bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting the inflammasome: clearance of Methicillin-Resistant Staphylococcus aureus in skin and soft tissue infections (162.14)
Authors: Artlett Carol , Sassi-Gaha Sihem , Purohit Mitali , Rest Richard , Thacker James
Published: January 2023

We reported an endogenously derived inflammasome activating lipopeptide (1-peptidyl-2-arachidonoyl-3-stearoyl glyceride) with the amino acid sequence for the peptide being acALYDKGYTSKEQKDCVGI (acALY18) and determined that the peptide alone was responsible for the biological activity. Inflammasomes are important for the activation of the innate immune response by detection of danger signals in response to pathogen infection and sterile injury. The inflammasomes modulate the activation of IL-1β and IL-18, by controlling the activation of caspase-1 that cleaves these cytokines for secretion. In fibroblasts, the activation of the inflammasome mediated signaling events by acALY18 was inhibited by Z-YVAD(OMe)-FMK (caspase-1 inactivator) and secretion of IL-1β and IL-18 was abolished. Inflammasome activation in primary human fibroblasts suggests that acALY-18 could be prophylactically useful for the prevention of hospital-acquired infections. Therefore, we conducted a pilot study consisting of two cohorts of 30 mice each infected with MRSA +/- 100 μg/Kg acALY18. In both cohorts, acALY18 reduced the pathogen load in the tissues resulting in smaller lesions and an increased healing rate whereas the untreated mice had much larger lesions with a thick, purulent exudate and no signs of healing. This data suggests that in vivo administration of acALY-18 could induce innate immune responses that would be prophylactically useful for the prevention of hospital-acquired infections.
Cellular mechanisms underlying LPS-induced bactericidal activity in Dictyostelium discoideum (162.15)
Authors: Snyder Michelle , Pflaum Katherine , Gerdes Kimberly , Yovo Kossi , Callahan Jennifer , Walk Alexander
Published: January 2023

Cells of the innate immune system detect pathogen-associated molecular patterns (PAMPs) through highly conserved pattern-recognition machinery. The conservation of this machinery allows for its characterization in model organisms. The unique model organism Dictyostelium discoideum can exist as single-celled amoebae that phagocytize bacteria for nutritional uptake. However, it has not been determined if D. discoideum uses pattern-recognition machinery to detect PAMPs. Here we show that D. discoideum mounts a response against the bacterial cell wall PAMP, lipopolysaccharide (LPS). LPS treatment results in an increased clearance of phagoctyzed bacteria by D. discoideum. LPS-induced bactericidal activity appears to be dependent on MAPK and Tir domain-containing protein A (TirA)-mediated pathways. Recent results suggest that increased bactericidal activity upon treatment with LPS may involve the induction and maturation of autophagosomes. We show here, by following the delivery of the autophagosomal marker Atg8 to degradative compartments, that Staphylococcus aureus induces autophagosomal maturation in D. discoideum only upon addition of LPS. We are currently characterizing the signaling pathways underlying bacterial induction of autophagy in D. discoideum. Characterization of the cellular events induced upon microbial pattern recognition in D. discoideum should provide valuable insight into conversed cellular mechanisms underlying microbial detection and killing.
The cytosolic sensors RIG-I and DAI can mediate glial inflammatory responses to neurotropic viruses (162.17)
Authors: Furr Samantha , Grdzelishvili Valery , Marriott Ian
Published: January 2023

The rapid onset of severe neuroinflammation is a defining feature of viral encephalitis. Glial cells are ideally positioned to respond to CNS pathogens and produce potent inflammatory mediators following exposure to neurotropic viruses. However, the mechanisms by which glia perceive viruses remain unclear. Recently the cytosolic proteins RIG-I and DAI have been reported to function as intracellular sensors of RNA and DNA viruses, respectively, but their role in virally-induced neuroinflammation has not been defined. We have shown that primary glial cells express such receptors constitutively and that their expression can be further induced following viral challenge. In the present study we demonstrate that resident CNS cells express these cytosolic pattern recognition receptors in situ and we show that their expression is elevated in murine models of viral encephalitis. Importantly, we have utilized specific ligands for RIG-I and DAI to demonstrate that these viral sensors can mediate the production of inflammatory/neurotoxic mediators by microglia and astrocytes. Furthermore, we have employed siRNA approaches to demonstrate the importance of RIG-I and DAI in such responses to the neurotropic viruses, VSV and HSV-1, respectively. Together, our studies indicate that these novel cytosolic viral sensors may play a critical role in the recognition of viral pathogens and the initiation of the potentially lethal neuroinflammation associated with acute viral CNS infection.
RON receptor tyrosine kinase, a negative regulator of inflammation, is decreased during SIV associated central nervous system disease. (162.18)
Authors: Cary Daniele , Henderson Andrew , Clements Janice
Published: January 2023

Expressed on tissue resident macrophages, the receptor tyrosine kinase, RON, functions to maintain inflammation homeostatisis by activating genes that promote wound repair and resolve inflammation; while repressing genes that perpetuate tissue damage and cell death. Chronic HIV infection is associated with dysregulated inflammation that contributes to organ diseases, and we hypothesize that altered RON expression contributes to the development of HIV associated CNS disease. We utilized a well characterized SIV model in macaques to explore the temporal regulation of RON in the brain during the course of infection. Following prolonged SIV infection, RON expression decreased below levels observed in controls. RON expression inversely correlated with the development of CNS disease; RON was maintained in animals that did not develop CNS disease and was reduced in macaques that demonstrated CNS disease symptoms. Arginase expression was reduced in the brain during late infection whereas expression of the inflammatory genes, interleukin 12p40 and TNFα were elevated. Our data support a model in which during acute SIV infection in the brain, active inflammation is controlled by RON, however, following prolonged infection, RON expression is decreased, genes that quell inflammation are repressed, and inflammatory mediators are induced to directly promote CNS disease.
Altered oral mucosal homeostasis in Type 1 diabetes (162.19)
Authors: Wallet Shannon , Alonso Tess , Lalane Charles , Catalfamo Dana , Neiva Kathleen
Published: January 2023

In type 1 diabetes (T1D) alterations in mucosal barrier function and immunity are implicated in disease pathogenesis. We have reported that TLR2-inducing homeostatic responses are disrupted in oral epithelial cells (HOK) from T1D individuals. The objective of the study was to delineate mechanisms of TLR2-induced homeostasis altered in T1D. miRNA, protein phosphorylation and cytokine expression were evaluated in HOK challenged with TLR2 or TLR4 agonists. Expression of cytokines was evaluated using a multiplex assay. Total RNA was probed for miR146a, mir133, miR9 and miR155 using qPCR. The expression and phosphorylation of MyD88, TRAF6, IRAK1, and IRAK4 along with the expression of PTPN5, PTPN7 and PTPRR were quantified using western blot. In HOK from non-diabetic participants, TLR2-induced expression of TGFβ and IL10 in the absence of TNFα, IL6 and IL8 was concomitant with increased expression of miR146a and miR155 as well as PTPN5 and PTPRR along with decreased expression and phosphorylation of TRAF6 and IRAK4 when compared to TLR4 stimulation. Upon TLR2 ligation of HOK from T1D, miR146a, miR155, PPTN5 and PTPRR expression was unaltered when compared to TLR4 ligation. Therefore, miRNA regulation of TLR2-induced homeostatic responses as well as phosphatase regulation of TLR signaling cascades are altered in human T1D HOK resulting in over-expression and phosphorylation of TLR signaling intermediates allowing for exacerbated epithelial mediated inflammatory responses.
Heparan sulfate-induced pancreatic inflammatory response is TLR4 dependent (162.2)
Authors: Akbarshahi Hamid , Axelsson Jakob , Said Katarzyna , Fischer Hans , Malmström Anders , Andersson Roland
Published: January 2023

Degraded extracellular matrix can stimulate the innate immune system via the Toll-Like Receptor-4 (TLR4). In the pancreas, heparan sulfate (HS) on syndecan anchored in the ductal epithelium, can be cleaved off by enzymes and potentially activate TLR4 signalling. To investigate these signalling events, we infused heparan sulfate (HS) into the pancreatic duct of the mice. Phosphate-buffered saline and lipopolysaccharide (LPS)were used as controls. The wild type C57BL/6J mice were sacrificed after 1, 3, 6, 9, and 48 hours. To study the downstream TLR4 signalling pathway of HS in the pancreas, genetically engineered mice lacking TLR4, MyD88 or IRF3 were used. Neutrophil sequestration and myeloperoxidase activity in the pancreas were increased 9 hours following HS challenge. However, in TLR4, MyD88, and IRF3 knockout mice the neutrophil recruitment and myeloperoxidase activity were abrogated in the HS group while the LPS response was only abolished in TLR4 and MyD88 knockouts. In wild type mice, the monocyte chemoattractant protein -1 increased at 3 hours after infusion while RANTES increased after 9 hours. In conclusion, we have shown that HS is capable of initiating an innate immune response in the pancreas distinctly different from that induced by LPS. Furthermore, the data shows that the inflammatory response is TLR4 dependent and mediated through both MyD88-dependent and independent pathways. Thus, heparan sulfate is potentially involved in the ductal defense in the pancreas.
Cannabinoids Receptor-2 (CB2) agonist ameliorates spontaneous colitis by attenuating neutrophils, mast cells, natural killer cells and inducing endogenous cannabinoids (162.3)
Authors: Singh Udai , Singh Narendra , Singh Balwan , Price Robert , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

Inflammatory bowel disease (IBD) is a chronic intestinal inflammation caused by hyperactivated effector immune cells that produce pro-inflammatory cytokines and an influx of neutrophils. It is well known that endocannabinoids mediate protection against intestinal inflammation. Here, we investigate the activation of endocannabinoid receptors -2 (CB2) by JWH-133, a potent CB2 agonist- mediated inhibition of chronic colitis in IL-10-/- mice. In the present study, we report that JWH-133 effectively attenuated the overall clinical score, reversed colitis-associated pathogenesis including the decrease in body weight, and increased the expression of endocannabinoid-2 receptors in the colon. After JWH-133 treatment, the percentage of CD4+ T cells in the spleen of colitis mice was restored to normal levels and there was a decrease in these cells in the intestinal lamina propria (LP) and mesenteric lymph nodes (MLN). Similarly, the percentage of mast, natural killer (NK1.1) and activated T cells in the LP of colitis mice declined after JWH-133 treatment. JWH-133 also reduced the number and percentage of neutrophils that were induced during chronic colitis progression. These findings suggest that JWH-133 ameliorates chronic colitis by inducing endogenous CB2 receptors at sites of inflammation and reduces Th1-, mast-, NK1.1 cells, and neutrophils from the spleen and LP. These results support the idea that the CB2 receptor agonist may be developed as a novel therapeutic target for IBD.
IL-25 enhances the accumulation and function of lung resident TH2 memory/effector cells during allergic inflammation (163.14)
Authors: Liu Bo , Chen Chun-Yu , Ohta Shoichiro , Angkasekwinai Pornpimon , Burwinkel Karen , Izuhara Kenji , Liu Yong-Jun , Wang Yui-Hsi
Published: January 2023

IL-25 is a TH2-promoting cytokine, however, its role in regulating the maintenance and function of TH2 memory/effector cells in vivo remains elusive. We have developed a novel mouse model to study the regulation of TH2 memory/effector cells after re-exposure to OVA antigen. Phenotypic analyses reveal that the recalled TH2 memory/effector cells in lung express distinct level of IL-25 receptor (IL-25R), ST2, and OX40. Notably, the frequency of local IL-25R+CD4+ TH2 memory/effector cells in inflamed lung, but not other lymphoid tissues, is elevated after increased numbers of OVA antigen re-exposure. These lung resident IL-25R+CD4+ TH2 memory/effector cells are much more potent to produce TH2 cytokines than the TH2 cells in other lymphoid tissues. Enforced expression of IL-25R by T cells in mice resulted in increased accumulation of local antigen-specific TH2 memory/effector cells, leading to more prominent airway inflammation, compare to those of littermate controls in our novel mouse model of allergic lung diseases. These results suggest that IL-25 may promote allergic inflammation by enhancing the accumulation and function of lung resident IL-25R+CD4+ TH2 memory/effector cells in vivo.
Skin histopathology, including a significant itch response, associated chemically-induced contact hypersensitivity reactions in the mouse are, in part, a consequence of skin infiltrating eosinophils (163.16)
Authors: Condjella Rachel , Protheroe Cheryl , Ochkur Sergei , Zellner Katie , Colbert Dana , Scott Gregory , Jacoby David , Fryer Allison , Lee Nancy , Lee James
Published: January 2023

Atopic dermatitis and allergic contact dermatitis are two of the most common skin diseases. In both diseases, eosinophils have been suggested as potential contributors of the pathologies linked with these skin conditions. Our goal was to establish a causative relationship between the inflammatory response of chemically induced contact hypersensitivity and eosinophil-mediated activities. Wild type and eosinophil-less PHIL mice were sensitized and challenged with 2,4-dinitrochlorobenzene (DNFB) or alternatively trimellitic anhydride (TMA). Time lapsed videography was used to capture the number of itching events associated with treatment. Immunohistochemistry for eosinophils and nerves was performed to correlate the effect of dermatitis on innervation and eosinophil accumulation in these mice. Our studies showed that in both the DNFB and TMA models a robust eosinophil infiltrate occurred that was accompanied by copious levels of eosinophil degranulation. More importantly, these studies demonstrated that metrics of inflammation (e.g., induced ear swelling (i.e., thickness)) was reduced in PHIL mice relative to wild type. Our observations were also directly correlative with a loss of nerve growth and branching in PHIL mice. These data implicate eosinophils as contributors to the pathologies linked with chemically-induced contact hypersensitivity, including the induced itch responses associated with these models.
Opposing roles for fyn and lyn kinases in FcγR signaling and IgG-mediated systemic anaphylaxis (163.17)
Authors: Falanga Yves , Finkelman Fred , Odom Sandra , Rivera Juan , Oskeritzian Carole , Ryan John
Published: January 2023

Mast cells (MCs) are key players in the innate immune system and atopic diseases. Once activated, they degranulate and produce cytokines, resulting in the recruitment of other immune cells and the clearance of the infectious agent. In this study we focused on MC activation via the immunoglobulin G (IgG) receptors, FcγR. We found that croslinkage of mouse mast cells with an antibody recognizing the low affinity receptors FcγRIIb and FcγRIII induced tyrosine phosphorylation of both Lyn and Fyn. We used Fyn- or Lyn-deficient (KO) mice to demonstrate that FcγR, like the IgE receptor, FcϵRI, triggers mast cell degranulation and cytokine release via a Fyn- and Lyn-dependent manner. Lyn KO mast cells displayed increased FcγR-mediated degranulation and cytokine release, while a decrease was observed using Fyn KO MC. We show that the role for Fyn and Lyn downstream of FcγR is not restricted to the MC lineage, as similar effects were found in basophils and macrophages. Furthermore, these effects were consistent in vivo, when measuring IgG-mediated passive systemic anaphylaxis (PSA). Collectively, these data demonstrate Fyn and Lyn activation during FcγR signaling and highlight their homeostatic contribution to IgG-mediated inflammation.
Walnut extract induces near-fatal anaphylactoid shock via the complement C3 dependent pathway in naïve mice (163.2)
Authors: Gangur Venu , Parvataneni Sitaram , Gonipeta Babu
Published: January 2023

Walnut is a major tree nut associated with severe allergic/anaphylactic reactions in humans. Here we surprisingly found that oral administration of walnut extract to naïve mice resulted in a sharp drop in rectal temperature associated with near-fatal clinical presentation within 1 hour. This reaction was independent of mice strain as both BALB/c and C57BL/6 developed reactions. The reactions were not due to LPS because LPS receptor mutant C3H/Hej mice also exhibited reactions. The reactions were not allergic because low/non-IgE responder SJL mice and IgE receptor knockout (KO) mice also exhibited such reactions. Furthermore, these mice had no detectable antibodies (IgE or IgG1) to walnut. We found that the acute near-fatal reactions were associated with severe hypotension and bradycardia. Surprisingly, the complement C3 KO mice and the C3a receptor KO mice were resistant to walnut induced hypotensive shock responses. These data demonstrate for the first time that walnut extract induces near-fatal acute anaphylactoid shock via the innate complement system in naïve mice.
Invariant NKT cells are crucial for airway inflammation induced by environmental antigens (163.22)
Authors: Wingender Gerhard , Rogers Paul , Batzer Glenda , Lee Myung , Bai Dong , Khurana Archana , Kronenberg Mitchell , Horner Anthony
Published: January 2023

Invariant NKT (iNKT) cells are a unique subset of T lymphocytes that rapidly carry out effector functions. Here we report that a majority of sterile house dust extracts (HDEs) tested contained antigens capable of activating mouse and human iNKT cells. HDEs had adjuvant-like properties in an ovalbumin-induced asthma model, that were dependent on Vα14i NKT cells, as vaccinated animals deficient for iNKT cells displayed significantly attenuated immune responses and airway inflammation. Furthermore, the administration of HDEs together with ovalbumin mutually augmented the synthesis of cytokines by Vα14i NKT cells and by conventional CD4+ T cells in the lung, demonstrating a profound immune response synergy for both Th2 cytokines and IL-17A. These data demonstrate that iNKT cell antigens are far more widely dispersed in the environment than previously anticipated. Furthermore, as the antigenic activity in different houses varied greatly, they further suggest that iNKT cell responses to ambient antigens, particular to certain environments, might promote sensitization to conventional respiratory allergens.
PARP-1 deficiency blocks IL-5 expression through calpain-dependent degradation of STAT-6 in a murine asthma model (163.6)
Authors: Naura Amarjit , Datta Rahul , Zerfaoui Mourad , Errami Youssef , Oumouna Mustapha , Kim Hogyoung , Jihang Ju , Ronchi Virginia , Haas Arthur , Boulares Hamid
Published: January 2023

We recently showed that PARP-1 plays a role in allergen (Ovalbumin)-induced airway eosinophilia, potentially through a specific effect on IL-5 production. Here we have explored the mechanism by which PARP-1 regulates IL-5 production using a murine model of allergic airway inflammation and splenocytes derived from wild type or PARP-1-/- mice. We show that PARP-1 knockout-associated reduction in IL-5 upon allergen exposure occurs at the mRNA level. Such an effect appears to take place after IL-4 receptor activation as PARP-1 inhibition exerted no effect on JAK1/JAK3 activation. STAT-6 protein was severely downregulated in spleens of PARP-1-/- mice without any effect on mRNA levels, suggesting an effect on protein integrity rather than gene transcription. Interestingly, the degradation of STAT-6 in PARP-1-/- mice required allergen stimulation. The dowregulation of STAT-6 coincided with mRNA and protein reduction of GATA-3 and occupancy of its binding site on the IL-5 gene-promoter. IL-4 was sufficient to induce STAT-6 downregulation in both PARP-1-/- mice and isolated splenocytes. Such degradation may be mediated by calpain, but not by proteasomes. Our results demonstrate a novel function of PARP-1 in regulating IL-5 expression during allergen-induced inflammation and explain the underlying mechanism by which PARP-1 inhibition results in IL-5 reduction.
Regulation of Allergic Airways Disease by CD4+ T cells engineered to overexpress Interleukin-17 (163.7)
Authors: Nakada Emily , Kinyanjui Margaret , Fixman Elizabeth
Published: January 2023

Background: Clinical data have associated Th17 cells, IL-17 and neutrophilia with severe asthma. The objective of these experiments is to assess how T cell specific overexpression of IL-17 modifies OVA-induced airway inflammatory responses using an adoptive transfer murine model. Methods: Recombinant retroviruses encoding either EGFP alone (pAP2) or both EGFP and IL-17 were used to transduce CD4+ T cells from OVA-sensitized donor BALB/c mice, re-stimulated with OVA ex vivo for targeted retroviral transduction. CD4+ T cells were isolated using magnetic selection and adoptively transferred intraperitoneally into naïve mice on day 0. Recipients were then anaesthetized and challenged intranasally with OVA daily for 5 days. Mice were sacrificed 24h after the final challenge. Results: Recombinant retroviruses efficiently transduced CD4+ T cells after re-stimulation with OVA such that 0.17% of T cells transduced with control virus expressed IL-17 vs 4.65% of T cells transduced with IL-17pAP2. Supernatants from IL-17pAP2 transduced cells produced abundant IL-17, which was undetectable in pAP2 transduced cells. Following adoptive transfer and challenge, IL-17 levels in the bronchoalveolar lavage fluid (BALF) of recipients from the IL-17pAP2 group were greater than those from the pAP2 control group. BALF levels of inflammatory cells were increased in each group. This model will allow us to define how allergic airway inflammation is modified by T cell specific IL-17 overproduction.
Gender specific increase of IL-17A in a murine model of asthma in the absence of T-bet (163.8)
Authors: Koch Sonja , Übel Caroline , Finotto Susetta
Published: January 2023

Recent studies in humans have suggested that females may be more susceptible to allergic asthma than male. In addition, studies in Balb/c mice revealed that females have higher serum IgE levels, Th2 cytokines and an increased number of CD4+ T-cells. Mice lacking the Th1-specific transcriptional factor T-bet (T-bet-/-) show a severe phenotype of asthma with increased AHR, eosinophils and Th2 type cytokines in their airways. We therefore analyzed gender specific immune-responses in T-bet-/- mice in a murine model of allergic asthma. To this aim, we first looked at the Th2 inducing cytokine IL-6 and found it induced in the airways of naïve T-bet-/- mice. IL-6 inhibits T regulatory cells and together with TGFβ1 induces Th17 cells. We then studied the expression of different cytokines involved in the development of the Th17-pathway in a gender dependent manner. We found that total lung cells isolated from asthmatic female T-bet-/- mice release increased levels of IL-6, TGFβ1 and IL-17 compared to the male littermates. Consistently, in these mice, other cytokines of the Th17-pathway like IL-23 and IL-21 were also found induced in the lung of female T-bet-/- mice as compared to the levels measured in the lung of males. These results might explain why females are more susceptible than males to some aspects of asthma and therefore gender specific studies in asthma might help to better design therapies for this disease.
Identification and analysis of lipid mediators in a mouse model of allergic contact dermatitis (163.9)
Authors: Chen Huan-Yuan , Yang Jun , Inceoglu Ahmet Bora , Wang Li-Chieh , Yu Lan , Hammock Bruce , Liu Fu-Tong
Published: January 2023

Allergic contact dermatitis (CD) is a type of inflammatory skin disease resulting from delayed-typed hypersensitivity to haptenic antigens. The antigens are presented to T cells by dendritic cells in the skin and result in activation and recruitment of other immune cells to the skin. The inflammatory response is frequently accompanied by production of metabolic mediators. Here we sought to identify mediators involved in allergic CD by using a mouse model. C57BL/6 mice were sensitized with dinitrofluorobenzene or oxazolone and challenged with the same antigen on the ear. The treated ears were swollen and exhibited accumulation of leukocytes. The affected ears were subjected to targeted quantitative metabolomic analysis by mass spectrometry to identify lipid metabolites that displayed significant changes. A cyclooxygenase product, thromboxane A2, was remarkably induced by more than 10 fold compared to vehicle control. A number of other bioactive lipid metabolites were induced by more than two fold. We found that mice treated with aspirin or sEHi showed reduced ear thickness compared to vehicle treatment. Our data suggest that the cyclooxygenase and soluble epoxide hydrolase pathways are involved in the development of allergic CD by modulating the inflammatory skin response. Inhibitors of these two pathways and antagonists of prostanoid receptors for the elevated metabolites are expected to be effective therapeutically to treat allergic CD.
Astrocyte-specific blockade of the IL-17 pathway reduces disease severity of experimental autoimmune encephalomyelitis (164.11)
Authors: Yan Yaping , Ding Xiaoli , Li Ke , Rostami Abdomohamad , Zhang Guang-xian
Published: January 2023

IL-17 is crucial for the progression of experimental autoimmune encephalomyelitis. Through neutoectoderm-specific knockout studies, it has been shown that astrocyte-restricted ablation of IL-17 induced, Act1-mediated signaling ameliorates autoimmune encephalomyelitis. However, it is difficult to translate these results into therapy because of: 1) complex functions of Act1; 2) potentially severe side effects from constitutively repressed Act1 expression; and 3) impracticality of knockout model for human therapy. To overcome these weaknesses, we have established a GFAP promoter driving miR-30 based shRNA cassette targeting Act1 to knockdown expression of Act1 and block the IL-17-IL-17R pathway in astrocytes, and tested its therapeutic effect on ongoing EAE. Intracerebroventricular (i.c.) injection of 2×106 IU/mouse of GFAP-miR-shAct1-EGFP lentivirus significantly inhibited disease progression and severity of ongoing chronic EAE in MOG-immunized C57BL/6 mice. Further analysis showed that astrocyte-specific blockade of the IL-17 pathway greatly reduced the number of mononuclear cells in central nervous system, partially due to reduced expression of chemokines, such as CXCL1, CXCL2, CCL20 and MMP3, which are necessary for the recruitment of peripheral leukocyte infiltration during EAE pathogenesis. These results suggest that astrocyte-specific blockade of the IL-17 pathway can be a promising way to treat neuroinflammatory diseases.
RTL551-mediated treatment of EAE involves reduction in CD226 and T-bet expressing pathogenic CD4+ T-cells (164.12)
Authors: Sinha Sushmita , Miller Lisa , Vandenbark Arthur , Halina Offner
Published: January 2023

Recombinant T cell receptor ligands (RTLs) reverse clinical and histological signs of EAE in an antigen specific manner, and are currently in clinical trials for treatment of multiple sclerosis. To evaluate possible regulatory mechanisms, we tested effects of RTLs on expression of two pathogenic T-cell maturation markers, CD226 and T-bet, by CD4+ Th1/Th17+ cells in a MOG-35-55 peptide-induced model of EAE. A single injection of RTL551 (two-domain I-Ab covalently linked to MOG-35-55 peptide), but not the control RTL550 (“empty” two-domain I-Ab without a bound peptide), reduced clinical signs of EAE, prevented trafficking of cells outside the spleen, significantly reduced expression of CD226 and T-bet on blood-borne MOG-35-55-specific CD4+ T-cells and inhibited expansion of CD44 expressing CD4 cells in blood and spleen. Concomitantly, RTL551 selectively reduced absolute numbers of T-bet+ CD4+ T-cells and IL-17 and IFN-γ secretion in the CNS. These novel data demonstrate that a major effect of RTL therapy is to prevent maturation of memory T-cells that mediate clinical signs and CNS inflammation in EAE.
Highly specific and reliable biomarker of human MM cells in NOD/SCID animal models through the cancer/testis antigen AKAP-4: new opportunities for pharmacological studies (165.17)
Authors: Chiriva Maurizio , Mirandola Leonardo , Yu Yuefei , Jenkins Marjorie , Cobos Everardo , John Constance
Published: January 2023

Despite recent findings for the treatment of multiple myeloma (MM) it still remains a deadly disease. In the effort to develop new and more effective therapies for human tumors, murine animal models have been proven critical for a variety of neoplasias. Unfortunately, although recent studies have described different animal models of human MM, none is available for tracking the tumor progression with a specific biomarker employing human MM cells. This lack hampers the development of new approaches for treating MM. Here we describe a human MM model in NOD/SCID mice that allows for highly sensitive and specific monitoring of disease progression through the endogenously-expressed tumor specific cancer/testis antigen AKAP-4. The human MM cells U266 were subcutaneously injected in NOD/SCID animals. AKAP-4 expression resulted to be a reliable and sensitive antigen to monitor tumor growth and spread by different and independent techniques, namely flow-cytometry, E.L.I.S.A., Western blot and RT-PCR. The relevance of our findings stems from the suitability of this innovative model for a thorough pre-clinical evaluation of experimental pharmacological strategies for the treatment of human MM.
Pro-apoptotic effect of increasing miR15a/16-1 expression by different strategies in B-CLL cells (165.28)
Authors: Kasar Siddha , Underbayev Chingiz , Yuan Yao , Hanlon Matthew , Mehta Smruti , Neuberger Eric , Raveche Elizabeth
Published: January 2023

Chronic Lymphocytic Leukemia (CLL) is a chronic lymphoid malignancy characterized by accumulation of mature CD5+ B-cells in peripheral lymphoid organs, bone marrow and peripheral blood and is incurable with conventional therapy. We have previously shown that similar to human B-CLL cells, NZB mice (de novo mouse model of CLL) have 50% less level of miR15a/16-1 (pro-apoptotic). We hypothesized that exogenously increasing their expression would lead to increased apoptosis in tumor cells. Using a lentiviral delivery system, we were able to stably increase the level of these two microRNAs in LNC (NZB derived B-CLL cell line). The apoptosis observed in miR-GFP transduced LNC was twice as much as in LNC transduced with GFP lentivirus (control). In addition to the miR defect, B-CLL cells have a very high level of B-cell specific activator protein (BSAP) and this prevents their terminal differentiation. Recent reports have suggested that BSAP and miR15a/16-1 form an autoregulatory loop. We found that the level of the two oncoproteins BSAP and c-Myb were reduced in the miR-GFP transduced cells as compared to the control. Alternatively, we found that the miR defect can also be corrected by knocking down BSAP (using RNAi) leading to a cell cycle arrest. The second strategy is clinically relevant, since developing a BSAP inhibitor is more feasible than systemic microRNA delivery. Based on our findings we propose that targeting BSAP could be used as a novel therapeutic strategy for B-CLL.
An adenosinergic loop contributes to the generation of a microenvironment favoring growth and expansion of CLL cells (165.39)
Authors: Serra Sara , Horenstein Alberto , Tripodo Claudio , Vaisitti Tiziana , Audrito Valentina , Rossi Davide , Gottardi Daniela , Gaidano Gianluca , Malavasi Fabio , Deaglio Silvia
Published: January 2023

CD39 and CD73 are surface enzymes with extracellular catalytic sites. CD39 hydrolyses ATP/ADP to AMP, which is then converted to adenosine (ADO) by CD73. ADO may re-enter the cell or engage different types of purinergic receptors, eliciting potent immunosuppressive responses. The working hypothesis is that CD39+/CD73+ leukemic cells from chronic lymphocytic leukemia (CLL) patients produce ADO, generating environmental conditions favoring growth, through the inhibition of the host immune response. Results indicate that while CD39 is constantly expressed, CD73 is present in 30% of a cohort of 300 CLL patients, where it associates with markers of aggressive disease. Differential CD73 expression is confirmed by immunohistochemical analyses of BM and LN biopsies, with the highest intensity scored by cells within the lymph nodes, where the proliferative core of the leukemia resides. Furthermore, CD73 is expressed at high levels in proliferation centers and by CLL cells surrounded by CD3+ T lymphocytes. CD39+/CD73+ CLL cells produce ADO from ADP, in a time- and dose-dependent way. RT-PCR data indicate that T and CLL lymphocytes express higher levels of a functional A2A receptor, as compared to age-matched healthy controls. These results suggest the existence of an adenosinergic loop, active in selected CLL patients, which likely contributes to determining conditions that favour the development, maintenance and progression of the leukemia.
Low dose irradiation reduces lipid uptake in tumor resident dendritic cells which in combination with tumor specific CD8 T cells induces regression and prevents relapse (165.43)
Authors: Lerret Nadine , Rogozinska Magdalina , Jaramillo Andres , Marzo Amanda
Published: January 2023

Total body irradiation has been shown to enhance the efficacy of adoptively transferred tumor cells. The mechanisms responsible for this have largely been attributed to removal of cytokine sinks, suppressive cells and increase in danger signals. We now show that irradiation results in the down regulation of the macrophage scavenger receptor 1 on dendritic cells within the tumor and as a consequence reduces lipid uptake by tumor resident dendritic cells. The tumor antigen targeted in our model is Mammaglobin-A, expressed by approximately 80% of human breast tumors. We show that only the adoptive transfer of Mammaglobin-A2.4 specific CD8 T cells leads to long-term tumor regression and in combination with low-dose irradiation prevents tumors from reoccurring. These data demonstrate that irradiation reduces lipid uptake by dendritic cells and increases the efficacy of adoptively transferred Mammaglobin-A specific CD8 T cells providing a potential therapy for the treatment and/or prevention of breast cancer.
Oncogenic role for miR-17-92 paralog clusters in AIDS-related non-Hodgkin’s lymphomas (165.45)
Authors: Thapa Dharma , Martinez-Maza Otoniel
Published: January 2023

HIV infected individuals are at an increased risk for developing non-Hodgkin’s lymphomas (AIDS-NHL). Two key factors, immune deficiency, which allows the outgrowth of Epstein-Barr virus (EBV) infected cells, and chronic B cell activation, which promotes the development of oncogenic molecular lesions, are thought to contribute to the development of AIDS-NHL. Recently, microRNAs (miRNA) have been shown to play critical roles in cancers, including in B cell NHLs. miRNAs are small noncoding RNA molecules that can bind to mRNAs and inhibit protein expression. We hypothesized that miRNAs are deregulated in AIDS-NHLs, that these deregulated miRNAs serve as useful biomarkers, and also provide insights into the molecular mechanism of NHL pathogenesis, especially in the setting of immune deficiency. We did a microarray based miRNA profiling of 26 primary cases of AIDS-NHLs from four lymphoma subtypes, Burkitt’s lymphoma (BL, n=6), primary central nervous system lymphoma (PCNSL, n=6), diffuse large B cell lymphoma (DLBCL, n=9), and primary effusion lymphoma (PEL, n=5) and compared the miRNA profile to each other and to normal B cells subsets (naïve, germinal center, and memory) isolated from tonsils. We show that: (1) normal tonsillar B cells exhibit a differentiation stage specific miRNA profile; (2) AIDS-BL and DLBCL overexpressed miRNAs from the miR-17-92 paralog clusters; and (3) members of miR-17-92 paralog clusters target the cell cycle inhibitor p21/CDKN1A.
Growth and constitutive expression of CCL2 and CCL5 by murine mammary carcinomas are dependent upon Myd88 function (165.6)
Authors: Kurt Robert , Egunsola Adetutu , Zawislak Carolyn , Akuffo Afua , Chalmers Samantha , Ewer Jason , Vail Caroline , Lombardo Jeffrey , Perez Dana
Published: January 2023

Previously we reported that lipopolysaccharide (LPS) treatment of murine mammary carcinomas resulted in decreased growth of the tumors in vivo, but not in vitro. Here we explored whether the effects of LPS were dependent upon TLR4 and Myd88 using RNA interference. Two clones with reduced TLR4 mRNA and protein expression and two clones with reduced Myd88 mRNA and protein expression were generated, as well as a clone expressing shRNA specific for lacZ as a control. Significantly, tumors with reduced TLR4 or Myd88 expression no longer exhibited decreased growth rates in vivo following LPS treatment indicating that the decreased growth rate of the 4T1 tumor following LPS treatment was mediated through effects downstream of TLR4 and Myd88. Perhaps more significantly, tumor cells with reduced levels of TLR4 or Myd88 exhibited slower growth rates in vitro and in vivo relative to the parental 4T1 and lacZ controls, and reducing Myd88 expression was sufficient to decrease CCL2 and CCL5 expression. To determine whether inhibiting Myd88 function was enough to alter tumor growth and CCL2 and CCL5 expression we used a Myd88 homodimerization inhibitory peptide. The data revealed that inhibiting Myd88 function was sufficient enough to impair growth of the tumor cells, and decrease constitutive expression of CCL2 and CCL5. These data imply that Myd88 is necessary for growth of murine mammary carcinomas, and expression of at least two proinflammatory chemokines.
Regulatory B10 cells within the peritoneal cavity delay colitis onset by altering local T cell-specific IFNγ production (166.1)
Authors: Maseda Damian , Smith Susan , Poe Jonathan , Tedder Thomas
Published: January 2023

B cells mediate multiple functions that influence immune and inflammatory responses. A rare subset of splenic CD1dhiCD5+ B cells with capacity to produce IL-10 (B10 cells) has been found to regulate T cell responses, inflammation and autoimmunity. Relative to the spleen, higher frequencies of B cells produce IL-10 in the peritoneal cavity (PC) upon a combined in vitro LPS, PMA and Ionomycin stimulation for 5 hours. In this study, the phenotypic and functional characteristics of IL10-competent B10 cells within the PC (PC B10 cells) were characterized. PC B10 cells share several phenotypic markers with splenic B10 cells, and were found within the phenotypically defined B1a (46%), B1b (28%) and B2 (5%) subsets. Adoptive transfer of PC B10 cells significantly delayed disease onset and severity in both the IL10-/- mouse model of spontaneous colitis and the model of induced colitis through transfer of CD4+CD25-CD45RBhi into Rag-/- mice. Overall, when PC B10 cells were transferred i.p. into both disease model recipient mice, the number of activated and IFNγ-producing CD4+ cells significantly decreased in the PC and mesenteric lymph nodes. Thereby, PC B10 cells significantly modulate T cell pathogenicity during colitis.
Changes in T cell subsets in 12 patients enrolled in a tolerance induction protocol with combined kidney and hematopoietic cell transplantation (169.34)
Authors: Dejbakhsh-Jones Sussan , Takahashi Kazuhiro , Jensen Kent , Busque Stephan , Scandling John , Shizuru Judy , Lowsky Robert , Engleman Edgar , Strober Samuel
Published: January 2023

The goal of the study was to determine the changes in T cell subsets for up to 3 years in12 patients given HLA matched combined kidney and hematopoietic cell transplants using total lymphoid irradiation (TLI) and anti-thymocyte globulin (ATG) as posttransplant conditioning. Eight of the 12 patients were successfully withdrawn from their immunosuppressive drugs. After the completion of the conditioning regimen the absolute number of CD3+T cells in the blood fell 10 by 100 fold in all patients as compared to the pretransplant levels. The greatest reduction was among the naïve CD4+ and CD8+ T cells. Among the CD4+ T cells the percentage of the naïve cells fell from about 40% pretransplant to about 2% at the nadir with a gradual increase thereafter to pretransplant levels during the end of the second year. In contrast, the percentage of Treg cells among CD4+ T cells increased about 3 fold after conditioning, and the ratio of Treg:naive CD4+ cells increased about 20 fold at that time. The change in the ratio approached the pretransplant value at about 6 months as the percentage of Treg cells declined and naive cells rose. During this period, there was upregulation of the expression of the PD-1 activation marker on both Treg and CD4+ Tcon cells, and transient increase in the ratio of NK T cells to naive T cells. Although T cell subsets returned to the pretransplant levels in the patients completely withdrawn from immunosuppressive drugs, no rejections were observed.
Inhibition of STAT1 modulates GVHD after allogeneic BMT through induction of S100A8/A9 (169.38)
Authors: Nasholm Nicole , Bouchkouj Najat , Fry Terry , Capitini Christian
Published: January 2023

T cell depletion (TCD) of donor bone marrow (BM) reduces graft-versus-host-disease (GVHD) after allogeneic blood and marrow transplantation (alloBMT), but can result in poor T cell reconstitution and relapse. We have shown that transplantation of TCD BM deficient in the gamma interferon (IFNγ) receptor allows delayed donor lymphocyte infusions (DLI) without GVHD. We have replicated these results with TCD BM deficient in STAT1, which mediates IFNα and IFNγ signals, and demonstrated expanded plasmacytoid dendritic cells (pDCs). In this study, we further characterized the mechanism of GVHD resistance. After MHC-matched, minor histocompatibility antigen-mismatched alloBMT, TCD STAT1-deficient BM prevents DLI-induced GVHD. Treatment of wild type BM donors and recipients with the glucagon-like peptide-1 analog, Exenatide, which mediates off-target inhibition of STAT1, attenuates GVHD and increases splenic B220+ cell recovery. TCD IRF9-deficient BM, which lacks IFNα signaling, does not prevent GVHD, confirming that IFNγ signaling via STAT1 is the primary pathway of GVHD resistance. Selective deletion of STAT1 using CD11c cre/STAT1flox BM is sufficient to attenuate GVHD-associated weight loss, further implicating a DC subset. Although S100 proteins are not typically found in pDCs, pDCs from TCD STAT1-deficient BM recipients express increased S100A8. Finally, TCD S100A8/A9-deficient BM causes more severe weight loss after DLI, confirming the relevance of S100 proteins in GVHD.
Regulatory dendritic cell mediated allo-antigen specific tolerance for acute graft versus host disease in young and older mice (169.42)
Authors: Scroggins Sabrina , Legge Kevin , Schlueter Annette
Published: January 2023

Graft-versus-host disease (GVHD) occurring after allogeneic bone marrow transplant (BMT) is often fatal. When compared to younger recipients, older BMT recipients experience increased morbidity and mortality. Following BMT, in our model, older (≥14 month) and young (3-4 month) mice succumb to GVHD, but older mice experience faster onset of weight loss and earlier mortality. Prophylactic treatment with syngeneic regulatory DC (DCreg) has been shown to decrease mortality of GHVD and maintain the graft vs. tumor response. DCreg were generated as previously described from age-matched bone marrow. In vitro analysis of young and older DCreg revealed no difference in co-stimulatory or inhibitory marker expression following culture. Following BMT and DCreg treatment most young DCreg treated BMT mice exhibited a brief period of weight loss and survived at least 45 days post-BMT with no evidence of GVHD; older mice had increased morbidity and decreased long-term survival than young mice. Analysis of inhibitory molecules on DCreg one day following adoptive transfer showed significantly lower expression of PD-L1 and PIR B on older DCreg than younger. To assess the donor immune system’s capacity to respond to infection while remaining free of GVHD, DCreg treated BMT mice were challenged with influenza. DCreg treated BMT mice displayed similar morbidity to control mice and GVHD relapse was not observed. Future studies will continue to investigate the mechanisms of DCreg mediated tolerance.
Th1 memory cells induce lethal allogeneic disease in recipient bone marrow and spleen (169.43)
Authors: Chewning Joseph , Zhang Weiwei , Schoeb Trenton , Weaver Casey
Published: January 2023

Graft versus host disease (GVHD) represents a major cause of morbidity and mortality in patients undergoing hematopoietic stem cell transplantation (HSCT). We sought to determine if Th1 memory CD4 cells were capable of inducing disease in allogeneic recipients. Studies were performed using the murine MHC Class II-disparate HSCT model, C57BL/6 (B6) to B6.C-H-2bm12 (B6→bm12). Initial studies using naïve CD4 cells in the B6→bm12 model revealed bone marrow aplasia present in diseased mice, in addition to classic GVHD tissue findings. FACS CD4 phenotyping of the transferred cells isolated directly from diseased tissues revealed a marked increase in IFN-γ in the allogeneic mice compared to syngeneic recipients. Using a previously described transgenic Ifng reporter mouse containing the Thy1.1 gene knocked in-frame into the first exon of the Ifng gene, we were able to isolate IFN-γ-producing Th1 cells with magnetic bead separation. Adoptive transfer studies of allogeneic Thy1.1-positive cells into bm12 allogeneic mice resulted in fatality in a dose-dependent fashion. FACS phenotyping of Thy1.1-expressing cells in mice demonstrating illness revealed persistence of the Th1 phenotype and tissue histology showed severe disease of bone marrow and spleen in the absence of classic GVHD findings. Serial blood counts confirmed decreases in all hematologic parameters in allogeneic Th1 recipients. These results have important implications for future studies in graft manipulation for human HSCT.