Macrophages and Epithelial Cells Mutually Interact through NLRP3 to Clear Infection and Enhance the Gastrointestinal Barrier

Activation of the nod-like receptor protein 3 (NLRP3) leads to the release of the proinflammatory cytokine IL-1β, which then facilitates pathogen control by macrophages. The role of NLRPs in controlling infection of epithelial cells is not well understood. Our hypothesis was that activation of the NLRP3 inflammasome in colonic epithelial cells would promote macrophage-mediated epithelial recovery after infection with the pathogen Citrobacter rodentium. We devised a co-culture model using mouse colonic epithelial cells (CMT-93) and macrophages (J774A.1) during infection with C. rodentium. Inflammasome was activated using LPS and ATP and inhibited by YVAD. We assessed cytokine secretion (ELISA), macrophage recruitment and pathogen penetration (immunofluorescence), and epithelial barrier integrity (transepithelial electrical resistance). Macrophages were recruited to the apical membrane of epithelial cells, associated with tight junctions, promoted epithelial barrier recovery, and displaced C. rodentium. While NLRP3 was expressed in infected epithelial cells, IL-18 or IL-1β secretion remained unchanged. Supernatants from infected epithelial cells promoted infection clearance by macrophage; while this was inflammasome-independent, ATP significantly improved epithelial barrier recovery. The inflammasome appears to promote epithelial barrier function, independent of IL-18 and IL-1β secretion. Inflammasome activation in macrophages plays a dual role of promoting pathogen clearance and improving epithelial barrier integrity.

Effector-mediated subversion of proteasome activator (PA)28αβ enhances lysosomal targeting of Legionella pneumophila within cytokine-activated macrophages

Legionella pneumophila causes Legionnaires' Disease via replication within host macrophages using an arsenal of hundreds of translocated virulence factors termed effector proteins. Effectors are critical for intracellular replication but can also enhance pathogen clearance in mammalian hosts via effector-triggered immunity. The effector LegC4 confers a fitness disadvantage on L. pneumophila within mouse models of Legionnaires' Disease and uniquely potentiates the antimicrobial activity of macrophages activated with either tumor necrosis factor (TNF) or interferon (IFN)-γ. Here, we investigated the mechanism of LegC4 function. We found that LegC4 binds proteasome activator (PA)28α, a subunit of the PA28αβ (11S) proteasome regulator, and that the LegC4 restriction phenotype is abolished within PA28αβ-deficient macrophages. PA28αβ facilitates ubiquitin-independent proteasomal degradation of oxidant-damaged proteins. Impaired proteasome activity results in compensatory upregulation of lysosomal degradation pathways to relieve oxidative proteotoxic stress. We found that LegC4 impairs the resolution of oxidative proteotoxic stress and enhances phagolysosomal fusion with the Legionella-containing vacuole. PA28αβ has been traditionally associated with antigen presentation and adaptive immunity; however, our data support a model whereby suppression of PA28αβ by LegC4 impairs resolution of oxidative proteotoxic stress, which culminates in the lysosomal killing of L. pneumophila within activated macrophages. This work provides a solid foundation on which to evaluate induced proteasome regulators as mediators of cell-autonomous immunity.

Probiotics in Counteracting the Role of Neutrophils in Cancer Metastasis

Neutrophils are known for their role geared towards pathogen clearance by different mechanisms that they initiate, primarily by the release of neutrophil extracellular traps (NETs). However, their immune-surveillance capacity accompanied with plasticity in existing as interchangeable subsets, discovered recently, has revealed their property to contribute to complex cancer pathologies including tumor initiation, growth, angiogenesis and metastasis. Although there is a growing body of evidence suggesting a critical balance between the protumoral and antitumoral neutrophil phenotypes, an in-depth signaling pathway analysis would aid in determination of anticipatory, diagnostic and therapeutic targets. This review presents a comprehensive overview of the potential pathways involved in neutrophil-triggered cancer metastasis and introduces the influence of the microbial load and avenues for probiotic intervention.

Specificity and Heterogeneity of Trained Immunity in Hematopoietic Stem and Progenitor Cells

When innate immune cells and hematopoietic stem and progenitor cells (HSPCs) are exposed to pathogenic agents, they develop heightened responses to subsequent infection through a process called trained immunity. After exposure to a pathogen, bone marrow derived macrophages (BMDMs) generated from trained HSPCs are capable of enhanced pathogen clearance and cytokine production, while exhibiting persistent metabolic rewiring. Because some models of HSPC trained immunity are dependent on interferon gamma (IFNγ) signaling, and our lab has described extensive changes in HSC self-renewal and differentiation upon IFNγ exposure, we hypothesized that persistent IFNγ signaling induced by chronic infection results in reprogramming of HSPCs, causing improved non-specific immunity. To test our hypothesis, we generated a chimeric mouse model of trained immunity by transplanting control or M. avium-exposed HSPCs into naïve recipient mice. Mice that received M. avium trained HSPCs had decreased bacterial load, less splenomegaly, and fewer granulomas upon subsequent M. avium infection, indicating improved immunity. Furthermore, BMDMs generated from mice trained with a single dose of recombinant IFNγ (rIFNγ) exhibited increased pathogen clearance and metabolic profiles ex vivo. To test if rIFNγ training was sufficient to induce HSPC trained immunity in vivo, we isolated HSPCs from rIFNγ-exposed mice and challenged the recipients 4 months later. These in vivo experiments demonstrated that rIFNγ training was insufficient to induce substantial protection against subsequent M. avium challenge, but still induced BMDM metabolic rewiring four months post HSPC training. Collectively, our studies indicate that there are degrees of training that occur upon IFNγ exposure, likely related to the concentration and duration of the primary stimulus. To assess the specificity of cross protection of HSPC trained immunity, we utilized our chimeric mouse model and tested two different training and infection pathogens: M. avium and influenza. When we challenged M. avium-trained HSPC recipients with influenza, we found that although there was mildly decreased lung histopathology and increased production of IFNγ and TNFα, mice succumbed to infection like untrained controls. When we swapped the order of pathogens, we observed that mice receiving influenza-trained HSPCs produced BMDMs with increased killing capability and systemically higher IL-6 and RANTES levels, but these features were insufficient to significantly reduce bacterial CFU counts upon M. avium challenge. These transplant experiments indicate that trained immunity encoded in HSPCs is pathogen specific. To dissect the mechanism of M. avium-induced trained immunity in HSPCs, we performed RNAseq analysis on M. avium-trained HSPCs post-transplant and cross referenced it with RNAseq and WGBS data on primary M. avium-exposed HSPCs. These studies showed consistent differences in cellular signaling, metabolism, immunity, and antigen processing and presentation in the trained HSPCs, indicating that epigenetic and transcriptional reprogramming induced by M. avium exposure is durable following transplant and secondary challenge. To ascertain whether transcriptional changes are homogeneous throughout the HSC compartment, we completed scRNA-seq on naïve and M. avium-exposed hematopoietic cells. We found that genes upregulated upon M. avium exposure in HSCs, including Batf2 and Cxcl9, were induced in a subset of HSPCs, indicating that there is a heterogeneous response to training within the HSPC pool. Strikingly, the trained immunity signature was maintained in neutrophils and macrophages but lost in mature B cells, indicating specific propagation of genetic signatures induced by training in certain lineages. Finally, we found an emerging population of HSCs with B cell gene signatures upon M. avium exposure. Emergence of this HSPC subpopulation may suggest the development of a cell that acts as a direct intermediate between HSC and B cells following training. Our work shows that trained immunity induced by M. avium and persistent IFNγ signaling is pathogen-specific and heterogeneous among primitive HSPCs. Emergence of specific responder cell populations within the HSPC pool may be responsible for enhanced protection against specific infection stimuli, whereas the presence of non-responders may insure long term health of the HSPC pool. Disclosures No relevant conflicts of interest to declare.

886 Targeting VSIG4, a novel macrophage checkpoint, repolarizes suppressive macrophages which induces an inflammatory response in primary cell in vitro assays and fresh human tumor cultures

BackgroundVSIG4 (V-set immunoglobulin-domain-containing 4) is a B7 family related protein with known roles as a complement receptor involved in pathogen clearance as well as a negative regulator of T cell activation by an undetermined mechanism.1–3 VSIG4 is expressed in tumor associated macrophages (TAMs) with exquisite specificity. In cancer, increased expression of VSIG4 has been associated with worse survival in multiple indications, including non-small cell lung cancer, multiple myeloma, ovarian cancer, and glioma, suggesting an important role in tumor immune evasion.3–6 Based upon computational analysis of transcript data across thousands of primary cancer and normal tissue samples, we hypothesized that VSIG4 has an important regulatory role in promoting M2-like immune suppressive macrophages in the tumor microenvironment, and that targeting VSIG4 via a monoclonal antibody could relieve VSIG4-mediated macrophage suppression by repolarizing TAMs to an inflammatory phenotype capable of coordinating an anti-tumor immune response.MethodsThe ability of anti-VSIG4 antibodies to repolarize M2-like macrophages and induce T cell activation was assessed in vitro and ex vivo, by measuring production of inflammatory mediators. In vitro assays were performed primarily with M-CSF plus IL-10 driven monocyte-derived M2c macrophages from healthy donors. Ex vivo assays were performed with fresh, patient-derived tumor samples in culture. To determine whether targeting VSIG4 can lead to an anti-tumor effect in vivo, syngeneic mouse models were dosed with anti-mouse VSIG4 antibodies and characterized for changes in tumor volume and immune cell populations.ResultsIn in vitro and ex vivo assays anti-VSIG4 antibodies repolarize M2 macrophages and induce an immune response culminating in T cell activation. Targeting VSIG4 upregulates pro-inflammatory cytokines in M2c macrophages, as well as upregulates pro-inflammatory myeloid-derived cytokines and T cell-derived cytokines in M2c macrophages co-cultured with autologous T cells in the presence of staphylococcal enterotoxin B (SEB) activation. To assess targeting VSIG4 in a relevant translational model, fresh, patient-derived tumor samples were treated ex vivo with anti-VSIG4. Across multiple tumor types, anti-VSIG4 treatment resulted in a significant upregulation of cytokines involved in TAM repolarization and T cell activation, and chemokines involved in immune cell recruitment, at levels greater than observed by treatment with anti-PD-1 or a clinical macrophage repolarizing agent (anti-ILT-4). In vivo, tumor growth inhibition is observed in syngeneic mouse models dosed with anti-mouse-VSIG4 alone and in combination with anti-PD-1.ConclusionsTaken together, these data suggest that VSIG4 represents a promising new target capable of stimulating an anti-cancer response via multiple key immune mechanisms.Referencesvan Lookeren Campagne M, Verschoor A. Pathogen clearance and immune adherence “revisited”: immuno-regulatory roles for CRIg. Semin Immunol 2018;37:4–11.Xu S, Sun Z, Li L, Liu J, He J, Song D, Shan G, Liu H, Wu X. Induction of T cells suppression by dendritic cells transfected with VSIG4 recombinant adenovirus. Immunol Lett 2010;128(1):46–50.Liao Y, Guo S, Chen Y, Cao D, Xu H, Yang C, Fei L, Ni B, Ruan Z. VSIG4 expression on macrophages facilitates lung cancer development. Lab Invest 2014;94(7):706–715.Roh J, Jeon Y, Lee A, Lee S, Kim Y, Sung C, Park C, Hong J, Yoon D, Suh C, Huh J, Choi I, Park C. The immune checkpoint molecule V-set Ig domain-containing 4 is an independent prognostic factor for multiple myeloma. Oncotarget 2017;8(35):58122–58132.Xu T, Jiang Y, Yan Y, Wang H, Lu C, Xu H, Li W, Fu D, Lu Y, Chen J. VSIG4 is highly expressed and correlated with poor prognosis of high-grade glioma patients. Am J Transl Res 2015;7(6):1172–1180.Byun J, Jeong D, Choi I, Lee D, Kang M, Jung K, Jeon Y, Kim Y, Jung E, Lee K, Sung M, Kim K. The significance of VSIG4 expression in ovarian cancer. Int J Gynecol Cancer 2017;27(5):872–878.Ethics ApprovalAll legal and ethical requirements were met with regards to the humane treatment of animals described in the study. The animal study was conducted in compliance with CRL IACUC under IACUC No. I033.

Therapeutic Effect of an Antibody-Derived Peptide in a Galleria mellonella Model of Systemic Candidiasis

The synthetic peptide T11F (TCRVDHRGLTF), with sequence identical to a fragment of the constant region of human IgM, and most of its alanine-substituted derivatives proved to possess a significant candidacidal activity in vitro. In this study, the therapeutic efficacy of T11F, D5A, the derivative most active in vitro, and F11A, characterized by a different conformation, was investigated in Galleria mellonella larvae infected with Candida albicans. A single injection of F11A and D5A derivatives, in contrast with T11F, led to a significant increase in survival of larvae injected with a lethal inoculum of C. albicans cells, in comparison with infected animals treated with saline. Peptide modulation of host immunity upon C. albicans infection was determined by hemocyte analysis and larval histology, highlighting a different immune stimulation by the studied peptides. F11A, particularly, was the most active in eliciting nodule formation, melanization and fat body activation, leading to a better control of yeast infection. Overall, the obtained data suggest a double role for F11A, able to simultaneously target the fungus and the host immune system, resulting in a more efficient pathogen clearance.

Bacterial Subversion of Autophagy in Cystic Fibrosis

Cystic fibrosis (CF) is a genetic disease affecting more than 70,000 people worldwide. It is caused by a mutation in the cftr gene, a chloride ion transporter localized in the plasma membrane of lung epithelial cells and other organs. The loss of CFTR function alters chloride, bicarbonate, and water transport through the plasma membrane, promoting the production of a thick and sticky mucus in which bacteria including Pseudomonas aeruginosa and Burkholderia cenocepacia can produce chronic infections that eventually decrease the lung function and increase the risk of mortality. Autophagy is a well-conserved lysosomal degradation pathway that mediates pathogen clearance and plays an important role in the control of bacterial infections. In this mini-review, we describe the principal strategies used by P. aeruginosa and B. cenocepacia to survive and avoid microbicidal mechanisms within the autophagic pathway leading to the establishment of chronic inflammatory immune responses that gradually compromise the lung function and the life of CF patients.

Diversity of Macrophages in Lung Homeostasis and Diseases

Lung macrophages play important roles in the maintenance of homeostasis, pathogen clearance and immune regulation. The different types of pulmonary macrophages and their roles in lung diseases have attracted attention in recent years. Alveolar macrophages (AMs), including tissue-resident alveolar macrophages (TR-AMs) and monocyte-derived alveolar macrophages (Mo-AMs), as well as interstitial macrophages (IMs) are the major macrophage populations in the lung and have unique characteristics in both steady-state conditions and disease states. The different characteristics of these three types of macrophages determine the different roles they play in the development of disease. Therefore, it is important to fully understand the similarities and differences among these three types of macrophages for the study of lung diseases. In this review, we will discuss the physiological characteristics and unique functions of these three types of macrophages in acute and chronic lung diseases. We will also discuss possible methods to target macrophages in lung diseases.

Negative regulation of IMD contributes to disease tolerance during systemic bacterial infection in Drosophila

Disease tolerance is an infection phenotype where hosts show relatively high health despite harbouring elevated pathogen loads. Compared to the mechanisms of immune clearance our knowledge of the mechanisms underlying increased tolerance remains incomplete. Variation in the ability to reduce immunopathology may explain why some hosts can tolerate higher pathogen burdens with reduced pathology. Negative immune regulation would therefore appear to be a clear candidate for a mechanism underlying disease tolerance but this has not been tested directly for bacterial infections. Here, we examined how the negative regulation of the immune deficiency (IMD) pathway affects disease tolerance in Drosophila melanogaster when infected with the gram-negative bacterial pathogen Pseudomonas entomophila. We find that UASRNAi-mediated reduced expression of the negative regulators of IMD (pirk & caudal) severely reduced the ability to tolerate infection in both males and females across a wide range of infectious doses. While flies unable to regulate the IMD response exhibited higher expression of antimicrobial peptides and lower bacterial loads as expected, this was not accompanied by a proportional reduction in mortality. Instead, tolerance (measured as fly survival relative to its microbe load) was drastically reduced, likely due to the combination of increased immunopathology and cytotoxicity of elevated AMP expression. Our results therefore highlight that in addition to regulating an efficient pathogen clearance response, negative regulators of IMD also contribute to disease tolerance.