Impact of socioeconomic status on healthy immune responses in humans.

Individuals with low socioeconomic status (SES) are at greater risk of contracting and developing severe disease compared with people with higher SES. Age, sex, host genetics, smoking and cytomegalovirus (CMV) serostatus are known to have a major impact on human immune responses and thus susceptibility to infection. However, the impact of SES on immune variability is not well understood or explored. Here, we used data from the Milieu Intérieur project, a study of 1000 healthy volunteers with extensive demographic and biological data, to examine the effect of SES on immune variability. We developed an Elo-rating system using socioeconomic features such as education, income and home ownership status to objectively rank SES in the 1000 donors. We observed sex-specific SES associations, such as females with a low SES having a significantly higher frequency of CMV seropositivity compared with females with high SES, and males with a low SES having a significantly higher frequency of active smoking compared with males with a high SES. Using random forest models, we identified specific immune genes which were significantly associated with SES in both baseline and immune challenge conditions. Interestingly, many of the SES associations were sex stimuli specific, highlighting the complexity of these interactions. Our study provides a new way of computing SES in human populations that can help identify novel SES associations and reinforces biological evidence for SES-dependent susceptibility to infection. This should serve as a basis for further understanding the molecular mechanisms behind SES effects on immune responses and ultimately disease.

Human Granzyme K Is a Feature of Innate T Cells in Blood, Tissues, and Tumors, Responding to Cytokines Rather than TCR Stimulation.

NK cells and CD8 T cells use cytotoxic molecules to kill virally infected and tumor cell targets. While perforin and granzyme B (GzmB) are the most commonly studied lytic molecules, less is known about granzyme K (GzmK). However, this granzyme has been recently associated with improved prognosis in solid tumors. In this study, we show that, in humans, GzmK is predominantly expressed by innate-like lymphocytes, as well as a newly identified population of GzmK+CD8+ non- mucosal-associated invariant T cells with innate-like characteristics. We found that GzmK+ T cells are KLRG1+EOMES+IL-7R+CD62L-Tcf7int, suggesting that they are central memory T and effector memory T cells. Furthermore, GzmK+ cells are absent/low in cord blood, suggesting that GzmK is upregulated with immune experience. Surprisingly, GzmK+ cells respond to cytokine stimuli alone, whereas TCR stimulation downregulates GzmK expression, coinciding with GzmB upregulation. GzmK+ cells have reduced IFN-γ production compared with GzmB+ cells in each T cell lineage. Collectively, this suggests that GzmK+ cells are not naive, and they may be an intermediate memory-like or preterminally differentiated population. GzmK+ cells are enriched in nonlymphoid tissues such as the liver and adipose. In colorectal cancer, GzmK+ cells are enriched in the tumor and can produce IFN-γ, but GzmK+ expression is mutually exclusive with IL-17a production. Thus, in humans, GzmK+ cells are innate memory-like cells that respond to cytokine stimulation alone and may be important effector cells in the tumor.

Evolutionary selection identifies critical immune-relevant genes in lung cancer subtypes.

In an evolving population, proliferation is dependent on fitness so that a numerically dominant population typically possesses the most well adapted phenotype. In contrast, the evolutionary "losers" typically disappear from the population so that their genetic record is lost. Historically, cancer research has focused on observed genetic mutations in the dominant tumor cell populations which presumably increase fitness. Negative selection, i.e., removal of deleterious mutations from a population, is not observable but can provide critical information regarding genes involved in essential cellular processes. Similar to immunoediting, "evolutionary triage" eliminates mutations in tumor cells that increase susceptibility to the host immune response while mutations that shield them from immune attack increase proliferation and are readily observable (e.g., B2M mutations). These dynamics permit an "inverse problem" analysis linking the fitness consequences of a mutation to its prevalence in a tumor cohort. This is evident in "driver mutations" but, equally important, can identify essential genes in which mutations are seen significantly less than expected by chance. Here we utilized this new approach to investigate evolutionary triage in immune-related genes from TCGA lung adenocarcinoma cohorts. Negative selection differs between the two cohorts and is observed in endoplasmic reticulum aminopeptidase genes, ERAP1 and ERAP2 genes, and DNAM-1/TIGIT ligands. Targeting genes or molecular pathways under positive or negative evolutionary selection may permit new treatment options and increase the efficacy of current immunotherapy.

Liver Immunology, Immunotherapy, and Liver Cancers: Time for a Rethink?

The complex immune system of the liver has a major role in tumor surveillance, but also partly explains why current immune therapies are poorly effective against liver cancers. Known primarily for its tolerogenic capacity, the hepatic immune repertoire also comprises diverse populations of armored immune cells with tumor surveillant roles. In healthy people, these work together to successfully identify malignant cells and prevent their proliferation, thus halting tumor formation. When frontline hepatic immune surveillance systems fail, compromised hepatic immunity, driven by obesity, infection, or other pathological factors, allows primary or secondary liver cancers to develop. Tumor growth promotes the normal tolerogenic immunological milieu of the liver, perhaps explaining why current immunotherapies fail to work. This review explores the complex local liver immune system with the hope of identifying potential therapeutic targets needed to best overcome immunological barriers in the liver to create an environment no longer hostile to immunotherapy for the treatment of liver cancer.

Mid-luteal uterine artery Doppler indices in the prediction of pregnancy outcome in nulliparous women undergoing assisted reproduction.

Traditionally, the assessment of endometrial receptivity at transvaginal ultrasound scan has been based on the thickness and the morphological appearance of the endometrium. The objective of this study was to prospectively evaluate endometrial thickness (ET), endometrial morphology and uterine artery Doppler parameters prior to assisted reproduction treatment (ART) in the prediction of pregnancy outcome. This was a prospective cohort study. ET, morphology and uterine artery Doppler (UtAD) pulsatility index (PI) and resistance index (RI) were measured in the mid-luteal stage of the menstrual cycle ultrasonographically, timed with urinary luteinizing hormone testing. A total of 50 women were included in the analysis. The clinical pregnancy rate (CPR) per embryo transfer was 42.0% (n = 21/50). Twenty nine women (58.0%) had an unsuccessful outcome. There were no differences in mean ± SD endometrial thickness (ET) (10.0 ± 1.8 mm vs. 10.5 ± 2.4; p = 0.43), or endometrial morphology (100% (n = 21) vs 100% (n = 29); p = 1.00) between the pregnant and not pregnant groups. Similarly, there were no differences in mean ± SD UtAD PI (2.17 ± 0.83 vs. 2.07 ± 0.81; p = 0.67 or mean ± SD UtAD RI (0.84 ± 0.10 vs. 0.81 ± 0.10; p = 0.30). Ultrasonographic endometrial assessment did not differentiate between those who would have a subsequent clinical pregnancy.

Predator-Prey in Tumor-Immune Interactions: A Wrong Model or Just an Incomplete One?

Tumor-immune interactions are often framed as predator-prey. This imperfect analogy describes how immune cells (the predators) hunt and kill immunogenic tumor cells (the prey). It allows for evaluation of tumor cell populations that change over time during immunoediting and it also considers how the immune system changes in response to these alterations. However, two aspects of predator-prey type models are not typically observed in immuno-oncology. The first concerns the conversion of prey killed into predator biomass. In standard predator-prey models, the predator relies on the prey for nutrients, while in the tumor microenvironment the predator and prey compete for resources (e.g. glucose). The second concerns oscillatory dynamics. Standard predator-prey models can show a perpetual cycling in both prey and predator population sizes, while in oncology we see increases in tumor volume and decreases in infiltrating immune cell populations. Here we discuss the applicability of predator-prey models in the context of cancer immunology and evaluate possible causes for discrepancies. Key processes include "safety in numbers", resource availability, time delays, interference competition, and immunoediting. Finally, we propose a way forward to reconcile differences between model predictions and empirical observations. The immune system is not just predator-prey. Like natural food webs, the immune-tumor community of cell types forms an immune-web of different and identifiable interactions.

Bovine innate immune phenotyping via a standardized whole blood stimulation assay.

Cattle vary in their susceptibility to infection and immunopathology, but our ability to measure and longitudinally profile immune response variation is limited by the lack of standardized immune phenotyping assays for high-throughput analysis. Here we report longitudinal innate immune response profiles in cattle using a low-blood volume, whole blood stimulation system-the ImmunoChek (IChek) assay. By minimizing cell manipulation, our standardized system minimizes the potential for artefactual results and enables repeatable temporal comparative analysis in cattle. IChek successfully captured biological variation in innate cytokine (IL-1ß and IL-6) and chemokine (IL-8) responses to 24-hr stimulation with either Gram-negative (LPS), Gram-positive (PamCSK4) bacterial or viral (R848) pathogen-associated molecular patterns (PAMPs) across a 4-month time window. Significant and repeatable patterns of inter-individual variation in cytokine and chemokine responses, as well as consistent high innate immune responder individuals were identified at both baseline and induced levels. Correlation coefficients between immune response read-outs (IL-1ß, IL-6 and IL-8) varied according to PAMP. Strong significant positive correlations were observed between circulating monocytes and IL-6 levels for null and induced responses (0.49-0.61) and between neutrophils and cytokine responses to R848 (0.38-0.47). The standardized assay facilitates high-throughput bovine innate immune response profiling to identify phenotypes associated with disease susceptibility and responses to vaccination.

The impact of accurately timed mid-luteal endometrial injury in nulligravid women undergoing their first or second embryo transfer.

INTRODUCTION: Endometrial injury or 'scratch' preceding an assisted reproductive therapy (ART) cycle has recently been shown not to improve livebirth rates among women undergoing ART. The objective of this study was to compare pregnancy outcomes in nulliparous women who underwent an accurately timed mid-luteal scratch biopsy prior to ART with those who did not. METHODS: This was a prospective cohort study. Women were recruited between October 2016 and February 2018 inclusive. Women who met the inclusion criteria and who did not undergo an endometrial scratch in the study period were used as a comparison group. Patients underwent a cycle of ART in the menstrual cycle following endometrial scratch. RESULTS: Ninety-eight women were eligible for participation in the study. There were no differences in rates of implantation (35.7% (n = 20/56) vs. 35.4% (n = 17/48); p = 1.00), clinical pregnancy (40.0% (n = 20/50) vs. 39.5% (n = 17/43); p = 1.00) or live birth (34.0% (n = 17/50) vs. 25.6% (n = 11/43); p = 0.50) per embryo transfer between those who underwent a scratch and those who did not. CONCLUSION: Endometrial scratch is a simple, inexpensive and low-risk procedure. However, in this relatively small cohort study, no differences in rates of implantation, clinical pregnancy or live birth in women with primary infertility were determined between those who underwent a scratch and those who did not.

Artificial selection for host resistance to tumour growth and subsequent cancer cell adaptations: an evolutionary arms race.

BACKGROUND: Cancer progression is governed by evolutionary dynamics in both the tumour population and its host. Since cancers die with the host, each new population of cancer cells must reinvent strategies to overcome the host's heritable defences. In contrast, host species evolve defence strategies over generations if tumour development limits procreation. METHODS: We investigate this "evolutionary arms race" through intentional breeding of immunodeficient SCID and immunocompetent Black/6 mice to evolve increased tumour suppression. Over 10 generations, we injected Lewis lung mouse carcinoma cells [LL/2-Luc-M38] and selectively bred the two individuals with the slowest tumour growth at day 11. Their male progeny were hosts in the subsequent round. RESULTS: The evolved SCID mice suppressed tumour growth through biomechanical restriction from increased mesenchymal proliferation, and the evolved Black/6 mice suppressed tumour growth by increasing immune-mediated killing of cancer cells. However, transcriptomic changes of multicellular tissue organisation and function genes allowed LL/2-Luc-M38 cells to adapt through increased matrix remodelling in SCID mice, and reduced angiogenesis, increased energy utilisation and accelerated proliferation in Black/6 mice. CONCLUSION: Host species can rapidly evolve both immunologic and non-immunologic tumour defences. However, cancer cell plasticity allows effective phenotypic and population-based counter strategies.

Persistent fatigue following SARS-CoV-2 infection is common and independent of severity of initial infection.

Fatigue is a common symptom in those presenting with symptomatic COVID-19 infection. However, it is unknown if COVID-19 results in persistent fatigue in those recovered from acute infection. We examined the prevalence of fatigue in individuals recovered from the acute phase of COVID-19 illness using the Chalder Fatigue Score (CFQ-11). We further examined potential predictors of fatigue following COVID-19 infection, evaluating indicators of COVID-19 severity, markers of peripheral immune activation and circulating pro-inflammatory cytokines. Of 128 participants (49.5 ± 15 years; 54% female), more than half reported persistent fatigue (67/128; 52.3%) at median of 10 weeks after initial COVID-19 symptoms. There was no association between COVID-19 severity (need for inpatient admission, supplemental oxygen or critical care) and fatigue following COVID-19. Additionally, there was no association between routine laboratory markers of inflammation and cell turnover (leukocyte, neutrophil or lymphocyte counts, neutrophil-to-lymphocyte ratio, lactate dehydrogenase, C-reactive protein) or pro-inflammatory molecules (IL-6 or sCD25) and fatigue post COVID-19. Female gender and those with a pre-existing diagnosis of depression/anxiety were over-represented in those with fatigue. Our findings demonstrate a significant burden of post-viral fatigue in individuals with previous SARS-CoV-2 infection after the acute phase of COVID-19 illness. This study highlights the importance of assessing those recovering from COVID-19 for symptoms of severe fatigue, irrespective of severity of initial illness, and may identify a group worthy of further study and early intervention.

Qualitative and quantitative differences in endometrial inflammatory gene expression precede the development of bovine uterine disease.

The transcriptome of the endometrium early postpartum was profiled to determine if inflammatory gene expression was elevated in cows which subsequently developed uterine disease. Endometrial cytobrush samples were collected at 7 days postpartum (DPP) from 112 Holstein-Friesian dairy cows, from which 27 were retrospectively chosen for RNA-seq on the basis of disease classification [ten healthy and an additional 17 diagnosed with cytological endometritis (CYTO), or purulent vaginal discharge (PVD)] at 21 DPP. 297 genes were significantly differentially expressed between cows that remained healthy versus those that subsequently developed PVD, including IL1A and IL1B (adjusted p < 0.05). In contrast, only 3 genes were significantly differentially expressed in cows which subsequently developed CYTO. Accounting for the early physiological inflammatory status present in cows which do not develop disease enhanced the detection of differentially expressed genes associated with CYTO and further expression profiling in 51 additional cows showed upregulation of multiple immune genes, including IL1A, IL1B and TNFA. Despite the expected heterogeneity associated with natural infection, enhanced activation of the inflammatory response is likely a key contributory feature of both PVD and CYTO development. Prognostic biomarkers of uterine disease would be particularly valuable for seasonal-based dairy systems where any delay to conception undermines sustainability.

Searching for Goldilocks: How Evolution and Ecology Can Help Uncover More Effective Patient-Specific Chemotherapies.

Deaths from cancer are mostly due to metastatic disease that becomes resistant to therapy. A mainstay treatment for many cancers is chemotherapy, for which the dosing strategy is primarily limited by patient toxicity. While this MTD approach builds upon the intuitively appealing principle that maximum therapeutic benefit is achieved by killing the largest possible number of cancer cells, there is increasing evidence that moderation might allow host-specific features to contribute to success. We believe that a "Goldilocks Window" of submaximal chemotherapy will yield improved overall outcomes. This window combines the complex interplay of cancer cell death, immune activity, emergence of chemoresistance, and metastatic dissemination. These multiple activities driven by chemotherapy have tradeoffs that depend on the specific agents used as well as their dosing levels and schedule. Here we present evidence supporting the idea that MTD may not always be the best approach and offer suggestions toward a more personalized treatment regime that integrates insights into patient-specific eco-evolutionary dynamics.

Human PSC-Derived Hepatocytes Express Low Levels of Viral Pathogen Recognition Receptors, but Are Capable of Mounting an Effective Innate Immune Response.

Hepatocytes are key players in the innate immune response to liver pathogens but are challenging to study because of inaccessibility and a short half-life. Recent advances in in vitro differentiation of hepatocyte-like cells (HLCs) facilitated studies of hepatocyte-pathogen interactions. Here, we aimed to define the anti-viral innate immune potential of human HLCs with a focus on toll-like receptor (TLR)-expression and the presence of a metabolic switch. We analysed cytoplasmic pattern recognition receptor (PRR)- and endosomal TLR-expression and activity and adaptation of HLCs to an inflammatory environment. We found that transcript levels of retinoic acid inducible gene I (RIG-I), melanoma differentiation antigen 5 (MDA5), and TLR3 became downregulated during differentiation, indicating the acquisition of a more tolerogenic phenotype, as expected in healthy hepatocytes. HLCs responded to activation of RIG-I by producing interferons (IFNs) and IFN-stimulated genes. Despite low-level expression of TLR3, receptor expression was upregulated in an inflammatory environment. TLR3 signalling induced expression of proinflammatory cytokines at the gene level, indicating that several PRRs need to interact for successful innate immune activation. The inflammatory responsiveness of HLCs was accompanied by the downregulation of cytochrome P450 3A and 1A2 activity and decreased serum protein production, showing that the metabolic switch seen in primary hepatocytes during anti-viral responses is also present in HLCs.

The Immune Consequences of Lactate in the Tumor Microenvironment.

The tumor microenvironment consists of complex and dynamic networks of cytokines, growth factors, and metabolic products. These contribute to significant alterations in tissue architecture, cell growth, immune cell phenotype, and function. Increased glycolytic flux is commonly observed in solid tumors and is associated with significant changes in metabolites, generating high levels of lactate. While elevated glycolytic flux is a characteristic metabolic adaption of tumor cells, glycolysis is also a key metabolic program utilized by a variety of inflammatory immune cells. As such lactate and the pH changes associated with lactate transport affect not only tumor cells but also immune cells. Here we provide an overview of lactate metabolic pathways and the effects lactate has on tumor growth and immune cell function. This knowledge provides opportunities for synergistic therapeutic approaches that combine metabolic drugs, which limit tumor growth and support immune cell function, together with immunotherapies to enhance tumor eradication.

Improved filtration method to isolate pure populations of primary bovine endometrial epithelial and stromal cells for immunological studies.

OBJECTIVES: Isolation and culture of distinct primary endometrial cells are key to reliable in-vitro models to investigate the uterine immune response and optimse new disease interventions. Details on the isolation method and purity of distinct cell populations is lacking in currently available protocols leading to inconsistent results across laboratories. METHODS: Bovine endometrial tissue from non-pregnant bovine uteri were collected immediately post-mortem and separated using differential size filtering. Isolations (n = 15) yielded an average of 3.1 × 105 ± 0.7 × 105 epithelial cells and 1.88 × 106 ± 5.44 × 105 stromal fibroblasts per uterine horn. Following expansion in culture, the purity of cell populations was confirmed using morphology and positive staining for cytokeratin and vimentin which identifies epithelial and stromal fibroblast populations, respectively. Using PCR, cDNA from both cell populations was negative for CD45, a marker of immune cells. RESULTS: On challenge with a bacterial PAMP (LPS), epithelial and stromal fibroblasts showed a marked increase in the expression of the inflammatory mediators IL8, IL6, S100A8 and S100A9, with both cell populations displaying distinct expression profiles. Here we provide a detailed methodology on the culture of primary bovine endometrial epithelial and stromal cells and demonstrate these cells provide a physiologically relevant model for studies of endometrial inflammation and its regulation.

Chemotherapy and repeat resection abrogate the prognostic value of neutrophil lymphocyte ratio in colorectal liver metastases.

BACKGROUND: Evolution in surgical and oncological management of CRLM has called into question the utility of clinical risk scores. We sought to establish if neutrophil lymphocyte ratio (NLR) has a prognostic role in this patient cohort. METHODS: From 2005 to 2015,379 hepatectomies were performed for CRLM, 322 underwent index hepatectomy, 57 s hepatectomies were performed. Clinicopathological data were obtained from a prospectively maintained database. Variables associated with longterm survival following index and second hepatectomy were identified by Cox regression analyses and reviewed along with 30-day post-operative morbidity and mortality. RESULTS: Following index hepatectomy 1-,3-and 5-year survival was 90.7%, 68.1% and 48.6%. Major resection, positive margins and >5 tumours were negatively associated with survival. Those with elevated NLR(>5) had a median survival of 55 months, compared to 70 months with lower NLR(p = 0.027). Following neoadjuvant chemotherapy, no association between NLR and survival was demonstrated (p = 0.93). Furthermore, NLR >5 had no impact on prognosis following repeat hepatectomy. Tumour diameter >5 cm (p = 0.04) was the sole predictor of poorer survival (p = 0.049). CONCLUSION: Despite elevated NLR correlating with shorter survival following index hepatectomy, this effect is negated by neoadjuvant chemotherapy and second hepatectomy for recurrent disease. This data would not support the use of NLR in the preoperative decision algorithm for patients with CRLM.

Hepatic Tumor Microenvironments and Effects on NK Cell Phenotype and Function.

The liver is a complex organ with critical physiological functions including metabolism, glucose storage, and drug detoxification. Its unique immune profile with large numbers of cytotoxic CD8+ T cells and significant innate lymphoid population, including natural killer cells, γ δ T cells, MAIT cells, and iNKTcells, suggests an important anti-tumor surveillance role. Despite significant immune surveillance in the liver, in particular large NK cell populations, hepatic cell carcinoma (HCC) is a relatively common outcome of chronic liver infection or inflammation. The liver is also the second most common site of metastatic disease. This discordance suggests immune suppression by the environments of primary and secondary liver cancers. Classic tumor microenvironments (TME) are poorly perfused, leading to accumulation of tumor cell metabolites, diminished O2, and decreased nutrient levels, all of which impact immune cell phenotype and function. Here, we focus on changes in the liver microenvironment associated with tumor presence and how they affect NK function and phenotype.

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells.

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.  Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

The hepatitis C virus (HCV) protein, p7, suppresses inflammatory responses to tumor necrosis factor (TNF)-α via signal transducer and activator of transcription (STAT)3 and extracellular signal-regulated kinase (ERK)-mediated induction of suppressor of cytokine signaling (SOCS)3.

Viruses use a spectrum of immune evasion strategies that enable infection and replication. The acute phase of hepatitis C virus (HCV) infection is characterized by nonspecific and often mild clinical symptoms, suggesting an immunosuppressive mechanism that, unless symptomatic liver disease presents, allows the virus to remain largely undetected. We previously reported that HCV induced the regulatory protein suppressor of cytokine signaling (SOCS)3, which inhibited TNF-α-mediated inflammatory responses. However, the mechanism by which HCV up-regulates SOCS3 remains unknown. Here we show that the HCV protein, p7, enhances both SOCS3 mRNA and protein expression. A p7 inhibitor reduced SOCS3 induction, indicating that p7's ion channel activity was required for optimal up-regulation of SOCS3. Short hairpin RNA and chemical inhibition revealed that both the Janus kinase-signal transducer and activator of transcription (JAK-STAT) and MAPK pathways were required for p7-mediated induction of SOCS3. HCV-p7 expression suppressed TNF-α-mediated IκB-α degradation and subsequent NF-κB promoter activity, revealing a new and functional, anti-inflammatory effect of p7. Together, these findings identify a molecular mechanism by which HCV-p7 induces SOCS3 through STAT3 and ERK activation and demonstrate that p7 suppresses proinflammatory responses to TNF-α, possibly explaining the lack of inflammatory symptoms observed during early HCV infection.-Convery, O., Gargan, S., Kickham, M., Schroder, M., O'Farrelly, C., Stevenson, N. J. The hepatitis C virus (HCV) protein, p7, suppresses inflammatory responses to tumor necrosis factor (TNF)-α via signal transducer and activator of transcription (STAT)3 and extracellular signal-regulated kinase (ERK)-mediated induction of suppressor of cytokine signaling (SOCS)3.

Non-canonical Inflammasome-Mediated IL-1ß Production by Primary Endometrial Epithelial and Stromal Fibroblast Cells Is NLRP3 and Caspase-4 Dependent.

Inflammation of the post-partum uterus is a normal physiological event, crucial for tissue involution and repair. However, in the bovine, some cows fail to resolve this inflammation, resulting in endometritis, which compromises fertility. Earlier work has identified upregulated expression of the potent inflammatory cytokine IL-1ß early post-partum, in cows which subsequently develop endometritis. As a result, targeting IL-1ß expression holds potential as a novel treatment for this disease, yet the regulatory mechanisms contributing to IL-1ß expression in the bovine endometrium remain unknown. To investigate this, endometrial tissue samples were obtained 7 and 21 days post-partum (DPP) from cows that were diagnosed with endometritis at 21 DPP and cows that experienced a physiological level of inflammation throughout involution. IL-1ß was measured by qPCR, ELISA, and immunohistochemistry. Seven DPP, endometrial IL-1ß protein levels were significantly higher in animals that proceeded to develop endometritis at 21 DPP. IL-1ß production could be detected in luminal and glandular epithelium, in underlying stromal fibroblasts as well as infiltrating immune cells. To investigate the mechanisms regulating IL-1ß expression, primary endometrial epithelial cells, stromal fibroblasts and PBMCs were stimulated with LPS and the inflammasome activator nigericin. Stromal fibroblast cells were particularly potent producers of IL-1ß. Basolateral LPS stimulation of polarized epithelial cells induced IL1B mRNA and a previously undescribed IL-1ß protein isoform, with preferential protein secretion into the apical compartment. Key inflammasome components [nod-like receptor protein 3 (NLRP3), nima-related kinase-7 (NEK7), apoptosis speck like protein containing a CARD (ASC), and gasdermin-D] were expressed by endometrial cells following stimulation. Endometrial cell stimulation in the presence of NLRP3 receptor (MCC950) and pan-caspase (Z-VAD-FMK) inhibitors blocked IL-1ß production, demonstrating its dependence on the NLRP3 inflammasome and on caspase activity. Furthermore, caspase-4 specific siRNA prevented IL-1ß production, confirming that inflammasome activation in endometrial cells is caspase-4 but not caspase-1 dependent, as shown in other species. Identifying the tissue- and species-specificity of inflammasome assembly and activation has critical relevance for our understanding of inflammation and suggests new therapeutic targets to enhance the resolution of inflammatory pathologies including endometritis in cattle.