic pathways (LCN2, CMPK2, ARG2, SOD2, ERO1-α [or endoplasmic reticulum oxidoreductase1-α], CH25H, FABP4, and AK4), and proteins involved in membrane tracking and protein processing (ARL4D, NEURL3, PTX3, SERPINB2 [or PAI-2], and SERPINE1 [or PAI-1]). Interferon-sensitive genes (ISGs) are known for their role in cell-intrinsic immunity against diverse pathogens, such as viruses and intracellular bacterial species including Chlamydia, Mycobacteria, Listeria, Salmonella, and Toxoplasma [32]. We found several ISG sincluding Gbp5 (a member of IFN-inducible subfamily of GTPases) [33] and Parp12 [34-37] that were highly induced in the Cm infected OE-WT cells, but differentially regulated during TLR3 deficiency.
Some of the more moderately up-regulated genes encoding the pro-apoptotic proteins (FAS and XAF1), proteins related to MHC Class 1 antigen presentation (PSMB-8, PSMB-9 and TAP1), solute carrier (SLC) transporters (SLC7A2 and SLC15A3), and the JAK2 protein [38]. Other genes moderately up regulated included those encoding the TNF-α mediated pro-inflammatory proteins KRT16 (keratin), activator protein-1 (AP-1), family members of MAFF (MafbZIP transcription factor F) and FOSL1 (Fos-like 1or FRA1) [39], TRAF1, NF-κBia (inhibitor of nuclear factor κappa-B or IκB-α), NF-κB/RELB, MKK6 (Mitogen-activated protein kinase 6), and GRP (Gastrin releasing peptide). Finally, our data also showed some moderate but significantly up-regulated expression of genes encoding various receptors and signaling molecules including KISS1R, IL15R-α, IL1RL, CD14, PIK3R5 (phosphatidylinositol 3-kinase, regulatory subunit 5), and the RCL1(RNA terminal phosphate cyclase like-1)protein that is involved in eukaryotic 18S RNA biogenesis [40]. However, much like the highly up regulated genes, these moderately induced genes in Cm-infected OE-WT cells were differentially regulated during TLR3-deficiency. Interestingly, not all genes induced during Cm infection of WT-OE cells were attenuated or down regulated during TLR3 deficiency. Results depicted in Table 1 show that transcription levels for Isg15, Mx1, Gm4951, Oas1b, Mpeg1, Zfp296, Zbp1, and Klhl2 were not reduced in the Cm-infected OE-TLR3KO cells relative to Cm-infected OE-WT cells; instead, their expression levels were significantly increased during TLR3-deficiency. Isg15, Mx1, Oas1b and Mpeg1 are type I and II interferon responsive genes and have been implicated in the host protection against various pathogenic organisms [32, 41-45].
Table 2 shows 11 genes that were down-regulated more than 2.5-fold during Cm infection of the OE-WT cells. Aldh1a1, Ankrd35, Chchd10, Daam2, Fbln5, Fbxo31, Gcnt1, Lum, Ppm1e, Ptprv and Txnip were all down regulated during Cm infection in wild-type OE cells; however, their expression levels in OE-TLR3KO cells was several-fold higher. Interestingly, 8 of the 11 genes (Aldh1a1, Ankrd35, Chchd10, Daam2, Fbln5, Fbxo31, Gcnt1, and Ppm1e) have been reported to be prognostic markers in a variety of metastatic malignancies and other cancer-related diseases [46-54]. Ptprv and Txnip exhibit tumor-suppressor function and has involvement in cell proliferation and apoptosis [55, 56]. The Lum gene encodes the extracellular matrix protein lumican (LUM) that has a role in the bacterial clearance [57, 58], and its expression was upregulated 5.108-fold in Cm-infectedOE-TLR3KO cells when compared to Cm infected OE-WT cells.
Based on the gene expression data, a set of cellular path-ways predicted to be either activated or inhibited during Cm infection was identified by IPA software. Table 3 lists what are predicted to be the most significantly changed pathways based on the expression levels of the input genes. As indicated, most of the Cm infection-induced pathways in the OE-WT cells were found to be significantly inhibited in the Cm-infected OE-TLR3KO cells. Pathways regulating the role of PRRs in recognition of bacteria and viruses, LPS/IL-1 mediated inhibition of RXR function, and NF-κB signaling were not inhibited in the Cm-infected OE-TLR3KO cells when compared to Cm-infected WT-OE cells like the other pathways listed in Table 3; however, activation of these pathways was significantly lower in the OE-TLR3KO cells. The lower portion of Table 3 shows the canonical pathways that were predicted to be inactivated in Cm-infected OE-WT cells e.g., LXR/RXR activation, PPAR signaling, and PPARα/RXRα activation pathways. In contrast, LXR/RXR activation and PPAR signaling pathways are predicted to be activated in the Cm-infected OE-TLR3KO cells when compared to the Cm-infected OE-WT cells.
Upstream regulator analysis
IPA predicts upstream transcriptional regulators based on experimentally observed relationships between regulators and genes in the dataset. The calculated z-score predicts either activation or inhibition of regulators on the basis of the relationship with dataset genes and direction of expression change of input genes. There are 7 categories: immunological (cytokines and chemokines), chemical, kinase, transcription factor, trans membrane receptor, translation regulator, and transporter, where several Cm activated and inhibited upstream regulators are grouped together and listed in Supplementary Table 3 (Excel file). Among the 7 regulator categories, cytokine is the top-most significantly activated upstream regulator during Cm infection of OE cells, and the group includes IFN-γ, TNF, IL-1β, OSM, IL-1α, IL-17A, TNFSF-11, IL-1, IL-6, CD40LG, TNFSF-12, CSF-2, IL-18, IL-2, EDN1, CCL5, IL-15, MIF, IL-17F, CXCL2, IL-5, CXCL3, C5, CCL2, CXCL12, IL-7, IL-33, CXCL-8, TNFSF-14, TNFSF-13B, and IL-36A. Our data showed that these 31 cytokines were activated ≥ 2.5-fold in Cm-infected OE-WT cells when compared to the Mock-infected controls. In corroboration with our previously published results [25, 26, 59], many of these cytokines were predicted to be either severely reduced or inhibited in Cm-infected OE-TLR3KO cells relative to Cm-infected OE-WT cells in IPA. It is noteworthy that one particular cytokine (IL-10) was different from the other cytokines listed in Supplementary Table 3 in that it was predicted to be down regulated in the Cm-infected OE-WT cells relative to its expression in the Mock control OE-WT cells, but predicted to be activated in Cm-OE-TLR3KO cells relative to Cm-infected OE-WT cells. We previously reported significant increases in the genital tract secretion of IL-10 of TLR3-deficient mice when compared to wild-type control mice [59]; thus, our findings in the IPA support our in vivo data regarding this pleiotropic regulatory cytokine [60]. We hypothesized that IL-10 plays a role in attenuating the in vivo synthesis of IFN-β during in TLR3-deficient mice and likely promotes the increased Cm-replication that we have observed in the TLR3-deficient mice.
Upstream regulator analyses showed 61 chemical regulators that were either activated or inhibited in Cm-infected OE-WT cells when compared to Mock-infected OE-WT cells, but were differentially regulated in the Cm-infected OE-TLR3KO cells. The chemical categories encompass endogenous non-mammal, endogenous mammal, other, drug, reagent, toxicant, and kinase inhibitors. Further analyses showed that 17 kinase regulators were activated i.e.; CHUK, IKBKB, IKBKG, MAP3K7, RIPK2, MAPKAPK2, JAK2, JAK1, MAP3K14, MAPK8, PRKCD, MAP2K1, PRKCA, SRC, RET, GSK3B, SPHK1, whereas; one kinase (MAPK1) was found to be downregulated in Cm-infected OE-WT cells. Among transcription factors (TFs), 16TFs: NF-κB [complex], REL-A, JUN, STAT4, FOXL2, CEBPB, EGR1, TP63, ECSIT, HMGB1, IRF6, FOXO1, CEBPA, HIF1A, CEBPE, and JUNB) were activated and one TF (ZFP36) was inhibited. Other upstream regulators activated during Cm infection of OE-WT cells included 10 transmembrane receptors (TLR4, TLR3, TLR2, TLR5, CD40, CD14, TNFRSF1A, ICAM1, IL17RA, and TNFRSF1B), one translation regulator (EIF4E), and two transporters (LCN2 and TNNI3).The remaining upstream regulators spanning the 7 categories shown in Supplementary Table 3 were all inhibited in Cm-infected OE-WT cells when compared to Mock-infected OE-WT cells. However, they were also differentially regulated when compared to Cm-infected OE-TLR3KO cells in that they were either inhibited to a lesser degree, or the pathways were activated instead.
Disease pathogenesis and cellular function
Based on the gene expression input data, IPA predicted the pathways associated with disease pathogenesis and cellular function that are most affected in the cell during Cm infection of OE cells (Table 4). The inflammatory response and growth of epithelial tissue were not surprisingly the most affected pathways that are predicted to be affected when Chlamydia invades epithelial cells lining the female genital tract. As shown in Table 4, these pathways are the most highly up regulated in the Cm-infected OE-WT cells when compared to the Mock-infected OE-WT controls. As indicated, these two pathways are predicted to be significantly diminished or inhibited in the Cm-infected OE-TLR3KO cells when compared to the Cm-infected OE-WT cells. In support of our previously published reports showing that TLR3-deficiency leads to increased genital tract pathology in mice during Cm-infection [59], IPA showed that the pathways associated with organ inflammation and tissue necrosis were predicted to be significantly increased in the Cm-infected OE-TLR3KO cells when compared to Cm-infected OE-WT cells. Collectively, transcriptome and IPA results show role for TLR3 in modulating host cellular responses to Cm infection that extend beyond inflammation and fibrosis, and implicate TLR3 as a major component in the cellular response to Chlamydia infection in genital tract epithelial cells.
We previously showed that Cm infection induces IFN-β secretion in OE cells in a mostly TLR3-dependent manner, and that TLR3 deficiency lead to the dysregulation in syntheses of a plethora of other innate inflammatory modulators including IL-6, CXCL10, CXCL16 and CCL5. Although the expression of these cytokines induced by Chlamydia was severely diminished in TLR3 deficient OE cells and mice, replication of Cm during TLR3-deficiency was more robust than in OE-WT cells and mice. These data suggested that TLR3 had a biological impact on the innate immune response to Chlamydia infection; however, the impact of TLR3 signaling on the global cellular response to Chlamydia infection in OE cells remained unanswered. In the present study, we performed transcriptomics on wild-type and TLR3-deficient OE cells and conducted IPA to understand the complexity of genes and pathways in murine OE cells that are affected during Cm infection. In this regard, we are hoping to identify the spectrum of pathways that are either activated or inhibited during Cm infection, in order to ascertain those critical cellular mechanisms that can be exploited for the development of better treatments for Chlamydial disease and therapeutic agents.
Microarray results uncovered a multitude of genes that were either up-or down-regulated ≥ 2.5-fold in Cm infected OE-WT cells when compared to the mock-infected controls, and revealed that many of those genes were differentially regulated during TLR3-deficiency when comparing transcriptome data generated in Cm-infected OE-WT cells to results generated in the Cm-infected OE-TLR3KO cells. Among the most highly up-regulated genes during Cm infection of OE cells, the overwhelming majority were pro-inflammatory cytokines and chemokines (Table 1 and Supplementary Table 3, excel file). Our data showed that many of these pro-inflammatory mediators were either severely attenuated in their expression levels during TLR3 deficiency, or that their expression was down regulated below basal expression levels. Because epithelial cells lining the genital tract serve as immunological sentinels tomicrobial pathogens, and are responsible for initiating the primary phases of the host immune response [19, 21, 23], a comprehensive understanding of the cascade of cellular events that are triggered in OE cell during Cm infection would significantly enhance our knowledge and increase our understanding of the cellular mechanisms induced during Chlamydial pathogenesis.
Cytokines and chemokines are crucial in regulating a variety of molecular and cellular events specifically inflammation, scarring, and fibrosis. We found that CCL5 (RANTES), CXCL2 (MIP2-α), CSF3, CXCL5 (ENA-78), IL-6, CCL4 (MIP-1b), CCL2 (MCP-2) and CCL7 (MCP-3) were among the highest up-regulated cytokine and chemokines. CCL5 showed the highest up-regulation (80.053-fold) in WT-OE cells during Cm infection in comparison to mock-infected WT-OE cells, corroborating results from previous studies [26, 61]. CCL5 is a chemotactic factor that is secreted by a variety of cells including epithelial cells, and functions by recruiting leukocytes to the inflamed sites via binding to its CCR3 receptor. CCL5 also recruits macrophages to the site of inflammation by binding the CCR5 receptor. CCL5 is also a key chemokine in the induction of other Th1 cytokines, and has a functional role in the humoral immune responses against chlamydial pathogens [62, 63]. The C-X-C motif chemokine ligand 2, CXCL2 (MIP2-α) was also shown to be highly induced and was the next highest upregulated chemokine behind CCL5 in the microarray analyses. CXCL2 (along with CCL4) has been demonstrated to be involved in the recruitment of both ymphocytes and neutrophils to the inflamed sites in mouse model of Chlamydia infection [64, 65]. Other significantly upregulated cytokine and chemokine markers were colony stimulating factor 3 (CSF3), IL-6, CXCL5, CCL2 and CCL7. Induction of CSF3 and CXCL5 has been reported to be involved in attraction of neutrophils and other acute inflammatory cells against Chlamydia infection [18, 21, 66-68]. IL-6 is an important mediator of fever and major component of the acute inflammatory response; however, the role IL-6 synthesized during chlamydial genital tract immunopathology in mice is somewhat lesser defined and variable [69, 70]. CCL2 (MCP-1) and CCL7 (MCP-3) were also up-regulated by Cm infection in OE-WT cells, and was shown to play a crucial role in recruiting neutrophils, macrophage/monocytes, and dendritic cells to the site of chlamydial infection by other researchers [18, 68]. Because all of these highly up regulated cytokines and chemokines were severely down-regulated in TLR3-deficient OE cells when compared to WT-OE cells during Cm infection, the microarray results provides more comprehensive evidence that TLR3 deficiency results in a significantly impaired inflammatory immune response to Cm infection in OE cells, and supports our findings that TLR3 alters the pathogenicity of Chlamydia infection in vivo [59]. In addition to the vast array of inflammatory mediators that were affected by Cm infection in OE cells, there were other classes of genes that were significantly up-regulated as part of the cellular response to infection. IPA data showed that Cm infection in OE-WT cells induced genes encoding the glycoprotein laminin subunit gamma-2(LAMC2), follistatin (FST), matrix metalloproteinase (MM10),epiregulin (EREG), autotaxin (ATX or ENPP2)], fibroblast growth factor23 (FGF23), metalloreductase (STEAP4), MAdCAm, and ICAM1,which are known to function in remodeling of extracellular matrix (ECM) proteins [31]. The ECM provides structural and biochemical support of surrounding cells, and although the actual composition of ECM varies between multicellular structures, properties including cell adhesion, cell-to-cell communication, and cell differentiation are common functions of the ECM [71, 72]. The transcriptome data show that Cm infection in OE-WT cells substantially up regulates these ECM genes, and thereby can trigger or disrupt cellular processes that are essential for processes including cellular growth, wound healing, and fibrosis, but are highly suggestive that Chlamydia infection can also have impact on processes such as cell migration, gene expression, and cellular differentiation in macrophages and other hematopoietically-derived cells [73,74]. Our findings support the investigations of others who have implicated several members of this subset of ECM proteins in various disorders associated with genital tract Chlamydia infections such as oviduct fibrosis and scarring, uterus and oviduct distention, and tubal damage caused by ectopic pregnancy [75-80]. Because the transcriptome data show that TLR3-deficiency causes dysregulation in the gene expression levels of most of the ECM proteins identified by IPA, these data provides insight into a possible mechanism that expounds our recent report demonstrating thatTLR3 deficient mice suffer more severe genital tract pathology than wild-type mice during Cm infection [59].
Other genetic factors that were significantly up regulated during Cm infection of OE-WT cells included a subset of host-cell metabolism related genes encoding LCN2, CMPK2, ARG2, SOD2, ERO1-α, CH25H, FABP4, and AK4. Lipocalin-2 (LCN2) is an iron-sequestering multifunctional protein. Iron is an essential nutrient for many intracellular pathogens including Chlamydia. LCN2 binds to bacterial siderophores and thereby limiting the availability of iron for bacteria, inhibiting their growth, and thus protecting host from intracellular pathogens [81-83]. Intracellular pathogens including Chlamydia induce a strong Th1 immune response, which exerts pro-inflammatory effects by stimulating production of free radicals such as reactive oxygen species (ROS) and reactive nitrogen oxide species (RNOS) [84]. L-arginine is a crucial component for both inducible nitric oxide synthase (iNOS) and ARGase 1/2. It has been hypothesized that the enhanced expression of host SOD2 and ARG2 protects the host cells from the damaging ROS and RNOS [85-87], and both genes were found to be upregulated in our analyses. FABP4 (fatty acid binding protein 4) is primarily expressed in adipocytes and macrophages, and has roles in modulating immune responses and in lipid metabolism [88]. Walenna NF. et al demonstrated that C. pneumonia exploits host FABP4 for lipid metabolism in order to obtain ATP and lipids from the host cell, in order to facilitate its robust replication in adipocytes [89]. Not surprisingly, our findings show that TLR3 deficiency either severely attenuates or down regulated the expression of all of these metabolic factors, and presents the hypothesis that dysregulation of these metabolic factors will have a negative impact on the ability of the OE cells to control chlamydial replication. We showed in our previous reports that Cm replication in OE-TLR3KO cells was significantly more robust, and that the chlamydial inclusions were larger and more aberrantly shaped when compared to infection in OE-WT cells [26]. Collectively, our data show that TLR3 signaling does indeed play a role controlling chlamydial replication within the cell, and the data generated in IPA supports the hypothesis that TLR3 mediates this function by regulating the transcription of many of these host cell metabolism related genes.
Other genes that were highly up-regulated in the Cm-infected OE-WT cells but were differentially regulated in the Cm-infected OE-TLR3KO cells included genes that encode proteins that are involved in other areas of innate immunity such as ARL4D, NEURL3, PTX3, SERPINE1 and SERPINB2. These proteins include GTPases, ligases, and protease inhibitors that play important roles in actin remodeling, tissue repair, and ECM degradation [90-98]. In addition to the genes mentioned above, we observed significant increased expression levels of the interferon-responsive genes Gbp5 (a member of IFN-inducible subfamily of GTPases) and Parp12. GBP5 protects host against diverse pathogens [33, 99] and PARP12 localizes to the stress granules under stress condition, mediates cell survival/growth, and induces an anti-viral response by inhibiting protein translation at both viral and cellular protein levels [34-37]. We also saw up-regulated expression of the pro-apoptotic genes Fas and Xaf1 (XIAP associated factor 1) in our analysis, and increased transcription of genes encoding proteins involved in antigen processing including TAP1, LMP7, and LMP2.
We found 8 genes that were induced during Cm infection in OE-WT cells but were not reduced or inhibited in the Cm-infected OE-TLR3KO cells when compared to the Cm-infected WT-OE cells. In contrast to the large majority of genes that were either down regulated or inhibited in TLR3's absence, Isg15, Mx1, Gm4951, Oas1b, Mpeg1, Zfp296 and Klhl2 were all significantly up regulated in Cm-infected TLR3-deficient OE cells when compared to Cm-infected OE-WT cells. Because the absence of TLR3 results in the increased expression of these genes during Chlamydia infection, these results suggest a repressor role for TLR3 in the transcription of these particular genes in OE cells. The repressor function of TLR3 was also observed in the expression of Aldh1a1, Ankrd35, Chchd10, Daam2, Fbln5, Fbxo31, Gcnt1, Lum, Ppm1e, Ptprv and Txnip in Cm-infected OE-WT cells. These genes were either significantly up regulated in the TLR3-deficient OE cells, or their expression levels no longer repressed as they were in OE-WT cells (see Table 2). Although the role of TLR3 as an activator of gene expression has been well described such as in the direct activation of IFN-β in response to dsRNA [100], or in the indirect activation of CCL5 via TLR3-induced IFN-β [101], a mechanism to describe TLR3 signaling as a repressor pathway remains unclear and requires further study.
IPA identified several inflammatory pathways such as acute phase response signaling, dendritic cell maturation, role of PRR in recognition of bacteria and viruses, IL-6 signaling, interferon signaling, TREM1 signaling, VDR/RXR-activation, p38 MAPK signaling, LPS-stimulated MAPK signaling, Th1 signaling, LPS/IL-1 mediated inhibition of RXR function, JAK/STAT signaling, HMGB1 signaling, PI3K/AKT signaling, Tec kinase signaling and TLR signaling pathway to be most crucial in Chlamydia pathogenesis. These pathways were predicted to be significantly activated in Cm-infected OE-WT cells, but were predicted to be either severely attenuated or inhibited in TLR3-deficient OE cells during Cm infection. In contrast, we also found some canonical pathways that were predicted to be inactivated in Cm-infected OE-WT cells such as LXR/RXR activation, PPAR signaling and PPARα/RXRα activation pathway, but were activated in OE cells by Cm during TLR3-deficiency (LXR/RXR activation and PPAR signaling). Not surprisingly, there were several genes affected by Cm infection that are common amongst the various activated and inhibited pathways suggesting that many of these gene products are pleiotropic in their function; however, their impact in their respective pathways were differentially impacted based on the presence or absence of TLR3. These IPA data corroborates the hypothesis that TLR3 functions as a regulator in cellular responses to Chlamydia infection in OE cells, and can function as either an activator or repressor of numerous pathways associated with the inflammatory immune response.
Finally, IPA can be used to identify a cascade of upstream transcriptional regulators from transcriptome data identifying the genes that are activated or repressed during Cm infection of OE cells. In our analyses, we identified a multitude of upstream regulators that were either activated or repressed during Cm infection of OE cells including immunological (cytokines and chemokines), chemical, kinase, transcription factor, transmembrane receptor, translation regulator, and transporter regulators. Among the most highly activated upstream regulators, cytokines were at the top of the list, suggesting Chlamydia was able to modulate the cellular immune responses by altering the expression patterns of some critical immune system molecules including cytokines. Additionally, the main anti-inflammatory-cytokine, IL-10, was shown to be down regulated in Cm-infected OE-WT cells whereas; it was significantly activated in the Cm-infected TLR3-deficient OE cells which supports our recently published in vivo results [59]. IL-10 has been identified as a key player in the establishment and perpetuation of viral persistence, is known to be negatively regulated by IFN-β production, and promotes a suppressive environment that diminishes the antiviral response [102-104]. Because the Cm-induced synthesis of IFN-β is severely diminished in TLR3-deficient OE cells, IPA showing that IL-10 synthesis was significantly up regulated during Cm infection of OE-TLR3KO cells fits the paradigm that its synthesis is regulated by TLR3-dependent IFN-β.
IPA results show that other upstream-activated regulators belonging to classes of kinases, transcription factors, transmembrane receptors, translation regulators, and transporters groups were also impacted by Cm infection in OE cells. As was demonstrated with the immunological regulators, these additional upstream regulators were also negatively regulated in the TLR3-deficient OE cells during Cm infection when compared with Cm-infected OE-WT cells. Further, IPA results show that the Cm infection induced regulators either activated or inhibited cellular pathways in OE-WT cells that were more likely to restrict chlamydial pathogenesis. The IPA data was suggestive that the upstream regulators affected during Cm infection of OE-WT cells were more likely to attenuate chlamydial replication and promote growth of epithelial tissue; whereas, Cm infection in TLR3-deficient OE cells showed activation or inhibition in upstream regulators controlling 3-4 times the number of pathways involved in organ inflammation, fibrosis, and necrosis of epithelial tissue than that in the Cm-infected OE-WT cells. Collectively, the transcriptome and IPA results further validates our previous findings that TLR3 could elicit and regulate host protective cellular responses that limit bacterial proliferation and genital tract pathologies caused by Chlamydia infection [26, 59], and implicate TLR3 as a potential therapeutic target for drug and/ or vaccine development.
The authors wish to thank Dr. Santosh K. Maurya of Sanford Burnham Prebys Medical Discovery Institute at Lake Nona, Orlando, Florida, USA for the IPA analysis. This work was supported by National Institutes of Health (NIH) Grant AI104944 and funding from IUSM/CTSI Biomedical Research Grant.
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Igietseme JU, Portis JL, Perry LL. Inflammation and clearance of Chlamydia trachomatis in enteric and nonenteric mucosae. Infect Immun. 2001; 69(3): 1832-40. doi: 10.1128/IAI.69.3.1832-1840.2001
Kelly KA, Chan AM, Butch A, Darville T. Two different homing pathways involving integrin beta7 and E-selectin significantly influence trafficking of CD4 cells to the genital tract following Chlamydia muridarum infection. Am J Reprod Immunol. 2009; 61(6): 438-45. doi: 10.1111/j.1600-0897.2009.00704.x
Refaat BA, Bahathiq AO, Sockanathan S, Stewart RL, Wells M, Ledger WL. Production and localization of activins and activin type IIA and IIB receptors by the human endosalpinx. Reproduction. 2004; 128(2): 249-55. doi: 10.1530/rep.1.00156
Refaat B, Al-Azemi M, Geary I, Eley A, Ledger W. Role of activins and inducible nitric oxide in the pathogenesis of ectopic pregnancy in patients with or without Chlamydia trachomatis infection. Clin Vaccine Immunol. 2009; 16(10): 1493-503. doi: 10.1128/CVI.00221-09
Jansen AFM, Schoffelen T, Textoris J, et al. Involvement of matrix metalloproteinases in chronic Q fever. Clin Microbiol Infect. 2017; 23(7): 487 e7- e13. doi: 10.1016/j.cmi.2017.01.022
Bellmann-Weiler R, Schroll A, Engl S, et al. Neutrophil gelatinaseassociated lipocalin and interleukin-10 regulate intramacrophage Chlamydia pneumoniae replication by modulating intracellular iron homeostasis. Immunobiology. 2013; 218(7): 969-78. doi: 10.1016/j.imbio.2012.11.004
Hop HT, Arayan LT, Huy TXN, et al. Lipocalin 2 (Lcn2) interferes with iron uptake by Brucella abortus and dampens immunoregulation during infection of RAW 264.7 macrophages. Cell Microbiol. 2018; 20(3). doi: 10.1111/cmi.12813
Zhao H, Konishi A, Fujita Y, et al. Lipocalin 2 bolsters innate and adaptive immune responses to blood-stage malaria infection by reinforcing host iron metabolism. Cell Host Microbe. 2012; 12(5): 705-16. doi: 10.1016/j.chom.2012.10.010
Swindle EJ, Metcalfe DD. The role of reactive oxygen species and nitric oxide in mast cell-dependent inflammatory processes. Immunol Rev. 2007; 217: 186-205. doi: 10.1111/j.1600-065X.2007.00513.x
Eisenreich W, Heesemann J, Rudel T, Goebel W. Metabolic host responses to infection by intracellular bacterial pathogens. Front Cell Infect Microbiol. 2013; 3: 24. doi: 10.3389/fcimb.2013.00024
Prusty BK, Bohme L, Bergmann B, et al. Imbalanced oxidative stress causes chlamydial persistence during non-productive human herpes virus coinfection. PLoS One. 2012; 7(10): e47427. doi: 10.1371/journal. pone.0047427
Wang C, van Ginkel FW, Kim T, et al. Temporal delay of peak T-cell immunity determines Chlamydia pneumoniae pulmonary disease in mice. Infect Immun. 2008; 76(11): 4913-23. doi: 10.1128/IAI.00569-08
Storch J, Thumser AE. Tissue-specific functions in the fatty acid-binding protein family. J Biol Chem. 2010; 285(43): 32679-83. doi: 10.1074/jbc.R110.135210
Walenna NF, Kurihara Y, Chou B, Ishii K, Soejima T, Itoh R, et al. Chlamydia pneumoniae exploits adipocyte lipid chaperone FABP4 to facilitate fat mobilization and intracellular growth in murine adipocytes. Biochem Biophys Res Commun. 2018; 495(1): 353-9. doi: 10.1016/j.bbrc.2017.11.005
Li CC, Wu TS, Huang CF, et al. GTP-binding-defective ARL4D alters mitochondrial morphology and membrane potential. PLoS One. 2012; 7(8): e43552. doi: 10.1371/journal.pone.0043552
Hu Y, Nguyen TT, Bui KC, Demello DE, Smith JB. A novel inflammationinduced ubiquitin E3 ligase in alveolar type II cells. Biochem Biophys Res Commun. 2005; 333(1): 253-63. doi: 10.1016/j.bbrc.2005.05.102
Jaillon S, Moalli F, Ragnarsdottir B, et al. The humoral pattern recognition molecule PTX3 is a key component of innate immunity against urinary tract infection. Immunity. 2014; 40(4): 621-32. doi: 10.1016/j.immuni.2014.02.015
Magrini E, Mantovani A, Garlanda C. The Dual Complexity of PTX3 in Health and Disease: A Balancing Act?. Trends Mol Med. 2016; 22(6): 497-510. doi: 10.1016/j.molmed.2016.04.007
Harslund J, Frees D, Leifsson PS, Offenberg H, Romer MU, Brunner N, et al. The role of Serpine-1 and Tissue inhibitor of metalloproteinase type-1 in early host responses to Staphylococcus aureus intracutaneous infection of mice. Pathog Dis. 2013; 68(3): 96-104. doi: 10.1111/2049-632X.12055
Major LD, Partridge TS, Gardner J, et al. Induction of SerpinB2 and Th1/Th2 modulation by SerpinB2 during lentiviral infections in vivo. PLoS One. 2013; 8(2): e57343. doi: 10.1371/journal.pone.0057343
Renckens R, Roelofs JJ, Bonta PI, et al. Plasminogen activator inhibitor type 1 is protective during severe Gram-negative pneumonia. Blood. 2007; 109(4): 1593-601. doi: 10.1182/blood-2006-05-025197
Schroder WA, Le TT, Major L, et al. A physiological function of inflammation-associated SerpinB2 is regulation of adaptive immunity. J Immunol. 2010; 184(5): 2663-70. doi: 10.4049/jimmunol.0902187
Shea-Donohue T, Zhao A, Antalis TM. SerpinB2 mediated regulation of macrophage function during enteric infection. Gut Microbes. 2014; 5(2): 254-8. doi: 10.4161/gmic.28093
Pilla-Moffett D, Barber MF, Taylor GA, Coers J. Interferon-Inducible GTPases in Host Resistance, Inflammation and Disease. J Mol Biol. 2016; 428(17): 3495-513. doi: 10.1016/j.jmb.2016.04.032
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Application: Spray HEAVEN FOR LEATHER directly onto the leather making sure to avoid soaking any stitching. Agitate the cleanser gently with a soft detailing brush to create light foam and remove dirt and grime. Allow the leather to absorb the foam and when the surface of the leather is dry, wipe with a clean, damp microfibre cloth.


text:
Q: Using Excel VBA to run a CMD command I would like to use Excel VBA to run a CMD command.
The basic cmd command is:
"C:\Program Files\example program.exe" -w C:\Program files\outputfile.file "dir1" "dir2" "dir n+1"

The first part is the location of the program that will merge the files together.
The second part is the file location of the outputted merged files.
And the "dir1".... is the files that will be merged together.
I have code that lists the files to be merged but struggling to get the CMD code to get it so it does what I want as mentioned above. I have tried the following:
   Sub RunCMD()

        Dim wsh As Object
        Set wsh = VBA.CreateObject("WScript.Shell")
        Dim waitOnReturn As Boolean: waitOnReturn = True
        Dim windowStyle As Integer: windowStyle = 1
        Dim locationofprogram as string
       'dir of program that will do the merge
        Dim outputfolder as string
       'dir of where the merged file will go
        Dim listoffiles as string
       'list of files to be merged

        wsh.Run "cmd.exe /S /C locationofprogram & " " & "-w" & " " & outputfolder & " " & listoffiles, windowStyle, waitOnReturn

    End Sub

Thanks for the help!

A: You can always create a .bat file, run the file, then delete it.
MyFile = "C:\Bat-File\Bat-File.bat"

fnum = FreeFile()
Open MyFile For Output As #fnum
Print #fnum, "C:\Program Files\example program.exe -w C:\Program files\outputfile.file dir1 dir2 dir n+1"
Close #fnum

Shell MyFile, vbNormalFocus

' wait 10 seconds to let bat file finnish
newHour = Hour(Now())
newMinute = Minute(Now())
newSecond = Second(Now()) + 10
waitTime = TimeSerial(newHour, newMinute, newSecond)
Application.Wait waitTime

' delete bat-file
kill MyFile

I can't test that example program runs without the " around the dir´s so you have to see what happens.
But if it does not work you need to double enclose the " or use & CHR(34) &

A: If in doubt, send the text to the Immediate Window to see if it still matches the syntax using:
 Debug.Print "cmd.exe /S /C " & locationofprogram & " " & "-w" & " " & outputfolder & " " & listoffiles

If that works, use:
 wsh.Run "cmd.exe /S /C " & locationofprogram & " " & "-w" & " " & outputfolder & " " & listoffiles, windowStyle, waitOnReturn

redpajama_set_name: RedPajamaStackExchange


text:
Cipla 476.80 (-0.7%)
IndusInd Bank 1331.60 (-0.03%)
Maruti Suzuki 7367.65 (-1.1%)
Pakistan Foreign Minister's letter to UN warns of security deterioration
Updated : February 23, 2019 03:01 PM IST
Pakistan's foreign minister has appealed to the U.N. Security Council to draw attention to Indian threats of force in the wake of the Pulwama suicide bombing that killed more than 40 Indian soldiers in disputed Kashmir.
Shah Mahmood Qureshi in a letter to the Security Council on Friday warned that the security situation in the region is deteriorating as India threatens to use force against Pakistan.
Pakistan has denied any involvement in the attack.
The Pulwama attack last week escalated tensions between the two nuclear-armed South Asian neighbours and India blames Pakistan. It was the worst attack on Indian forces since the start of the Kashmir insurgency in 1989.
Tags india India- Pakistan JeM Narendra Modi Pakistan Pulwama terror attack UN
Zomato agrees to buy Uber Eats in all-stock deal

redpajama_set_name: RedPajamaCommonCrawl


Valve's new digital card game Artifact is go for launch on November 28th. They've now put up the official website with some more details as well as confirming their Linux plans.
If there was any doubt in your mind about Linux support, I spoke to Valve today about the release just to make sure. They said this in reply to my question about same-day Linux support "Yes. Confirmed for Windows, Linux and Mac.". We've had confirmation previously, but it's always good to see it hasn't changed so close to release.
What's quite interesting, is that it will have a built-in tournament feature. Valve say that Artifact will offer "complete control in creating a tournament", so we might have to run a few of our own if there's enough interest from readers.
I still can't quite get my head around it having multiple lanes like Dota 2, it seems like something that will keep you on your toes for sure. I have no doubt it will be massively popular! I'm excited to try it myself, as it looks really good! Will you be checking it out?
See more on the official site and Steam. The official site still doesn't have a lot, I expect a lot more to be revealed as the days go on.
Wait. Collectible Cards? You meant collectible hat game didn't you?
They should have delayed the Windows version by half a year. I guess that would have doubled our market share right there.
Kimyrielle They should have delayed the Windows version by half a year. I guess that would have doubled our market share right there.
As I have said before, my opinion is that they should give a free version only for linux that you can get for a limited time of a couple of months for example. Whoever gets this can play the game for free but only on linux. If he wants to play on windows/mac or does not get that in the limited time of the offer he has then to buy the game. What do you think of that idea? If I were Valve I would do that to help linux market share, not the exclusivity thing.
Little off topic, but I have had the thought that they are keeping HL3 hostage. I think they have a very well planned concept of it and how they want it to be, albeit it may evolve constantly. I think HL3 could be the card they will pull out, if MS decides to lock down Windows and only allow apps through their own store. Valve could use this as a way of damage control, by only releasing HL3 for Linux and possibly MacOS, as I am certain people would switch OS the same day it was released.
... since I don't believe in "forcing" people, but instead encouraging people.
I can't really see a card game being exclusive to Linux being a big driving factor in people abandoning Windows/OSX, even if said card game is made by Valve.
Personally, I have no interest in this.


text:
Los Angeles Studio Future-Proofs VFX Workflows with AWS and Spins Up 40K+ Cores for Complex Ad Projects
Framestore has captivated audiences since 1986, growing from a modest London-based visual effects studio into a multi-award-winning creative studio with six offices around the world and a talent pool of more than 2,400 artists and producers. A consistently smart and innovative use of technology has been central to the company's core mission since it was conceived, and it has continued to abide by that tenet as their scope of work evolved to encompass high-profile feature films, commercials, episodic television, and immersive projects. AWS has been an on-going presence in that evolution, beginning with Framestore's migration of its internal production management system, Front, to the cloud with AWS when it became too unwieldy to host locally, and continuing with the studio's use of AWS for cloud rendering.
Front is a proprietary suite of asset management tools for the easy review, approval, and delivery of content that sits at the heart of Framestore's workflow for nearly all of its projects across offices (with the exception of feature films). Currently, Front is entirely hosted on Amazon Elastic Compute Cloud (EC2), with Amazon Simple Storage Service (S3) used to store more than 150 terabytes of assets. Amazon CloudFront content delivery network (CDN) helps expedite the delivery of video content at all the various stages of completion, from work-in-progress to final product.
"From a logistical standpoint, our primary goal is to get content from edit to the client's inbox as quickly as possible. We brought Front to AWS about ten years ago and have integrated more AWS services into our workflow as they became available," shared Framestore Global Operations Manager Alan Newitt. "This has allowed us to easily scale and not worry about storage as we've opened more offices. We have the infrastructure in place on AWS to just fire up what we need; it would have been far more challenging if we were trying to continually expand with on-premises resources."
Building on that legacy of success with AWS, Framestore now also uses Amazon EC2 Spot Instances for rendering content out of its Los Angeles studio. After winning multiple advertising projects with high compute needs in early 2018, Framestore looked for ways to extend its capacity beyond its on-premises data center. Additionally, the work was to be done for a confidential client with tight security restrictions. Working closely with the content production workflow specialists at AWS Thinkbox, the studio established a secure Direct Connect into AWS regions and is now able to spin up instances based on demand, hitting upwards of 40,000 simultaneous cores during their delivery crunches. This setup allows Framestore to scale as much as they need with the highest security at a low cost.
"It is pivotal for us to have the burst capacity that AWS gives us and in a secure environment. We wouldn't be able to complete many of our sensitive, high resource projects otherwise, and the expertise of the AWS Thinkbox team was integral in implementing our hybrid workflow," said David Bees, Framestore's Lead System Engineer in Los Angeles, who also oversees the facility's advertising content production pipeline. "The AWS console has been easy to work with and we've capitalized on the available APIs to streamline the process for our needs. It's great that the experience is so customizable, from selecting CPU specs in the cloud to hooking into proprietary tools like Front. We've even done some initial testing with virtual workstations on AWS with promising results."


According to BlockShopper.com, the following residential sales were reported for the week of April 14-20, 2019.
According to BlockShopper.com, there were six reported residential sales in West Des Moines for the week of April 7-13, 2019.
According to BlockShopper.com, there were 22 reported residential sales in West Des Moines for the week of March 31-April 6, 2019.
The median sale price of a home sold in the first quarter of 2019 in West Des Moines in Dallas County rose by $10,000 while total sales decreased by 35.1%.
The median sale price of a home sold in the first quarter of 2019 in West Des Moines in Polk County rose by $14,000 while total sales decreased by 9.2%.
The median sale price of a home sold in March 2019 in West Des Moines in Dallas County rose by $5,000 while total sales decreased by 13.8%.
The median sale price of a home sold in March 2019 in Waukee fell by $23,575 while total sales decreased by 45.7%.
The median sale price of a home sold in March 2019 in West Des Moines in Polk County rose by $7,000 while total sales decreased by 9.5%.
On April 4, 2019, Jeremy and Elizabeth Knipper purchased a home at 478 S. Blue Stone Way, W. Des Moines from Grayhawk Homes of Iowa Inc. for $381,570.
On April 3, 2019, Finn 5772 LLC sold their three bedroom, 2.5 bathroom home at 5772 Gallery Court, W. Des Moines to Bradley L. and Sherri Renberg for $6,150,000.
According to BlockShopper.com, there were 13 reported residential sales in West Des Moines for the week of March 17-23, 2019.
The median sale price of a home sold in February 2019 in West Des Moines in Dallas County rose by $3,800 while total sales decreased by 40.9%.
The median sale price of a home sold in February 2019 in Waukee fell by $54,100 while total sales remained unchanged with 21 homes sold.
The median sale price of a home sold in February 2019 in West Des Moines in Polk County rose by $18,000 while total sales decreased by 20.4%.
Real estate agents and husband-and-wife team Rick and Marcia Wanamaker of Iowa Realty say on their website that they provide "outstanding, no-pressure customer service."


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If you're reading this blog, I'm hoping you're somewhat familiar with the concept of user stories (if not, here's a good place to start).
A key concept around user stories is that they should ideally embody 7 properties, which are usually represented by the acronym INVEST. What's NOT often talked about is that creating stories that follow all 7 of the guidelines in the INVEST acronym simultaneously is actually pretty hard. The various "generally good" properties can often be in tension, and getting stories to follow one can often mean trading off another.
I don't think enough time is spent thinking about why following all the tenets of INVEST is difficult. I don't think teams always recognize clearly that they ARE making tradeoffs, and that they could make different ones. And I don't think all teams communicate well about those tradeoffs are, why they've made the choices they have, and understand what might cause them to reconsider.
This week, I want to talk about the most important tensions I see in INVEST. I want to raise awareness that some of these tradeoffs are hard, and present some ways of thinking about how to make the right tradeoffs for your project.
INDEPENDENT. Good stories should not have implicit dependencies on other stories. This is important because it gives us one of the key advantages of Agile - that our product owner should be free to choose the order in which to play the stories based on business value. When stories have dependencies, there's an implicit sequencing - you can't play story D until after A, B, and C.
NEGOTIABLE. Stories should describe the business problem rather than a specific implementation. This allows negotiation/back-and-forth between the skilled development team and the product owner on what the solution might look like. Non-negotiable stories lead to "because I said so" development, where the team does what they're told without having input. It's also a leading cause of "it's what I asked for but not what I need."
VALUABLE. Every story should have a clear tie to why it delivers business value. If a story doesn't deliver meaningful business value, why should we spend time on it? This is often expressed in the "so that..." or "in order to..." clause of the one-sentence story formulation. Stories that don't express value clearly are likely to be deprioritized by the product owner (or, if we actually do them, frustrate the product owner by doing something they don't care about). Related - "valuable" stories are our method for having "thin threads through the system" - end-to-end slices of useful end-user functionality. If we don't focus on keeping stories valuable, we can wind up splitting "build the database for X" from "build the UI for X" into different stories, neither of which is actually useful to an end-user.
ESTIMABLE. Stories should be sufficiently specific that the development team can estimate the relative complexity of implementing that story. Stories that can't be estimated are difficult to fit into iterations, because they take away our ability to determine what's "reasonable" in a given timeframe.
SMALL. Stories should be as small as reasonably possible. A single story that takes 4 weeks to implement is not only difficult to fit into a single iteration, but it also reduces our ability to track our own progress. It's much easier to track done/not done than it is to track "I'm 45% complete with this story." Keeping stories small makes planning easier, prioritization easier, execution easier.
TESTABLE. It should be obvious to everyone on the team (including the product owner) when a story is complete, and the team can move on to another story. This means we need agreed to, testable conditions of completeness. This is often expressed in terms of acceptance criteria (e.g. an "I will know I am done when...." list, a set of "Given/When/Then" criteria, or any other agreed-on formulation).
I believe there are a wealth of ways that these items can come into conflict with each other, but here are some of the more common tensions I've seen.
The single most common tension I see on teams is trading off Small vs. Valuable.
"Valuable" stories have a clear tie to something that delivers clear business value. i.e. we know why the story makes the user's life easier. "Valuable" is often the Product Owner's domain on the team - the Product Owner is often the person articulating the business value, and needs to understand it to help the team prioritize effectively.
The tension arises because, with a little creative thinking, any story can almost always be split into smaller pieces (i.e. there's no "smallest POSSIBLE size"). Teams can generally choose the granularity they want. However, the smaller we split the story, the harder it is to maintain a clear grasp on how each individual piece delivers business value. If we split far enough, we'll pass the point where our Product Owner can understand why a given piece of work is important to deliver value, and we won't be able to prioritize effectively.
For example, let's say we have a story like "As a customer, I want to view my shopping cart online prior to checkout, so that I know what I'm buying." But that story is estimated at six weeks. Maybe we break it up into "view order basics," "view items," "view item quantities," "view total price for each item," "view order subtotal," "view order taxes," and "view estimated shipping charges." OK, still reasonably clear how all those items are valuable. Then "view estimated shipping charges" has to break down into "view shipping charges for ground items only," "view shipping charges for air-eligible items," "view shipping charges when there are hazardous items," "view shipping charges for international items..."
At some point, we pass the threshold where the set of stories we break down into are so far removed from the thing the product owner actually wants ("I want to see my shopping cart contents before checkout") that it becomes difficult-to-impossible for us to clearly see the value proposition for each one. Asking the product owner to prioritze the 12 stories related to seeing the shopping card against the 15 stories for checkout and the 29 stories for selecting items is a very difficult task. On the other hand, asking the development team to work on a project that has only 3 huge stories (because they're the things the product owner cares about) is likely to have serious planning and workflow issues.
How do we reduce this kind of tension? First, if nothing else, be aware that the finer we carve stories into pieces, the harder each is to prioritize (and vice versa). Second, most teams I've seen be successful target a certain story size. If you're one of those teams, talk about how that's working in your retrospectives. Don't be afraid to suggest your stories are too large to execute (or so small that we can't get a clear prioritization). Third, ensure that WHEN you split stories, the team is focused on ensuring that each sub-story is valuable (e.g. do NOT split "build the UI" from "build the business logic.) Fourth, consider how you parallelize stories - is a story that's "too big" something that could have two developers (or two pairs if you're pairing) work on different pieces at the same time? And if so, does that make more sense than splitting into two smaller stories we need to torture to express as "valuable"?
I think a bigger issue here is whether User Stories are in fact really the right unit for BOTH prioritization and workflow, but that's my article for next week, so stay tuned.
A central principal for most Agile teams is to defer decision making until the "last responsible moment" - the moment when we have to make the decision in order to avoid future pain. Having negotiable stories is the embodiment of this principal - we deliberately elect to defer deciding on implementation details until we've come to the point where we can't reasonably proceed without those details.
As noted above, we can in principal split stories ad infinitum if we wish, and often will do so to ensure stories are small enough to fit our "standard" granularity. And (as we just discussed) one thing we want to ensure we do when splitting stories is to keep each of the substories valuable. It's very easy, however, for this to mean that we lose negotiability - as we split a story into smaller pieces, we start making decisions on how we're going to implement the story, and each piece becomes a story.
For example, we might have a story like "As a returning customer, I want to be recognized during a future puchase, so that I won't have to re-enter all my information." If that story is too large, we might break into stories like "As a returning customer, I want to provide a username and password when I check out, so that my previously used information can be retrieved," and "as a returning customer who logged in successfully, I want to select a previously used shipping address during checkout, so I don't have to re-enter the data," and "as a new customer, I want to be prompted to optionally register an account and provide a password during checkout, so that I can be recognized in the future.
All these sub-stories are potentially small, and all seem valuable. But notice that we have a lot more decisions made about how the feature will be implemented than we had in the original story. We're using a username and password (as opposed to, say, storing the information in a cookie on the user's machine for later retrieval). We added a step the checkout process to choose a password (as opposed to, say, sending an e-mail with the option to register post-purchase). We have the ability to select from multiple previously used shipping addresses (as opposed to the most recent one).
All these decisions might be correct, and this might be the best implementation. But by breaking down the story this way, we've clearly implied a number of details of the implementation, and so reduced the negotiability of the story. Creative ideas the developers might have (for example, using a third-party ID tool instead of rolling our own) may be squeezed out.
Sorry, Small. I know I'm picking on you, but you're hard to do well.
As mentioned above with "Small vs. Negotiable," one common artifact of the process of breaking a story into smaller, valuable pieces is to make certain technical decisions up front, thus reducing the overall negotiability of a feature. A related common practice is break a given story up into smaller pieces by creating smaller stories that need to be done in a required order to be meaningful, which means they're not longer independently prioritizable.
For example, if we're building what we've decided is a three-step registration wizard, we might break it up into the logical steps the user goes through, like "Provide personal contact details in the wizard," "Provide address details in the wizard," "Provide payment details in the wizard," "Check the data collected by the wizard for errors," and "Create the profile based on wizard data." As noted above, this breakdown is less negotiable, but lets say we talked it over with our developers, and we all agree this is the right way to do registration.
Now we might have a different problem, which is that the stories are assuming they need to be done in order. If the only way to get to the "Payment Details" is from a button on the "Provide personal contact details," then logically we need to do contact details first. And we probably can't do "check for errors" or "submit" until we've collected all the data.
The problem with creating "flows" of stories like this is two fold. First, we've reduced our ability to reconsider pieces of the flow - if we decide later "You know what? Storing payment data is a security risk we don't want to take, and it's not really hard to re-enter it, so let's cut that story," then we have to re-work (at least) the "check for errors" and "submit" stories, and maybe others. Second, by having flows, we can "trap" ourselves for development - if the stories MUST be done in an order, then we can't do story 2 until story 1 is done, and can't do story 3 until after story 2. This means we "single thread" on these stories, which means we can't have more than one in play at a time (since they depend on each other). This can lead to long lead times.
The first thing I'd consider to reduce this tension is thinking about whether we've actually split the stories the right way, or if there's a different split that allows more independence. The piece that's suspicious to me here is the separate "check for errors" and "submit." A possibly better split would be "collect personal information from a wizard and save it to a profile," "collect address information from a wizard and save it to a profile," and "collect payment information from a wizard and save it to a profile." Rather than needing to build up a whole profile before saving, add the mechanism with each piece to error check and save that piece. If we want to do the "payment" story first, so be it. We might have (at least temporarily) profiles that are just anonymous payment information - is that actually a problem, or just a different way of thinking about profiles? That's not to say EVERY dependency issue can be resolved by a different splitting of stories, but it's sometimes the case that we block ourselves by thinking too narrowly about our options (in my example, thinking of the "profile" as an atomic thing that we need to build completely before submitting).
When stories are negotiable, a range of potential solutions are possible, so long as we solve the underlying business problem. However, a "range of possible solutions" can be difficult-to-impossible to estimate.
For example, a story to "provide feedback to a user when they are missing required data elements on the registration form" could be as simple as re-displaying the form with the missing fields highlighted in red, or could be as complicated as providing a wizard to guide the user through the form, with step-by-step instructions translated into the user's language.
It would be nearly impossible for a reasonable development team to provide a single estimate that covers both ends of this spectrum that has any degree of accuracy. On the other hand, if we decide that, in order to be estimable, we will choose to estimate the first option (redisplay with a color highlight), then we've reduced negotiability and made a choice that we might regret later (when we have a great framework we need in 6 other places to provide text guidance, but we have to do something different here because "the story says to."
To resolve some of this tension, a good practice is to separate the necessary (the business problem and the acceptance criteria) from the assumed (the specific implementation we're assuming in order to estimate). I like to keep a separate "estimating assumptions" section in stories, where we clearly record what assumptions our estimate was based on. Related, we need to revisit these estimating assumptions - if it becomes clear that the way we're going to implement the story is different from what we assumed earlier, we should revisit whether the estimate still makes sense. Finally, we need to set the expectation with the team that solving the business problem is more important that justifying our estimate - if there's a better way to solve the problem than the one we assumed when we estimated, we will pursue the better solution rather than justify our estimate.
I actually see two separate sources of tension here I want to tease apart.
First, similar to the tension with Negotiable vs. Small, there's a temptation to assume a specific implementation in our acceptance criteria for a story, either to make the story more concrete, or because there's only one possible solution in the mind of the person writing the acceptance criteria, and they don't realize other solutions are possible.
Second, similar to the tension with Negotiable vs. Estimable, a story that's highly open to a variety of options is very difficult to plan specific test cases for. We can't plan what we're going to test if we don't know exactly what the code is going to do yet.
For the first tension, I think it's important to think about our acceptance criteria as different entities from the tests we're going to execute to ensure the code works properly. Acceptance criteria ought to be "properties that EVERY acceptable solution to the problem must have." It takes thought to make our acceptance tests implementation agnostic. It also introduces a translation step - when we test the actual implementation, we need a mapping from the abstract "thing we need" to the specific "what we're going to do."
GIVEN I have not provided all the required registration information, WHEN I attempt to register, THEN I should get clear feedback that my information is incomplete AND I should be told what additional information is required.
GIVEN that I have not filled our some required fields on my registration form, WHEN I submit the form, THEN I should see my form re-displayed with an error message at the top and the missing fields highlighted in red.
The first form is relatively implementation agnostic. Multiple implementations could satisfy it. However, it's not easily executable - to actually test this, we need to know what "required information" is, what "attempting to register" means, what "feedback" will look like, etc. The second formulation is considerably closer to a runnable test case. However, it assumes a number of implementation elements - a "form," some kind of "submit" action, "fields" to highlight, and a separate "error message" at the top.
The second tension is more a question of when we need to do the translation from an abstract "need this to be true" into "we will do the following to know that it's true." It's virtually impossible to practically test a user story if you don't know whether you're submitting a form full of data or retrieving information from a third-party repository. At some point, we need tests specific to our chosen implementation.
There are a few things I suggest to lessen this tension. First, just as I suggested separating the "necessary" from the "assumed" when talking about Negotiable vs. Estimable, I suggest separating Acceptance Criteria (describing the necessary) from Acceptance Tests (describing how we'll verify we meet the acceptance criteria). Ideally, we present the developers with the Acceptance Criteria, and work out the details of the Acceptance Tests with them when we're ready to implement the story and so have to pick a specific design. Related - just as it's often an anti-pattern to have too large a backlog of fully fleshed out user stories, we should resist the temptation to build significant test plans for stories we haven't yet begun to work on. One technique I've found successful with multiple teams is to have a "story kick-off" with the analyst, testers, and developers (and ideally product owner) when we start development on a story. This ensures we have a common understanding of what the goals of the story are, and that the developers can articulate their expected vision for the implementation of that story. This allows the testers to develop more concrete test cases to the actual design "just in time" when the actual design is known.
What I hope I've accomplished in an overly-long blog post is at least illustrate WHY there are potential tensions, and that no matter what you're doing on your team, you ARE making some of these tradeoffs. If things are going well, you're probably making the tradeoffs that work well for your team, so well done. If things are frustrating, hopefully I've suggested some places to look and some balances you might want to revisit.
Good stuff, Mike! Lately I have been advocating that there's a priority order when thinking about stories and Valuable and Testable are tier one, Small and Independent come second and Estimable and Negotiable come third. It doesn't mean that you can easily disregard the later, just that when breaking stories down, they should pass the "Valuable & Testable" test first and so on. Usually, on a second "Independent & Small" pass, stories are broken down and design decisions are made, and finally "Estimable & Negotiable" come into play in some way, shape or form to settle everything. Of course it may not work in some settings, but I have been quite happy with results.
The Power of "I Don't Care"