Enhancing T-cell recruitment in renal cell carcinoma with cytokine-armed adenoviruses.

Immunotherapy has emerged as a promising approach for cancer treatment, with oncolytic adenoviruses showing power as immunotherapeutic agents. In this study, we investigated the immunotherapeutic potential of an adenovirus construct expressing CXCL9, CXCL10, or IL-15 in clear cell renal cell carcinoma (ccRCC) tumor models. Our results demonstrated robust cytokine secretion upon viral treatment, suggesting effective transgene expression. Subsequent analysis using resistance-based transwell migration and microfluidic chip assays demonstrated increased T-cell migration in response to chemokine secretion by infected cells in both 2D and 3D cell models. Flow cytometry analysis revealed CXCR3 receptor expression across T-cell subsets, with the highest percentage found on CD8+ T-cells, underscoring their key role in immune cell migration. Alongside T-cells, we also detected NK-cells in the tumors of immunocompromised mice treated with cytokine-encoding adenoviruses. Furthermore, we identified potential immunogenic antigens that may enhance the efficacy and specificity of our armed oncolytic adenoviruses in ccRCC. Overall, our findings using ccRCC cell line, in vivo humanized mice, physiologically relevant PDCs in 2D and patient-derived organoids (PDOs) in 3D suggest that chemokine-armed adenoviruses hold promise for enhancing T-cell migration and improving immunotherapy outcomes in ccRCC. Our study contributes to the development of more effective ccRCC treatment strategies by elucidating immune cell infiltration and activation mechanisms within the tumor microenvironment (TME) and highlights the usefulness of PDOs for predicting clinical relevance and validating novel immunotherapeutic approaches. Overall, our research offers insights into the rational design and optimization of viral-based immunotherapies for ccRCC.

Expression and regulation of the CXCL9-11 chemokines and CXCR3 receptor in Atlantic salmon (Salmo salar).

Chemokines are cytokines that mediate leukocyte traffic between the lymphoid organs, the bloodstream, and the site of tissue damage, which is essential for an efficient immune response. In particular, the gamma interferon (IFN- γ) inducible chemokines CXCL9, CXCL10, and CXCL11, and their receptor CXCR3, are involved in T cell and macrophage recruitment to the site of infection. The nature and function of these chemokines and their receptor are well-known in mammals, but further research is needed to achieve a similar level of understanding in fish immunity. Thus, in this study, we seek to identify the genes encoding the components of the Atlantic salmon (Salmo salar) CXCL9, CXCL10, CXCL11/CXCR3 axis (CXCL9-11/CXCR3), predict the protein structure from the amino acid sequence, and explore the regulation of gene expression as well as the response of these chemokines and their receptor to viral infections. The cxcl9, cxcl10, cxcl11, and cxcr3 gene sequences were retrieved from the databases, and the phylogenetic analysis was conducted to determine the evolutionary relationships. The study revealed an interesting pattern of clustering and conservation among fish and mammalian species. The salmon chemokine sequences clustered with orthologs from other fish species, while the mammalian sequences formed separate clades. This indicates a divergent evolution of chemokines between mammals and fish, possibly due to different evolutionary pressures. While the structural analysis of the chemokines and the CXCR3 receptor showed the conservation of critical motifs and domains, suggesting preserved functions and stability throughout evolution. Regarding the regulation of gene expression, some components of the CXCL9-11/CXCR3 axis are induced by recombinant gamma interferon (rIFN-γ) and by Infectious pancreatic necrosis virus (IPNV) infection in Atlantic salmon cells. Further studies are needed to explore the role of Atlantic salmon CXCL9-11 chemokines in regulating immune cell migration and endothelial activation, as seen in mammals. To the best of our knowledge, there have been no functional studies of chemokines to understand these effects in Atlantic salmon.

Cervical Cancer Evades the Host Immune System through the Inhibition of Type I Interferon and CXCL9 by LIF.

PURPOSE: Cervical cancer is a viral-associated tumor caused by the infection with the human papilloma virus. Cervical cancer is an immunogenic cancer that expresses viral antigens. Despite being immunogenic, cervical cancer does not fully respond to immune checkpoint inhibitors (ICI). LIF is a crucial cytokine in embryo implantation, involved in maternal tolerance that acts as an immunomodulatory factor in cancer. LIF is expressed in cervical cancer and high levels of LIF is associated with poor prognosis in cervical cancer. EXPERIMENTAL DESIGN: We evaluated the impact of LIF on the immune response to ICI using primary plasmocytoid dendritic cells (pDC) and macrophage cultures, syngeneic animals and patient-derived models that recapitulate the human tumor microenvironment. RESULTS: We found that the viral proteins E6 and E7 induce the expression of LIF via the NFκB pathway. The secreted LIF can then repress type I interferon expressed in pDCs and CXCL9 expressed in tumor-associated macrophages. Blockade of LIF promotes the induction of type I interferon and CXCL9 inducing the tumor infiltration of CD8 T cells. This results in the sensitization of the tumor to ICI. Importantly, we observed that patients with cervical cancer expressing high levels of LIF tend to be resistant to ICI. CONCLUSIONS: Our data show that the HPV virus induces the expression of LIF to provide a selective advantage to the tumor cell by generating local immunosuppression via the repression of type I interferon and CXCL9. Combinatory treatment with blocking antibodies against LIF and ICI could be effective against cervical cancer expressing high levels of LIF.

Adeno-associated virus delivered CXCL9 sensitizes glioblastoma to anti-PD-1 immune checkpoint blockade.

There are numerous mechanisms by which glioblastoma cells evade immunological detection, underscoring the need for strategic combinatorial treatments to achieve appreciable therapeutic effects. However, developing combination therapies is difficult due to dose-limiting toxicities, blood-brain-barrier, and suppressive tumor microenvironment. Glioblastoma is notoriously devoid of lymphocytes driven in part by a paucity of lymphocyte trafficking factors necessary to prompt their recruitment and activation. Herein, we develop a recombinant adeno-associated virus (AAV) gene therapy that enables focal and stable reconstitution of the tumor microenvironment with C-X-C motif ligand 9 (CXCL9), a powerful call-and-receive chemokine for lymphocytes. By manipulating local chemokine directional guidance, AAV-CXCL9 increases tumor infiltration by cytotoxic lymphocytes, sensitizing glioblastoma to anti-PD-1 immune checkpoint blockade in female preclinical tumor models. These effects are accompanied by immunologic signatures evocative of an inflamed tumor microenvironment. These findings support AAV gene therapy as an adjuvant for reconditioning glioblastoma immunogenicity given its safety profile, tropism, modularity, and off-the-shelf capability.

Antagonism of the Platelet-Activating Factor Pathway Mitigates Inflammatory Adverse Events Driven by Anti-erythrocyte Antibody Therapy in Mice.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by low platelet counts primarily due to antiplatelet autoantibodies. Anti-D is a donor-derived polyclonal Ab against the rhesus D Ag on erythrocytes used to treat ITP. Unfortunately, adverse inflammatory/hypersensitivity reactions and a Food and Drug Administration-issued black box warning have limited its clinical use. This underscores the imperative to understand the inflammatory pathway associated with anti-erythrocyte Ab-based therapies. TER119 is an erythrocyte-specific Ab with anti-D-like therapeutic activity in murine ITP, while also exhibiting a distinct inflammatory signature involving production of CCL2, CCL5, and CXCL9 but not IFN-γ. Therefore, TER119 has been used to elucidate the potential mechanism underlying the adverse inflammatory activity associated with anti-erythrocyte Ab therapy in murine ITP. Prior work has demonstrated that TER119 administration is associated with a dramatic decrease in body temperature and inflammatory cytokine/chemokine production. The work presented in the current study demonstrates that inhibiting the highly inflammatory platelet-activating factor (PAF) pathway with PAF receptor antagonists prevents TER119-driven changes in body temperature and inhibits the production of the CCL2, CCL5, and CXCL9 inflammatory cytokines in CD-1 mice. Phagocytic cells and a functional TER119 Fc region were found to be necessary for TER119-induced body temperature changes and increases in CXCL9 and CCL2. Taken together, this work reveals the novel requirement of the PAF pathway in causing adverse inflammatory activity associated with anti-erythrocyte Ab therapy in a murine model and provides a strategy of mitigating these potential reactions without altering therapeutic activity.

Increased type 1 inflammation in gynecologic cervicovaginal samples in patients with APS-1.

BACKGROUND: Inborn errors of immunity offer important insights into mucosal immunity. In autoimmune polyendocrine syndrome type-1 (APS-1), chronic mucocutaneous candidiasis has been ascribed to neutralizing IL-17 autoantibodies. Recent evidence implicates excessive T-cell IFN-γ secretion and ensuing epithelial barrier disruption in predisposition to candidiasis, but these results remain to be replicated. Whether IL-17 paucity, increased type I inflammation, or their combination underlies susceptibility to chronic mucocutaneus candidiasis in APS-1 is debated. OBJECTIVE: Our aim was to characterize the immunologic features in the cervicovaginal mucosa of females with APS-1. METHODS: Vaginal fluid was collected with a flocked swab from 17 females with APS-1 and 18 controls, and cytokine composition was analyzed using Luminex (Luminex Corporation, Austin, Tex). Cervical cell samples were obtained with a cervix brush from 6 patients and 6 healthy controls and subjected to transcriptome analysis. RESULTS: The vaginal fluid samples from patients with APS-1 had IFN-γ concentrations comparable to those of the controls (2.6 vs 2.4 pg/mL) but high concentrations of the TH1 chemokines CXCL9 and CXCL10 (1094 vs 110 pg/mL [P < .001] and 4033 vs 273 pg/mL [P = .001], respectively), whereas the IL-17 levels in the samples from the 2 groups were comparable (28 vs 8.8 pg/mL). RNA sequencing of the cervical cells revealed upregulation of pathways related to mucosal inflammation and cell death in the patients with APS-1. CONCLUSION: Excessive TH1 cell response appears to underlie disruption of the mucosal immune responses in the genital tract of patients with APS-1 and may contribute to susceptibility to candidiasis in the genital tract as well.

GPR182 limits antitumor immunity via chemokine scavenging in mouse melanoma models.

For many solid tumors, immune checkpoint blockade therapy has become first line treatment, yet a large proportion of patients with immunologically cold tumors do not benefit due to the paucity of tumor infiltrating lymphocytes. Here we show that the orphan G Protein-Coupled Receptor 182 (GPR182) contributes to immunotherapy resistance in cancer via scavenging chemokines that are important for lymphocyte recruitment to tumors. GPR182 is primarily upregulated in melanoma-associated lymphatic endothelial cells (LECs) during tumorigenesis, and this atypical chemokine receptor endocytoses chemokines promiscuously. In GPR182-deficient mice, T cell infiltration into transplanted melanomas increases, leading to enhanced effector T cell function and improved antitumor immunity. Ablation of GPR182 leads to increased intratumoral concentrations of multiple chemokines and thereby sensitizes poorly immunogenic tumors to immune checkpoint blockade and adoptive cellular therapies. CXCR3 blockade reverses the improved antitumor immunity and T cell infiltration characteristic of GPR182-deficient mice. Our study thus identifies GPR182 as an upstream regulator of the CXCL9/CXCL10/CXCR3 axis that limits antitumor immunity and as a potential therapeutic target in immunologically cold tumors.

Anatomically distinct fibroblast subsets determine skin autoimmune patterns.

The skin serves as a physical barrier and an immunological interface that protects the body from the external environment1-3. Aberrant activation of immune cells can induce common skin autoimmune diseases such as vitiligo, which are often characterized by bilateral symmetric lesions in certain anatomic regions of the body4-6. Understanding what orchestrates the activities of cutaneous immune cells at an organ level is necessary for the treatment of autoimmune diseases. Here we identify subsets of dermal fibroblasts that are responsible for driving patterned autoimmune activity, by using a robust mouse model of vitiligo that is based on the activation of endogenous auto-reactive CD8+ T cells that target epidermal melanocytes. Using a combination of single-cell analysis of skin samples from patients with vitiligo, cell-type-specific genetic knockouts and engraftment experiments, we find that among multiple interferon-γ (IFNγ)-responsive cell types in vitiligo-affected skin, dermal fibroblasts are uniquely required to recruit and activate CD8+ cytotoxic T cells through secreted chemokines. Anatomically distinct human dermal fibroblasts exhibit intrinsic differences in the expression of chemokines in response to IFNγ. In mouse models of vitiligo, regional IFNγ-resistant fibroblasts determine the autoimmune pattern of depigmentation in the skin. Our study identifies anatomically distinct fibroblasts with permissive or repressive IFNγ responses as the key determinant of body-level patterns of lesions in vitiligo, and highlights mesenchymal subpopulations as therapeutic targets for treating autoimmune diseases.

Immunological Microenvironment Alterations in Follicles of Patients With Autoimmune Thyroiditis.

Autoimmune thyroiditis (AIT) is the most prevalent autoimmune endocrine disease, with a higher incidence in women than in men. Immunological abnormalities may lead to the impairment of ovarian folliculogenesis; however, whether the presence of AIT affects immunological microenvironment in follicles remains controversial. We performed a cross-sectional study including 122 patients, aged 20-40 years, who underwent IVF/ICSI treatment owing to isolated male or tube factor infertility. Patients were divided into AIT and control groups according to clinical presentation, thyroid function, and thyroid autoantibody measurements. Follicular fluid was collected and the distribution of cytokines/chemokines in follicular fluid was measured by flow cytometry using multiplex bead assays between the two groups. Based on differences in levels of intrafollicular chemokines and cytokines between the AIT and control groups, the relevant inflammatory cascade was further demonstrated. Among the 12 chemokines analyzed, three (CXCL9, CXCL10, and CXCL11) showed significantly elevated levels in the follicular fluid of patients with AIT. Among the 11 cytokines detected, compared with those in the control group, significantly higher levels of IFNγ were observed in patients with AIT. IFNγ dose-dependently stimulated the expression and secretion of CXCL9/10/11 in cultured primary granulosa cells. The percentage of CXCR3+ T lymphocytes was significantly elevated in the follicular microenvironment of patients with AIT. We concluded that the IFNγ-CXCL9/10/11-CXCR3+ T lymphocyte inflammatory cascade is activated in the follicular microenvironment of patients with AIT. These findings indicate that a considerable immune imbalance occurred in the follicular microenvironment of patients with AIT.

Targeting interferon-γ in hyperinflammation: opportunities and challenges.

Interferon-γ (IFNγ) is a pleiotropic cytokine with multiple effects on the inflammatory response and on innate and adaptive immunity. Overproduction of IFNγ underlies several, potentially fatal, hyperinflammatory or immune-mediated diseases. Several data from animal models and/or from translational research in patients point to a role of IFNγ in hyperinflammatory diseases, such as primary haemophagocytic lymphohistiocytosis, various forms of secondary haemophagocytic lymphohistiocytosis, including macrophage activation syndrome, and cytokine release syndrome, all of which are often managed by rheumatologists or in consultation with rheumatologists. Given the effects of IFNγ on B cells and T follicular helper cells, a role for IFNγ in systemic lupus erythematosus pathogenesis is emerging. To improve our understanding of the role of IFNγ in human disease, IFNγ-related biomarkers that are relevant for the management of hyperinflammatory diseases are progressively being identified and studied, especially because circulating levels of IFNγ do not always reflect its overproduction in tissue. These biomarkers include STAT1 (specifically the phosphorylated form), neopterin and the chemokine CXCL9. IFNγ-neutralizing agents have shown efficacy in the treatment of primary haemophagocytic lymphohistiocytosis in clinical trials and initial promising results have been obtained in various forms of secondary haemophagocytic lymphohistiocytosis, including macrophage activation syndrome. In clinical practice, there is a growing body of evidence supporting the usefulness of circulating CXCL9 levels as a biomarker reflecting IFNγ production.

The Pro-Inflammatory Chemokines CXCL9, CXCL10 and CXCL11 Are Upregulated Following SARS-CoV-2 Infection in an AKT-Dependent Manner.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that is the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Patients with severe COVID-19 may develop acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) and require mechanical ventilation. Key features of SARS-CoV-2 induced pulmonary complications include an overexpression of pro-inflammatory chemokines and cytokines that contribute to a 'cytokine storm.' In the current study an inflammatory state in Calu-3 human lung epithelial cells was characterized in which significantly elevated transcripts of the immunostimulatory chemokines CXCL9, CXCL10, and CXCL11 were present. Additionally, an increase in gene expression of the cytokines IL-6, TNFα, and IFN-γ was observed. The transcription of CXCL9, CXCL10, IL-6, and IFN-γ was also induced in the lungs of human transgenic angiotensin converting enzyme 2 (ACE2) mice infected with SARS-CoV-2. To elucidate cell signaling pathways responsible for chemokine upregulation in SARS-CoV-2 infected cells, small molecule inhibitors targeting key signaling kinases were used. The induction of CXCL9, CXCL10, and CXCL11 gene expression in response to SARS-CoV-2 infection was markedly reduced by treatment with the AKT inhibitor GSK690693. Samples from COVID-19 positive individuals also displayed marked increases in CXCL9, CXCL10, and CXCL11 transcripts as well as transcripts in the AKT pathway. The current study elucidates potential pathway specific targets for reducing the induction of chemokines that may be contributing to SARS-CoV-2 pathogenesis via hyperinflammation.

Characterization of Type I Interferon-Associated Chemokines and Cytokines in Lacrimal Glands of Nonobese Diabetic Mice.

Type I interferons (IFNs) are required for spontaneous lacrimal gland inflammation in the nonobese diabetic (NOD) mouse model of Sjögren's disease, but the consequences of type I IFN signaling are not well-defined. Here, we use RNA sequencing to define cytokine and chemokine genes upregulated in lacrimal glands of NOD mice in a type I IFN-dependent manner. Interleukin (IL)-21 was the highest differentially expressed cytokine gene, and Il21 knockout NOD mice were relatively protected from lacrimal gland inflammation. We defined a set of chemokines upregulated early in disease including Cxcl9 and Cxcl10, which share a receptor, CXCR3. CXCR3+ T cells were enriched in lacrimal glands with a dominant proportion of CXCR3+ regulatory T cells. Together these data define the early cytokine and chemokine signals associated with type I IFN-signaling in the development of lacrimal gland inflammation in NOD mice providing insight into the role of type I IFN in autoimmunity development.

Plasmacytoid Dendritic Cells Depletion and Elevation of IFN-γ Dependent Chemokines CXCL9 and CXCL10 in Children With Multisystem Inflammatory Syndrome.

Background: SARS-CoV-2 occurs in the majority of children as COVID-19, without symptoms or with a paucisymptomatic respiratory syndrome, but a small proportion of children develop the systemic Multi Inflammatory Syndrome (MIS-C), characterized by persistent fever and systemic hyperinflammation, with some clinical features resembling Kawasaki Disease (KD). Objective: With this study we aimed to shed new light on the pathogenesis of these two SARS-CoV-2-related clinical manifestations. Methods: We investigated lymphocyte and dendritic cells subsets, chemokine/cytokine profiles and evaluated the neutrophil activity mediators, myeloperoxidase (MPO), and reactive oxygen species (ROS), in 10 children with COVID-19 and 9 with MIS-C at the time of hospital admission. Results: Patients with MIS-C showed higher plasma levels of C reactive protein (CRP), MPO, IL-6, and of the pro-inflammatory chemokines CXCL8 and CCL2 than COVID-19 children. In addition, they displayed higher levels of the chemokines CXCL9 and CXCL10, mainly induced by IFN-γ. By contrast, we detected IFN-α in plasma of children with COVID-19, but not in patients with MIS-C. This observation was consistent with the increase of ISG15 and IFIT1 mRNAs in cells of COVID-19 patients, while ISG15 and IFIT1 mRNA were detected in MIS-C at levels comparable to healthy controls. Moreover, quantification of the number of plasmacytoid dendritic cells (pDCs), which constitute the main source of IFN-α, showed profound depletion of this subset in MIS-C, but not in COVID-19. Conclusions: Our results show a pattern of immune response which is suggestive of type I interferon activation in COVID-19 children, probably related to a recent interaction with the virus, while in MIS-C the immune response is characterized by elevation of the inflammatory cytokines/chemokines IL-6, CCL2, and CXCL8 and of the chemokines CXCL9 and CXL10, which are markers of an active Th1 type immune response. We believe that these immunological events, together with neutrophil activation, might be crucial in inducing the multisystem and cardiovascular damage observed in MIS-C.

NK/ILC1 cells mediate neuroinflammation and brain pathology following congenital CMV infection.

Congenital human cytomegalovirus (cHCMV) infection of the brain is associated with a wide range of neurocognitive sequelae. Using infection of newborn mice with mouse cytomegalovirus (MCMV) as a reliable model that recapitulates many aspects of cHCMV infection, including disseminated infection, CNS infection, altered neurodevelopment, and sensorineural hearing loss, we have previously shown that mitigation of inflammation prevented alterations in cerebellar development, suggesting that host inflammatory factors are key drivers of neurodevelopmental defects. Here, we show that MCMV infection causes a dramatic increase in the expression of the microglia-derived chemokines CXCL9/CXCL10, which recruit NK and ILC1 cells into the brain in a CXCR3-dependent manner. Surprisingly, brain-infiltrating innate immune cells not only were unable to control virus infection in the brain but also orchestrated pathological inflammatory responses, which lead to delays in cerebellar morphogenesis. Our results identify NK and ILC1 cells as the major mediators of immunopathology in response to virus infection in the developing CNS, which can be prevented by anti-IFN-γ antibodies.

CXCL9 secreted by tumor-associated dendritic cells up-regulates PD-L1 expression in bladder cancer cells by activating the CXCR3 signaling.

BACKGROUND: Tumor-associated dendritic cells (TADCs) can interact with tumor cells to suppress anti-tumor T cell immunity. However, there is no information on whether and how TADCs can modulate programmed death-ligand 1 (PD-L1) expression by cancer cells. METHODS: Human peripheral blood monocytes were induced for DCs and immature DCs were cultured alone, or co-cultured with bladder cancer T24 or control SV-HUC-1 cells, followed by stimulating with LPS for DC activation. The activation status of DCs was characterized by flow cytometry and allogenic T cell proliferation. The levels of chemokines in the supernatants of co-cultured DCs were measured by CBA-based flow cytometry. The impacts of CXCL9 on PD-L1, STAT3 and Akt expression and STAT3 and Akt phosphorylation in T24 cells were determined by flow cytometry and Western blot. RESULTS: Compared with the control DCs, TADCs exhibited immature phenotype and had significantly lower capacity to stimulate allogenic T cell proliferation, particularly in the presence of recombinant CXCL9. TADCs produced significantly higher levels of CXCL9, which enhanced PD-L1 expression in T24 cells. Pre-treatment with AMG487 abrogated the CXCL9-increased PD-L1 expression in T24 cells. Treatment with CXCL9 significantly enhanced STAT3 and Akt activation in T24 cells. CONCLUSIONS: TADCs produced high levels of CXCL9 that increased PD-L1 expression in bladder cancer T24 cells by activating the CXCR3-related signaling. Our findings may shed new lights in understanding the regulatory roles of TADCs in inhibiting antitumor T cell responses and promoting tumor growth.

Type 1 helper T cells generate CXCL9/10-producing T-bet+ effector B cells potentially involved in the pathogenesis of rheumatoid arthritis.

Efficacy of B-cell depletion therapy highlights the antibody-independent effector functions of B cells in rheumatoid arthritis (RA). Given type 1 helper T (Th1) cells abundant in synovial fluid (SF) of RA, we have determined whether Th1 cells could generate novel effector B cells. Microarray and qPCR analysis identified CXCL9/10 transcripts as highly expressed genes upon BCR/CD40/IFN-γ stimulation. Activated Th1 cells promoted the generation of CXCL9/10-producing T-bet+ B cells. Expression of CXCL9/10 was most pronounced in CXCR3+ switched memory B cells. Compared with peripheral blood, SFRA enriched highly activated Th1 cells that coexisted with abundant CXCL9/10-producing T-bet+ B cells. Intriguingly, anti-IFN-γ antibody and JAK inhibitors significantly abrogated the generation of CXCL9/10-producing T-bet+ B cells. B cell derived CXCL9/10 significantly facilitated the migration of CD4+ T cells. These findings suggest that Th1 cells generate the novel CXCL9/10-producing T-bet+ effector B cells that could be an ideal pathogenic B cell target for RA therapy.

STING Signaling Drives Production of Innate Cytokines, Generation of CD8+ T Cells and Enhanced Protection Against Trypanosoma cruzi Infection.

A variety of signaling pathways are involved in the induction of innate cytokines and CD8+ T cells, which are major players in protection against acute Trypanosoma cruzi infection. Previous data have demonstrated that a TBK-1/IRF3-dependent signaling pathway promotes IFN-ß production in response to Trypanosoma cruzi, but the role for STING, a main interactor of these proteins, remained to be addressed. Here, we demonstrated that STING signaling is required for production of IFN-ß, IL-6, and IL-12 in response to Trypanosoma cruzi infection and that STING absence negatively impacts activation of IRF-dependent pathways in response to the parasite. We reported no significant activation of IRF-dependent pathways and cytokine expression in RAW264.7 macrophages in response to heat-killed trypomastigotes. In addition, we showed that STING is essential for T. cruzi DNA-mediated induction of IFN-ß, IL-6, and IL-12 gene expression in RAW264.7 macrophages. We demonstrated that STING-knockout mice have significantly higher parasitemia from days 5 to 8 of infection and higher heart parasitism at day 13 after infection. Although we observed similar heart inflammatory infiltrates at day 13 after infection, IFN-ß, IL-12, CXCL9, IFN-γ, and perforin gene expression were lower in the absence of STING. We also showed an inverse correlation between parasite DNA and the expression of CXCL9, IFN-γ, and perforin genes in the hearts of infected animals at day 13 after infection. Finally, we reported that STING signaling is required for splenic IFN-ß and IL-6 expression early after infection and that STING deficiency results in lower numbers of splenic parasite-specific IFN-γ and IFN-γ/perforin-producing CD8+ T cells, indicating a pivotal role for STING signaling in immunity to Trypanosoma cruzi.

Commensal-driven immune zonation of the liver promotes host defence.

The liver connects the intestinal portal vasculature with the general circulation, using a diverse array of immune cells to protect from pathogens that translocate from the gut1. In liver lobules, blood flows from portal triads that are situated in periportal lobular regions to the central vein via a polarized sinusoidal network. Despite this asymmetry, resident immune cells in the liver are considered to be broadly dispersed across the lobule. This differs from lymphoid organs, in which immune cells adopt spatially biased positions to promote effective host defence2,3. Here we used quantitative multiplex imaging, genetic perturbations, transcriptomics, infection-based assays and mathematical modelling to reassess the relationship between the localization of immune cells in the liver and host protection. We found that myeloid and lymphoid resident immune cells concentrate around periportal regions. This asymmetric localization was not developmentally controlled, but resulted from sustained MYD88-dependent signalling induced by commensal bacteria in liver sinusoidal endothelial cells, which in turn regulated the composition of the pericellular matrix involved in the formation of chemokine gradients. In vivo experiments and modelling showed that this immune spatial polarization was more efficient than a uniform distribution in protecting against systemic bacterial dissemination. Together, these data reveal that liver sinusoidal endothelial cells sense the microbiome, actively orchestrating the localization of immune cells, to optimize host defence.

An immune-based biomarker signature is associated with mortality in COVID-19 patients.

Immune and inflammatory responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contribute to disease severity of coronavirus disease 2019 (COVID-19). However, the utility of specific immune-based biomarkers to predict clinical outcome remains elusive. Here, we analyzed levels of 66 soluble biomarkers in 175 Italian patients with COVID-19 ranging from mild/moderate to critical severity and assessed type I IFN-, type II IFN-, and NF-κB-dependent whole-blood transcriptional signatures. A broad inflammatory signature was observed, implicating activation of various immune and nonhematopoietic cell subsets. Discordance between IFN-α2a protein and IFNA2 transcript levels in blood suggests that type I IFNs during COVID-19 may be primarily produced by tissue-resident cells. Multivariable analysis of patients' first samples revealed 12 biomarkers (CCL2, IL-15, soluble ST2 [sST2], NGAL, sTNFRSF1A, ferritin, IL-6, S100A9, MMP-9, IL-2, sVEGFR1, IL-10) that when increased were independently associated with mortality. Multivariate analyses of longitudinal biomarker trajectories identified 8 of the aforementioned biomarkers (IL-15, IL-2, NGAL, CCL2, MMP-9, sTNFRSF1A, sST2, IL-10) and 2 additional biomarkers (lactoferrin, CXCL9) that were substantially associated with mortality when increased, while IL-1α was associated with mortality when decreased. Among these, sST2, sTNFRSF1A, IL-10, and IL-15 were consistently higher throughout the hospitalization in patients who died versus those who recovered, suggesting that these biomarkers may provide an early warning of eventual disease outcome.

miR-588 is a prognostic marker in gastric cancer.

In an effort to identify a novel microRNA (miRNA) as a gastric cancer (GC) treatment target and prognostic biomarker, we surveyed The Cancer Genome Atlas database and found that miR-588 expression is low in GC tissues. This was confirmed by real-time reverse transcription polymerase chain reaction assays of GC patient plasma samples and SGC7901 and MNK28 cells. A constructed miRNA-mRNA network showed that CXCL5, CXCL9, and CXCL10 are target genes of miR-588. Analysis of the miRWalk database revealed that miR-588 directly binds to CXCL5 and CXCL9. Overexpression of miR-588 reduced GC cell proliferation in vitro and in vivo. High expression of miR-588 inhibited Ki-67 expression in vivo. The FunRich database also showed that CXCL5, CXCL9, and CXCL10 are involved in immune responses, while the Database of Immune Cell Expression showed they are differentially expressed in CD8+ T cells. High expression of CXCL9 and CXCL10 correlated positively with infiltrating levels of CD4+ T and CD8+ T cells in stomach adenocarcinoma. High expression of miR-588, CXCL5, CXCL9, and CXCL10 was associated with prolonged survival of GC patients. These findings indicate that miR-588 is a biomarker for tumor-associated immune infiltration and a prognostic marker in GC patients.