Non-depleted Mice Research Articles

Dendritic epidermal T cells (DETC) are not required for control of skin Staph infections

Abstract Staphylococcal bacteria are a common cause of skin infections in mice and humans. γδ T cells and the cytokine IL-17 are important in controlling these infections. To test roles for IL-17 in autoimmune Ets1 knockout (KO) mice, we generated double knockout (DKO) mice lacking both Ets1 and IL-17RA. We found that DKO mice develop worse autoimmune disease than Ets1 KO mice and are also susceptible to spontaneous Staphylococcus xylosus skin infections. While IL-17RA KO mice also develop skin lesions, these lesions are found at a higher rate and increased severity in DKO mice. Furthermore, exogenous infection of DKO skin with bioluminescent Staphylococcus aureus results in a failure to clear the bacteria, while IL-17RA KO mice are able to clear. Given that γδ T cells are important in controlling Staph skin infections, we examined these populations and found that both Ets1 KO and DKO mice lack the dendritic epidermal T cells (DETC), while other γδ T cell subsets are present. Further analysis showed that DETC progenitors are present in the fetal thymus of Ets1 KO mice, but DETC are missing from the skin of P5 and adult mice lacking Ets1. We hypothesized that loss of DETC (due to absence of Ets1) might cooperate with loss of IL-17 signaling (due to loss of IL-17RA) to create susceptibility to skin infections. To determine if DETC have a specific contribution to the skin immune response to Staphylococcal bacteria, we depleted DETC from wild-type mice using antibody against TCR Vγ5 followed by S. aureus infection. We found that DETC are not required to clear skin Staph infections, since depleted mice cleared with the same kinetics as non-depleted mice. Studies are in progress to further examine the roles of other γδ T cell populations in clearance of skin Staph infections. Supported by grant from NIH (R01 AI122720)

Abstract 1825: A bacterial consortium engineered from feces of patients responding to anti-PD1 treatment mediates tumor response through IFNγ in non-small cell lung cancer

Abstract Introduction: We previously showed that non-small cell lung cancer (NSCLC) patients that respond (R) to immune checkpoint blockade (ICB) have a different microbial community structure than non-responders (NR) pre-treatment (Genome Med 2022). Importantly, we showed that pooled R microbiota transplantation into gnotobiotic xenograft mice decreased tumor growth compared to NR colonized mice following anti-PD1 therapy, a phenomenon associated with enrichment of the Bacteroides genus. However, the bacterial strains driving this synergistic effect with anti-PD1 therapy and mechanism of action are unknown. Method: Feces collected from R mice were used as source material for high-throughput microbial isolation performed with the GALT Prospector technology. Bacterial identification was performed with MALDI-TOF Biotyper and Sanger sequencing. The cell free supernatants and under 3 kDa small molecules of 183 Bacteroides isolates were screened for their ability to stimulate IFNγ production by primary CD8+ T cells by flow cytometry. A consortium composed of 6 IFNγ-stimulating isolates or NR feces was transplanted into a gnotobiotic mouse model of lung cancer treated with anti-PD1, with or without anti-IFNγ monoclonal antibody depletion. Tumors were harvested at endpoint for flow cytometric analysis, and blood serum for IFNγ ELISA. Bioactivity-guided fractionation coupled with LC-MS analysis was used to identify small molecules from the cell free supernatant of a single stimulating Bacteroides isolate. Potential bioactive small molecule characterization was performed using LC-MS/MS supported by computational pipelines for structural prediction followed by NMR for structural confirmation. Results: We cultured and identified 679 isolates from 30 unique species. The cell free supernatant from 6 out of 183 Bacteroides isolates stimulated IFNγ production from primary CD8+ T cells. Small molecules from the six stimulatory isolates’ supernatants significantly induced IFNγ production compared to 6 taxonomy-matched non-stimulatory isolates (P=0.039). A defined consortium composed of the six stimulatory isolates (R-consortium) was able to colonize germ free mice, and decreased tumor growth compared to NR feces-colonized mice (P=0.041). IFNγ depletion of R-consortium mice significantly increased tumor growth (P=0.012) compared to non-depleted mice. Intratumor IFNγ+ CD8+ T cell frequency and circulating serum IFNγ was elevated only in R-consortium mice. Bioassay guided fractionation revealed 5 small molecule metabolites produced by Bacteroides and their structure and bioactivity are currently being investigated. Conclusion: A microbial consortium engineered from R patients’ feces synergizes with anti-PD1 therapy to reduce lung cancer growth through an IFNγ-dependent mechanism which may be mediated by small molecule metabolites. Citation Format: Rachel C. Newsome, Joonseok Oh, Wenya Jian, Raad Z. Gharaibeh, Jason Crawford, Christian Jobin. A bacterial consortium engineered from feces of patients responding to anti-PD1 treatment mediates tumor response through IFNγ in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1825.

MP34-13 SYNERGY BETWEEN THE HOST IMMUNE SYSTEM AND BACTERIOPHAGES SUPPORTS PHAGE THERAPY FOR URINARY TRACT INFECTION

You have accessJournal of UrologyCME1 Apr 2023MP34-13 SYNERGY BETWEEN THE HOST IMMUNE SYSTEM AND BACTERIOPHAGES SUPPORTS PHAGE THERAPY FOR URINARY TRACT INFECTION Kathrin Bausch Kathrin BauschKathrin Bausch More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003268.13AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Urinary tract infections (UTI) are one of the most common bacterial infections affecting >150 million people world-wide, every year. Globally, extraintestinal pathogenic Escherichia coli (ExPEC), which include uropathogenic E. coli, increasingly display resistance to antibiotics. One ExPEC clonal group, sequence type 131 (ST131), has emerged as a pandemic multidrug-resistant pathogen. To continue to be able to effectively treat UTI, despite increasing rates of antibiotic resistance, bacteriophage therapy is attracting renewed interest. Although phage therapy has been used for decades, mechanisms underlying phage-mediated bacterial clearance in UTI remain largely unknown. METHODS: To address this question, we investigated phage treatment for UTI caused by the ST131 strain JJ2528 and its effects on the bladder immune response in a mouse model. RESULTS: Using this model, we found that female C57BL/6 mice infected with JJ2528 had significantly reduced bladder bacterial burdens compared to untreated mice when they received phage HP3 either intraperitoneally or intravesically. Additionally, we found phage therapy may protect memory responses. Our previous work demonstrates that a non-sterilizing adaptive immune response arises following an acute UTI, but elimination of bacteria by antibiotics ablates development of immune memory. We tested whether phage-treated mice, in which bacteria are cleared from the bladder with a similar kinetic to antibiotic treatment, had an impaired memory response. Surprisingly, bacterial burden in challenged animals was similar between control and phage-treated animals, suggesting that immune memory is intact. While bacteriophage therapy alone did not induce inflammation in the bladder, phage therapy in the context of infection induced a significant increase in neutrophil infiltration compared to control-treated mice. To determine the role of neutrophils in phage treatment, we used two models of neutrophil depletion and treated infected mice with phages. Neutrophil-depleted mice had a significantly higher bacterial burden compared to non-depleted mice, supporting that neutrophil infiltration is necessary for phage treatment efficacy. A possible mechanism behind this may be that the administration of phages and bacteria leads to increased ROS production and cell death in human neutrophils compared to bacteria alone. CONCLUSIONS: Our data demonstrate that phage treatment is effective in reducing a primary UTI and does not impair the development of immune memory to rUTI. Phage treatment induces a neutrophil infiltration that is essential for phage efficacy mediated by increased ROS production and “cell death”. This study supports that phage treatment of UTI is a potentially viable alternative to antibiotics for this very common infection. Source of Funding: none © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e464 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kathrin Bausch More articles by this author Expand All Advertisement PDF downloadLoading ...

Myeloid-derived suppressor cells are associated with impaired Th1 and Th17 responses and severe pulmonary paracoccidioidomycosis which is reversed by anti-Gr1 therapy.

Previous studies on paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America, revealed that host immunity is tightly regulated by several suppressive mechanisms mediated by tolerogenic plasmacytoid dendritic cells, the enzyme 2,3 indoleamine dioxygenase (IDO-1), and regulatory T-cells (Tregs). IDO-1 orchestrates local and systemic immunosuppressive effects through the recruitment and activation of myeloid-derived suppressor cells (MDSCs), a heterogeneous population of myeloid cells possessing a potent ability to suppress T-cell responses. However, the involvement of MDSCs in PCM remains uninvestigated. The presence, phenotype, and immunosuppressive activity of MDSCs were evaluated at 96h, 2 weeks, and 8 weeks of pulmonary infection in C57BL/6 mice. Disease severity and immune responses were assessed in MDSC-depleted and nondepleted mice using an anti-Gr1 antibody. Both monocytic-like MDSCs (M-MDSCs) and polymorphonuclear-like MDSCs (PMN-MDSCs) massively infiltrated the lungs during Paracoccidioides brasiliensis infection. Partial reduction of MDSC frequency led to a robust Th1/Th17 lymphocyte response, resulting in regressive disease with a reduced fungal burden on target organs, diminishing lung pathology, and reducing mortality ratio compared with control IgG2b-treated mice. The suppressive activity of MDSCs on CD4 and CD8 T-lymphocytes and Th1/Th17 cells was also demonstrated in vitro using coculture experiments. Conversely, adoptive transfer of MDSCs to recipient P. brasiliensis-infected mice resulted in a more severe disease. Taken together, our data showed that the increased influx of MDSCs into the lungs was linked to more severe disease and impaired Th1 and Th17 protective responses. However, protective immunity was rescued by anti-Gr1 treatment, resulting in a less severe disease and controlled tissue pathology. In conclusion, MDSCs have emerged as potential target cells for the adjuvant therapy of PCM.

γδ Tcells license immature B cells to produce a broad range of polyreactive antibodies.

Immature autoreactive B cells are present in all healthy individuals, but it is unclear which signals are required for their maturation into antibody-producing cells. Inducible depletion of γδ Tcells show that direct interaction between γδ Tcells and immature B cells in the spleen support an "innate" transition to mature B cells with a broad range of antigen specificities. IL-4 production of γδ Tcells and cell-to-cell contact via CD30L support B cell maturation and induce genes of the unfolded protein response and mTORC1 signaling. Eight days after invivo depletion of γδ Tcells, increased numbers of B cells are already stuck in the transitional phase and express increased levels of IgD and CD21. Absence of γδ Tcells leads also to reduced levels of serum anti-nuclear autoantibodies, making γδ Tcells an attractive target to treat autoimmunity.

Basophil depletion alters host immunity, intestinal permeability and mammalian host-to-mosquito transmission in malaria.

Abstract Malaria-induced bacteremia has been shown to result from intestinal mast cell (MC) activation (Chau et al. 2013, Potts et al. 2016, Céspedes et al. 2020). The appearance of MCs in the ileum and increased permeability to enteric bacteria are preceded by an early Th2-biased host immune response to infection, characterized by the appearance of IL-4, IL-10, mast cell protease (Mcpt)1 and Mcpt4, and an increase in circulating basophils and eosinophils. Given the functional similarities of basophils and MCs in the context of allergic inflammation and the capacity of basophils to produce IL-4 in large amounts, we sought to define the role of basophils in the development of increased intestinal permeability and in MC influx and the development of bacteremia in the context of malaria. Upon infection with non-lethal Plasmodium yoelii yoelii 17XNL, mice that lack basophils exhibited increased intestinal permeability and increased ileal MC numbers, without any increase in bacterial 16S ribosomal DNA copy numbers in the blood, relative to non-depleted mice. Analysis of cytokines, chemokines and MC-associated factors in the ileum revealed significantly increased TNFα and IL-13 at day 6 post infection (PI) in basophil-depleted mice compared to non-depleted mice. Moreover, network analysis revealed profound differences between depleted and non-depleted mice over time following infection in both systemic and local immune responses to parasites and translocated bacteria. Finally, basophil depletion was associated with significantly increased gametocytemia and parasite transmission to mosquitoes, suggesting that basophils play a previously undescribed role in controlling gametocytemia and in turn, mammalian host-to-mosquito transmission. This work was supported by NIH NIAID RO1 AI131609 to Shirley Luckhart.

The Actin-Binding Protein Cortactin Promotes Sepsis Severity by Supporting Excessive Neutrophil Infiltration into the Lung.

Sepsis is a systemic infection that can lead to multi-organ failure. It is characterised by an uncontrolled immune response with massive neutrophil influx into peripheral organs. Neutrophil extravasation into tissues depends on actin remodeling and actin-binding proteins such as cortactin, which is expressed ubiquitously, except for neutrophils. Endothelial cortactin is necessary for proper regulation of neutrophil transendothelial migration and recruitment to sites of infection. We therefore hypothesised that cortactin plays a crucial role in sepsis development by regulating neutrophil trafficking. Using a murine model of sepsis induced by cecal ligation and puncture (CLP), we showed that cortactin-deficient (KO) mice survive better due to reduced lung injury. Histopathological analysis of lungs from septic KO mice revealed absence of oedema, reduced vascular congestion and mucus deposition, and better-preserved alveoli compared to septic wild-type (WT) mice. Additionally, sepsis-induced cytokine storm, excessive neutrophil infiltration into the lung and oxidative stress were significantly reduced in KO mice. Neutrophil depletion 12 h after sepsis improved survival in WT mice by averting lung injury, similar to both neutrophil-depleted and non-depleted KO mice. Our findings highlight a critical role of cortactin for lung neutrophil infiltration and sepsis severity.

Contribution of Inflammation and Bone Destruction to Pain in Arthritis: A Study in Murine Glucose-6-Phosphate Isomerase-Induced Arthritis.

Arthritis is often characterized by inflammation and bone destruction. This study was undertaken to investigate the contribution of inflammation and bone destruction to pain. Inflammation, bone resorption, pain-related behaviors, and molecular markers (activating transcription factor 3 [ATF-3], p-CREB, and transient receptor potential vanilloid channel 1) in sensory neurons were measured in murine glucose-6-phosphate isomerase (G6PI)-induced arthritis, a model of rheumatoid arthritis. Depletion of Treg cells before immunization changed self-limiting arthritis into nonremitting arthritis with pronounced bone destruction. Zoledronic acid (ZA) was administered to reduce bone resorption. Compared to nondepleted mice, Treg cell-depleted mice exhibited arthritis with more severe bone destruction and higher guarding scores (P < 0.05; n = 10 mice per group) as well as more persistent thermal hyperalgesia (P < 0.05), but displayed similar mechanical hyperalgesia at the hindpaws (n = 18-26 mice per group). These pain-related behaviors, as well as an up-regulation of the neuronal injury marker ATF-3 in sensory neurons (studied in 39 mice), appeared before the clinical score (inflammation) became positive and persisted in Treg cell-depleted and nondepleted mice. In the late stage of arthritis, Treg cell-depleted mice treated with ZA showed less bone resorption (<50%; P < 0.01) and less thermal hyperalgesia (P < 0.01) than Treg cell-depleted mice without ZA treatment (n = 15 mice per group), but ZA treatment did not reduce the clinical score and local mechanical hyperalgesia. Pain-related behaviors precede and outlast self-limiting arthritis. In nonremitting arthritis with enhanced bone destruction, mainly local thermal, but not local mechanical, hyperalgesia was aggravated. The up-regulation of ATF-3 indicates an early and persisting affection of sensory neurons by G6PI-induced arthritis.

The Behavioral Sequelae of Social Defeat Require Microglia and Are Driven by Oxidative Stress in Mice.

Chronic social defeat (CSD) in male mice can produce anxiety and aberrant socialization. Animals susceptible to CSD show activation of microglia, which have elevated levels of oxidative stress markers. We hypothesized that microglia and reactive oxygen species (ROS) production contribute to the CSD stress-induced changes in affective behavior. First, we selectively depleted microglia (99%) by administering the CSF1R (colony-stimulating factor 1 receptor) antagonist PLX5622 before and during the 14 d CSD procedure. Microglia-depleted mice in contrast to nondepleted mice were protected from the stress effects measured by light/dark and social interaction tests. ROS production, measured histochemically following dihydroethidium administration, was elevated by CSD, and the production was reduced to basal levels in mice lacking microglia. The deleterious stress effects were also blocked in nondepleted mice by continuous intracerebral administration of N-acetylcysteine (NAC), a ROS inhibitor. In a second experiment, at the end of the CSD period, PLX5622 was discontinued to allow microglial repopulation. After 14 d, the brain had a full complement of newly generated microglia. At this time, the mice that had previously been protected now showed behavioral deficits, and their brain ROS production was elevated, both in all brain cells and in repopulated microglia. NAC administration during repopulation prevented the behavioral decline in the repopulated mice, and it supported behavioral recovery in nondepleted stressed mice. The data suggest that microglia drive elevated ROS production during and after stress exposure. This elevated ROS activity generates a central state supporting dysregulated affect, and it hinders the restoration of behavioral and neurochemical homeostasis after stress cessation.SIGNIFICANCE STATEMENT Chronic psychosocial stress is associated with psychiatric disorders such as depression and anxiety. Understanding the details of CNS cellular contributions to stress effects could lead to the development of intervention strategies. Inflammation and oxidative stress are positively linked to depression severity, but the cellular nature of these processes is not clear. The chronic social defeat (CSD) paradigm in mice produces mood alterations and microglial activation characterized by elevated reactive oxygen species (ROS) production. The depletion of microglia or ROS inhibition prevented adverse stress effects. Microglial repopulation of the brain post-CSD reintroduced adverse stress effects, and ROS inhibition in this phase protected against the effects. The results suggest that stress-induced microglial ROS production drives a central state that supports dysregulated affective behavior.

168. Intradermal Immunization Drives Humoral and Cellular Immunity to the Lung and Protects Against Acute P. aeruginosa Pneumonia

Background Pseudomonas aeruginosa is a leading cause of hospital-associated pneumonia, with 6,700 multidrug-resistant infections in the US annually. Evidence suggests that antibodies and CD4 Th1 and Th17 responses contribute to protection against P. aeruginosa infection. Recent work suggests that intradermal (ID) immunization with a vaccine adjuvanted with a double mutant of E. coli heat-labile toxin (dmLT) can direct protective immune responses to mucosal tissues such as the lungs. We sought to determine whether ID immunization with P. aeruginosa outer membrane proteins (OMPs) with dmLT could drive migration of CD4+ T cells and antibodies to the lungs and protect against P. aeruginosa pneumonia.MethodsWe immunized C57Bl/6 mice with 1 µg of OMPs with 1 µg dmLT. Control mice received OMPs or saline. Antibody and T-cell responses were assessed by ELISA and flow cytometry, respectively. We then evaluated the protective efficacy of the vaccine in a lethal acute pneumonia model. Immunized mice were challenged with 7 × 106 CFU of P. aeruginosa via oropharyngeal aspiration into the lungs. Finally, we examined whether memory CD4+ T cells was essential for protection by depleting immunized mice of vaccine-induced memory CD4+ T cells.ResultsMice immunized with OMPs and dmLT had a significantly greater concentration of anti-pseudomonal IgG in the serum and lungs and a significantly greater proportion of CD4+ T cells in the lung producing IFN-γ or IL-17A than mice immunized with OMPs alone or saline. ID immunization provided significant protection against P. aeruginosa pneumonia, with 78% of immunized mice surviving compared with 100% mortality in saline immunized controls. Memory CD4+ T-cell–depleted mice displayed reduced survival (40%) compared with nondepleted mice (80%), confirming that memory CD4 T+ cells contribute to OMP-dmLT vaccine-mediated protection.ConclusionThese results demonstrate that ID vaccination against P. aeruginosa protects against acute lethal P. aeruginosa pneumonia by stimulating antigen-specific IgG and Th1- and Th17-type CD4+ memory T cells in the pulmonary milieu. ID immunization with dmLT may reduce the global morbidity and mortality caused by multidrug-resistant respiratory pathogens.Disclosures All authors: No reported disclosures.

Antigen Uptake by Langerhans Cells Is Required for the Induction of Regulatory T Cells and the Acquisition of Tolerance During Epicutaneous Immunotherapy in OVA-Sensitized Mice.

The skin is a major immunologic organ that may induce protection, sensitization or tolerance. Epicutaneous immunotherapy (EPIT) has been proposed as an attractive strategy to actively treat food allergy and has been shown to induce tolerance in sensitized mice through the induction of Foxp3+ regulatory T cells (Tregs), especially CD62L+ Tregs. Among immune cells in the skin, dendritic cells are key players in antigen-specific immune activation or regulation. The role of different populations of skin DCs in tolerance induction remains to be elucidated. Using OVA-sensitized BALB/c mice, we demonstrated that the application of a patch containing OVA-A647 to the skin resulted in allergen uptake by Langerhans cells (LCs) and CD11b+ dermal cDC2 and subsequent migration into skin draining lymph nodes. These 2 populations induced Foxp3 expression in CD4+ cells in vitro. Only LCs induced LAP+ cells and CD62L+ Tregs. Using Langerin-eGFP-DTR mice, we analyzed the role of LCs in the mechanisms of tolerance induction by EPIT in vivo. Following complete depletion of LCs, a dramatic decrease in the number of OVA+ DCs and OVA+ CD11b+ dermal cDC2 was observed in skin draining lymph nodes 48 h after epicutaneous application. Likewise, 2 weeks of EPIT in non-depleted mice induced Foxp3+ Tregs, especially CD62L+, and LAP+ Tregs in skin draining lymph nodes and spleen, whereas no induction of Tregs was observed in LC-depleted mice. Following 8 weeks of treatment, EPIT-treated mice showed significant protection against anaphylaxis accompanied by a significant increase of Foxp3+ Tregs, especially CD62L+ Tregs, which was not seen in the absence of LCs. In summary, although both LCs and CD11b+ dermal cDC2s could induce regulatory T cells, the absence of LCs during EPIT impaired treatment efficacy, indicating their crucial role in skin-induced tolerance.

Involvement of CD8+ T cells in the development of renal hemorrhage in a mouse model of hemorrhagic fever with renal syndrome.

Hemorrhagic fever with renal syndrome (HFRS) is caused by hantavirus infection. Although host immunity is thought to be involved in the pathogenesis of HFRS, the mechanism remains to be elucidated. A mouse model of HFRS, which showed renal hemorrhage similar to that seen in patients, has been developed previously. In this study, we aimed to clarify whether CD4+ and CD8+ T cells are involved in the development of renal hemorrhage in the mouse model. At 2days before virus inoculation, CD4+ or CD8+ T cells in 6-week-old BALB/c mice were depleted by administration of antibodies. The CD4+ T cell-depleted mice developed signs of disease such as transient weight loss, ruffled fur and renal hemorrhage as in non-depleted mice. In contrast, the CD8+ T cell-depleted mice showed no signs of disease. After determination of CTL epitopes on the viral glycoprotein in BALB/c mice, the quantity of virus-specific CTLs was analyzed using an MHC tetramer. The quantity of virus-specific CTLs markedly increased in spleens and kidneys of virus-infected mice. However, the quantity in high-pathogenic clone-infected mice was comparable to that in low-pathogenic clone-infected mice. We previously reported that the high-pathogenic clone propagated more efficiently than the low-pathogenic clone in kidneys of mice during the course of infection. Therefore, there is a possibility that the balance between quantities of the target and effector is important for disease outcome. In conclusion, this study showed that CD8+ T cells are involved in the development of renal hemorrhage in a mouse model of HFRS.

Exploring the Functional Relevance of BTK Beyond Chronic Lymphocytic Leukemia (CLL) Cells: BTK Expression in Non-Malignant Immune Cells of the Microenvironment Mediates CLL Development and Progression In Vivo

Background:Bruton's tyrosine kinase (BTK) is a critical component of the BCR pathway, but BTK and/or BTK-related enzymes such as ITK, Tec, BMX and RLK are also expressed in T, NK and myeloid cells, all of which are important sources of CLL-associated immune dysfunction. Whether BTK and/or BTK-related enzymes within non-malignant immune cells have any effects on CLL progression and immune function is not known. This is highly relevant translationally, as newer generations of BTK inhibitors selectively target BTK while sparing BTK-related enzymes in other cells. As the effects of constitutively active BCR and BTK targeting can be adequately modelled in the TCL-1 and TCL-1xXID mouse models (Woyach et al. Blood 2014), we used these preclinical tools to investigate if BTK expression in non-malignant immune cells modulates CLL progression in vivo. Our aims were: (1) to characterize differences in phenotype and function of T, NK and myeloid cells in leukemia-free and leukemic WT and BTK-inactive mice; (2) to assess BTK-related PD-L1 expression; and (3) to evaluate the in vivo relevance of BTK in non-malignant immune cells to CLL progression.Methods:Single-cell suspension cells were obtained from spleens from CLL-free matched WT B6 and BTK-inactive XID mice (n=11 each) and leukemic TCL-1xXID and TCL-1 mice (n=10 each). Flow cytometry was used to characterize the phenotype of B, T, NK and myeloid cells and myeloid-derived suppressor cells (MDSCs), PD-L1 expression, and T-cell degranulation. For survival experiments, WT B6 (n=29) and XID (n=27) mice were injected with 5x106 splenocytes from leukemic TCL1 donors and monitored daily. For in vivo depletion of monocytes/ macrophages, WT and XID (both n=12) mice received clodronate i.p. every 3 days from day 1 after injection of 3x107 splenocytes from leukemic TCL1 donors, and were euthanized after 36 days of treatment. RT qPCR was conducted on BTK-deficient XLA cell lines.Results:To explore whether BTK expression in non-malignant immune cells affects CLL development in vivo, we first conducted adoptive transfer experiments into WT and BTK-inactive XID mice. While WT mice had a median survival of 52 days after CLL development, survival was almost twice as long (90 days) in XID mice, indicating that BTK inactivation in non-leukemic cells prolongs survival. To identify whether this could be explained by differences in immune cell populations, we next characterized phenotype and function of T, NK and myeloid cells in leukemia-free and leukemic BTK functional and BTK-inactive mice. This comparison revealed that CLL-induced T-cell changes were broadly recapitulated in BTK-inactive mice, while significantly more immature (p=0.0206) and mature (p=0.008) NK cells were maintained. In the myeloid compartment, BTK activity did not affect the overall quantity of CD11b+ myeloid cells, but led to significant qualitative CLL-associated changes: while mice with functional BTK exhibited a significant CLL-driven loss of neutrophils (p=0.0002) and expansion of F4/80+CD11b+ macrophages (p=0.0001), leukemic BTK-inactive mice largely maintained these populations, suggesting anti-tumor properties of these cells. We next depleted myeloid cells with presumed BTK-associated anti-tumor properties in CLL-engrafted mice using clodronate. After 36 days of treatment, differences in spleen weights, CLL load in spleen and blood, and percentage of proliferating CLL cells were lost between WT and XID mice (all p>0.05), indicating that monocytes/ macrophages in XID mice indeed have in vivo anti-tumor functions. Neutrophils were expanded in both WT (median 44.7% of CD11b+ cells) and XID mice (43.7% vs. 6.1% in non-depleted mice). Interestingly, PD-L1 expression as a surrogate marker for tumor-associated immune suppression was generally significantly increased on antigen-presenting cells from BTK-inactive leukemic mice (CLL cells p=0.0002; inflammatory monocytes p=0.0014; patrolling monocytes p=0.001; MDSCs p=0.0201). RT qPCR experiments using XLA cell lines with modified BTK expression revealed that absence of BTK was associated with significantly higher PD-L1 RNA expression (p=0.0075), indicating a direct mechanistic link between BTK and PD-L1.Conclusions: Our data indicate that BTK in myeloid and NK cells directly mediates CLL development and progression in vivo. Targeting BTK in those cells in addition to CLL cells might provide substantial clinical benefits. DisclosuresWoyach:Acerta: Research Funding; Karyopharm: Research Funding; Morphosys: Research Funding.

Noradrenergic modulation of evoked dopamine release and pH shift in the mouse dorsal hippocampus and ventral striatum

Rapid monoamine release in the dorsal hippocampus is not well characterized, despite its postulated role in modulating fast hippocampal circuit dynamics. We measured monoamine release in the dorsal hippocampus upon stimulation of the ventral tegmental area (VTA) with fast-scan cyclic voltammetry in anesthetized norepinephrine-depleted and non-depleted mice. Within the hippocampus, norepinephrine depletion altered the ability of α2 adrenergic compounds and transporter blockers to modulate the small, evoked monoamine signal. These manipulations also affected the pH shifts observed after stimulation in a drug-dependent manner. The evoked signal was potentiated by α2C adrenoceptor subtype antagonism, but was not affected by or α2A adrenoceptor antagonism. The same subtype-specific pattern was observed on evoked dopamine release in the ventral striatum. The pharmacological and anatomical evidence supports a contribution by dopamine to the VTA-evoked hippocampal monoamine signal, and confirms the interaction between the mesohippocampal and coeruleohippocampal systems. These results also reinforce the notion that α2C, but not α2A adrenoceptors regulate endogenous dopaminergic activity. We believe our findings hold implications for understanding the efficacy of α2 adrenergic agonists and antagonists that are used widely for therapeutic purposes.

Type 2 inflammatory response protects against Staphylococcus aureus-induced sepsis

Abstract Sepsis is defined as new organ dysfunction due to acute infection and has a mortality rate &amp;gt;20%. Recent studies indicate that type 2 cytokines, best known for their role in allergies, may be beneficial to the host during sepsis. We hypothesized that these responses protect against mortality during Staphylococcus aureus (S. aureus) induced sepsis. We tested this hypothesis in 2 different mouse models, using either house dust-mite (HDM) sensitization and challenge to activate an adaptive type 2 response, or pre-treatment with IL-33 via intratracheal instillation to induce an innate type 2 response. This was followed by intravenous infection with S. aureus to induce sepsis. Pre-treatment with either HDM or IL-33 protected against S. aureus-induced mortality, indicating that activation of type 2 responses is beneficial during acute infection. Analysis of lung immune cell populations by flow cytometry revealed increased levels of type 2 innate lymphoid cells (ILC2) in mice pre-treated with HDM or IL-33 compared to untreated controls. Further, we found a relative decrease in lung neutrophils and an increase in lung eosinophils in type 2-stimulated compared to untreated mice, suggesting that type 2 responses may suppress an overwhelming neutrophilic response. However, systemic depletion of neutrophils prior to infection resulted in accelerated mortality compared to non-depleted mice, indicating that neutrophils are necessary to protect against S. aureus infection, and that augmented eosinophilia may confer protection against mortality. We conclude that the type 2 response is protective during sepsis, possibly due to the activity of ILC2s and a delicate balance of eosinophils and neutrophils in the inflamed lung.

Dendritic Cell Protection from Cisplatin Nephrotoxicity Is Independent of Neutrophils.

Cisplatin is a very effective chemotherapeutic agent used against a wide range of solid tumors. A major adverse effect of cisplatin therapy is acute kidney injury (AKI). Neutrophils are reported to infiltrate and exacerbate injury in a wide range of sterile inflammatory models of tissue injury. Here, we studied the kinetics of neutrophil infiltration into kidneys and their role in cisplatin-mediated AKI. Mice treated with cisplatin showed an increase in circulating neutrophils 24 and 48 h after cisplatin administration. Cisplatin treatment caused an increase in kidney leukocytes with neutrophils accounting for the majority of the infiltrating leukocytes. The extent of neutrophil infiltration coincided with the severity of kidney injury and renal dysfunction. To examine the functional relevance of infiltrating neutrophils in cisplatin nephrotoxicity, we depleted neutrophils using a neutrophil-specific antibody (anti-Ly-6G). This antibody resulted in greater than 90% depletion of neutrophils in both the blood and kidney. Of note, depletion of neutrophils had no impact on the extent of cisplatin-induced AKI as compared to non-depleted mice. Earlier, we reported that dendritic cell depletion in CD11c-DTRtg mice causes exacerbation of AKI and a dramatic increase in renal neutrophils. Thus, we also examined the role of neutrophils in dendritic cell-depleted mice treated with cisplatin. Dendritic cell depletion exacerbated AKI in spite of neutrophil depletion. These data demonstrate that cisplatin nephrotoxicity is not mediated by neutrophils and that dendritic cells protect kidneys via neutrophil-independent mechanisms.

Neutrophil Extracellular Traps are Involved in the Innate Immune Response to Infection with Leptospira.

NETosis is a process by which neutrophils extrude their DNA together with bactericidal proteins that trap and/or kill pathogens. In the present study, we evaluated the ability of Leptospira spp. to induce NETosis using human ex vivo and murine in vivo models. Microscopy and fluorometric studies showed that incubation of human neutrophils with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 (LIC) resulted in the release of DNA extracellular traps (NETs). The bacteria number, pathogenicity and viability were relevant factors for induction of NETs, but bacteria motility was not. Entrapment of LIC in the NETs resulted in LIC death; however, pathogenic but not saprophytic Leptospira sp. exerted nuclease activity and degraded DNA. Mice infected with LIC showed circulating NETs after 2 days post-infection (dpi). Depletion of neutrophils with mAb1A8 significantly reduced the amount of intravascular NETs in LIC-infected mice, increasing bacteremia at 3 dpi. Although there was a low bacterial burden, scarce neutrophils and an absence of inflammation in the early stages of infection in the kidney and liver, at the beginning of the leptospiruric phase, the bacterial burden was significantly higher in kidneys of neutrophil-depleted-mice compared to non-depleted and infected mice. Surprisingly, interstitial nephritis was of similar intensity in both groups of infected mice. Taken together, these data suggest that LIC triggers NETs, and that the intravascular formation of these DNA traps appears to be critical not only to prevent early leptospiral dissemination but also to preclude further bacterial burden.

Relevance of Foxp3+ regulatory T cells for early and late phases of murine sepsis

The role of Foxp3(+) regulatory T (Treg) cells in the course of the early hyper-inflammatory and subsequent hypo-inflammatory phases of sepsis is ambiguous. Whereas Nrp1 expression has been reported to discriminate natural Treg cells from induced Treg cells, the Treg cell stability depends on the methylation status of foxp3-TSDR. To specifically evaluate the role of Foxp3(+) Treg cells in the early and late phases of sepsis, we induced sepsis by caecal ligation and puncture and subsequent Pseudomonas aeruginosa lung infection in a DEREG (DEpletion of REGulatory T cells) mouse model. We found an increase of Foxp3(+) Treg cells to all CD4(+) T cells during murine sepsis. Using a new methylation-sensitive quantitative RT-PCR method and deep amplicon sequencing, we demonstrated that natural (Nrp1(+) Foxp3(+) ) Treg cells and most induced (Nrp1(-) Foxp3(+) ) Treg cells are stable and exhibit unmethylated foxp3-TSDR, and that both Treg populations are functionally suppressive in healthy and septic mice. DEREG mice depleted of Foxp3(+) Treg cells exhibit higher disease scores, mortality rates and interleukin-6 expression levels than do non-depleted DEREG mice in early-phase sepsis, a finding indicating that Foxp3(+) Treg cells limit the hyper-inflammatory response and accelerate recovery. Treg cell depletion before secondary infection with P. aeruginosa 1 week after caecal ligation and puncture does not influence cytokine levels or the course of secondary infection. However, a moderate Treg cell recurrence, which we observed in DEREG mice during secondary infection, may interfere with these results. In summary, Treg cells contribute to a positive outcome after early-phase sepsis, but the data do not support a significant role of Treg cells in immune paralysis during late-phase sepsis.

In Vivo Induction of Functionally Suppressive Induced Regulatory T Cells from CD4+CD25- T Cells Using an Hsp70 Peptide.

Therapeutic peptides that target antigen-specific regulatory T cells (Tregs) can suppress experimental autoimmune diseases. The heat shock protein (Hsp) 70, with its expression elevated in inflamed tissue, is a suitable candidate antigen because administration of both bacterial and mouse Hsp70 peptides has been shown to induce strong immune responses and to reduce inflammation via the activation or induction of Hsp specific Tregs. Although two subsets of Tregs exist, little is known about which subset of Tregs are activated by Hsp70 epitopes. Therefore, we set out to determine whether natural nTregs (derived from the thymus), or induced iTregs (formed in the periphery from CD4+CD25- naïve T cells) were targeted after Hsp70-peptide immunization. We immunized mice with the previously identified Hsp70 T cell epitope B29 and investigated the formation of functional iTregs by using an in vitro suppression assay and adoptive transfer therapy in mice with experimental arthritis. To study the in vivo induction of Tregs after peptide immunization, we depleted CD25+ cells prior to immunization, allowing the in vivo formation of Tregs from CD4+CD25- precursors. This approach allowed us to study in vivo B29-induced Tregs and to compare these cells with Tregs from non-depleted immunized mice. Our results show that using this approach, immunization induced CD4+CD25+ T cells in the periphery, and that these cells were suppressive in vitro. Additionally, adoptive transfer of B29-specific iTregs suppressed disease in a mouse model of arthritis. This study shows that immunization of mice with Hsp70 epitope B29 induces functionally suppressive iTregs from CD4+CD25- T cells.

Macrophages and galectin 3 play critical roles in CVB3-induced murine acute myocarditis and chronic fibrosis

Macrophage influx and galectin 3 production have been suggested as major players driving acute inflammation and chronic fibrosis in many diseases. However, their involvement in the pathogenesis of viral myocarditis and subsequent cardiomyopathy are unknown. Our aim was to characterise the role of macrophages and galectin 3 on survival, clinical course, viral burden, acute pathology, and chronic fibrosis in coxsackievirus B3 (CVB3)-induced myocarditis. Our results showed that C3H/HeJ mice infected with CVB3 and depleted of macrophages by liposome-encapsulated clodronate treatment compared with infected untreated mice presented higher viral titres but reduced acute myocarditis and chronic fibrosis, compared with untreated infected mice. Increased galectin 3 transcriptional and translational expression levels correlated with CVB3 infection in macrophages and in non-depleted mice. Disruption of the galectin 3 gene did not affect viral titres but reduced acute myocarditis and chronic fibrosis compared with C57BL/6J wild-type mice. Similar results were observed after pharmacological inhibition of galectin 3 with N-acetyl-d-lactosamine in C3H/HeJ mice. Our results showed a critical role of macrophages and their galectin 3 in controlling acute viral-induced cardiac injury and the subsequent fibrosis. Moreover, the fact that pharmacological inhibition of galectin 3 induced similar results to macrophage depletion regarding the degree of acute cardiac inflammation and chronic fibrosis opens up the possibility of new pharmacological strategies for viral myocarditis.