bluuebunny/crossref_metadata_2025 huggingface dataset:
RNA splicing regulates the temporal order of TNF-induced gene expression (167.5)
Authors: Hao Shengli , Baltimore David
Published: January 2023

When cells are induced to express inflammatory genes by treatment with TNF the mRNAs for the induced genes appear in three distinct waves, defining gene Groups I, II and III or early, intermediate and late genes. To examine the basis for these different kinetic classes, we developed a PCR-based procedure to distinguish pre-mRNA synthesis from mRNA synthesis. It showed that the three Groups initiate transcription virtually simultaneously but that delays in splicing characterize Groups II and III. We examined the elongation times and polyadenylation timing, concluding that pre-mRNA synthesis is coordinate but splicing differences directly regulate the timing of mRNA production. Coupled to previous measurements of the half-life of mRNAs, the kinetic parameters can be combined in a general mathematical model of pre-mRNA conversion to mRNA that reproduces the experimental kinetics without recourse to differences in transcription.This model could be a generally relevant description of sequential transcriptional events in response to inducers.
19S ATPase subunits of the 26S proteasome play critical roles in transcription elongation. (167.7)
Authors: Greer Susanna , Maganti Nagini , Thakkar Meghna , Truax Agnieszka
Published: January 2023

Major histocompatibility class II molecules are cell surface glycoproteins that present extracellular peptides to CD4+ T cells and initiate adaptive immune responses. MHC II molecules are inducibly expressed in response to interferon gamma and are tightly regulated at the level of transcription by a master regulator, the class II transactivator, CIITA. We have recently shown that the 26S proteasome, itself a master regulator of proteins, regulates transcription initiation at interferon gamma inducible CIITApIV and MHC II genes in a degradation independent manner. The 26S proteasome consists of two major subunits, a 19S regulatory particle and a 20S proteolytic core. The 19S contains six ATPases which associate with MHC II and CIITA promoters and play important roles in transcription initiation by recruiting critical histone modifying enzymes. To determine if the 19S ATPases function in transcription elongation, 19S ATPase binding and activity in CIITApIV coding regions was analyzed. ChIP assays indicate significant binding of 19S ATPases in CIITA coding regions, and knockdown of 19S ATPases results in significant decreases in RNA pol II binding throughout CIITApIV coding regions. Further, knockdown of 19S ATPases has minimal impact on the expression of short CIITApIV transcripts but significant negative impact on long transcript expression, indicating roles for 19S ATPases in RNA pol II processivity at CIITApIV and likely other inducible mammalian genes.
Macrophage migration inhibitory factor enhances dengue virus replication through autophagy formation and ROS generation (168.10)
Authors: Chuang Yung Chun , Yeh Trai Ming
Published: January 2023

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine which has been identified as a risk factor in dengue virus (DENV) infection. Previous study showed MIF deficient mice exhibit lower virus titer than wild type mice during DENV infection. However, the mechanism is still unknown. Herein, we first examined whether MIF interfere DENV replication in vitro. By using flow cytometry assay, we found both MIF inhibitor ISO-1 or MIF knock down could reduce DENV replication in Huh 7 cells. Moreover, we found that reactive oxygen species (ROS) of Huh 7 cells induced by DENV infection were also decreased in the presence of ISO-1. ROS is an inducer of autophagy which has been showed to be involved in DENV replication. Thus, we hypothesize MIF facilitate DENV replication through ROS generation and autophagy formation. We found ISO-1, MIF knock down, or ROS scavenger N-acetyl-L-cysteine (NAC) could reduce autophagy during DENV infection by western blotting analysis. The LC3 punctae formation was also reduced in MIF knock down Huh 7 cells during DENV infection. Taken together, blocking of MIF by its inhibitor or antibody may not only prevent inflammation but also viral replication during DENV infection.
Toll-like receptor 2 is required for complete protection against lethal HSV-1 encephalitis in a murine lip scarification model (168.25)
Authors: Kollias Christina , Jennings Stephen
Published: January 2023

Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen capable of causing fatal encephalitis in rare instances. The immediate recognition of the virus by pattern recognition receptors at the infection site is crucial for establishing an antiviral state and limiting nervous system involvement. The importance of TLR3 and TLR9 in the type I interferon-dependent protection against HSV disease has been well documented, however, the role of TLR2 is less clear. This study aims to define the function of TLR2 in a natural infection model of mucocutaneous HSV-1 disease. TLR2 is expressed on the cell surface of various cell types, and ligation leads to an NF-B-dependent inflammatory response. Here we demonstrate that Tlr2-/- mice are significantly more susceptible to HSV-1 disease than WT controls. Whereas Tlr2-/- mice have indistinguishable levels of infectious virus at the inoculation site, the deficient animals display faster nervous system invasion and elevated viral loads in the trigeminal ganglia, brainstem, and brain, indicating that TLR2-expressing cells in the nervous system aid in controlling HSV-1 replication and spread. Mortality from HSV-1 CNS infection is the result of virus-mediated cytotoxicity in combination with the ensuing neuroinflammatory response. We are currently assessing the magnitude and kinetics of CNS inflammation as well as the activation and infiltration of immune cells to the CNS in order to delineate the mechanism of TLR2 protection.
LPS enhances HIV-1-induced maturation and programmed death ligand-1 expression on human plasmacytoid dendritic cells (168.27)
Authors: Patel Meher , Fitzgerald-Bocarsly Patricia , Dai Jihong
Published: January 2023

Elevated plasma levels of lipopolysaccharide (LPS) resulting from disruption of gut mucosal integrity during HIV infection has been implicated as a major factor contributing to immune activation that is a hallmark of HIV infection. The negative costimulatory molecule, Programmed Death-1 (PD-1), and its ligand, Programmed Death Ligand -1(PD-L1), negatively regulate the function of exhausted CD8 T cells in chronic HIV-1 infection leading to suppression of specific anti-viral T cell responses. In HIV-infected individuals, increased PD-L1 expression on APCs correlates directly with viral load, indicating a potential effect of the virus on PD-L1 expression. Since the mechanisms of PD-L1 up-regulation in these settings are not understood, we investigated how PD-L1 expression is regulated in plasmacytoid dendritic cells (pDC). We also investigated the influence of LPS on virally stimulated pDC maturation and PD-L1 expression. By kinetic studies, we observed that LPS enhanced HIV-1 induced PD-L1, CD40 and CD83 expression on pDC, and that PD-L1 expression was up-regulated on matured pDC. IFN-α expression preceded PD-L1 expression on pDC following HIV-1 stimulation, but IFN-a by itself only modestly increased PD-L1 expression, indicating that up-regulation of PD-L1 on pDC was specifically a primary response to HIV stimulation. Together, these results suggest that pDC adopt a tolerogenic phenotype following maturation during HIV-1 infection and LPS helps mediate this process.
Modeling childhood resistance to influenza mortality in mice (169.14)
Authors: Suber Freeman , Kobzik Lester
Published: January 2023

In the 1918 influenza pandemic, infected children between ages 5-14 had low mortality. To study the mechanisms for childhood resistance to disease mortality, we created a murine model of mortality difference from influenza (flu) in pre-pubertal (Pre-P) vs. post-pubertal (P) mice (onset is day 28). We compared mortality rates in C57BL/6 (Pre-P) and (P) mice infected with the same viral dose (1 HAU of A/PR/8/34). We also used gonad-intact and gonadectomized (Gx) mice that received hormone replacement to test the effect of sex steroids on morbidity and mortality during flu infection. Mortality was lower in Pre-P mice (22% for infections started on postnatal day 25 (P25), 16% at P26) when compared to P mice (71% at P28, n > 18, p<0.05). Data were similar for both sexes. In the sex steroid-ablated Gx mice, mortality was also lower (23 % vs. 64%, 28% vs. 50% for the castrated vs. sham-castrated, and ovariectomized (Ox) vs. sham-Ox mice, respectively). Gx female mice treated with estrogen (E) replacement (i.p. (E), 10ug/100ul) showed an increase in mortality (28% in Ova+vehicle (V) vs. 71%, 66% and 80% respectively, for the Ova+E, sham-Ova+V and sham-Ova+E mice, p=0.057). Histopathology showed severe lung injury with hyaline membranes in all E-intact, however, this pathology was absent in (V)-treated Gx mice. The model will be useful for further studies of how sex hormones, e.g. (E), and other host factors may contribute to mortality after influenza infection.
The IL-10-/- mouse as a model of chronic Lyme arthritis. (169.16)
Authors: Sonderegger F. Lynn , Ma Ying , Weis John , Weis Janis
Published: January 2023

IL-10 suppresses the development of arthritis in Borrelia burgdorferi infected mice. Infected IL-10-/- mice were previously found to have a robust and sustained interferon-inducible response in joint tissue, and elevated IFN-γ in sera. Treatment of IL-10-/- mice with anti-IFN-γ reduced arthritis severity and suppressed interferon-inducible transcripts to wild type levels, thereby linking dysregulation of IFN-γ to disease in the IL-10-/- mouse. Arthritis in IL-10 deficient mice was associated with elevated numbers of NK cell, NKT cell, α/β T cell, and macrophage infiltration of the infected joint. FACS lineage sorting revealed NK cells and CD4+ T cells as sources of IFN-γ in the joint tissue of IL-10-/- mice. These findings suggest the presence of a positive feedback loop in the joint tissue of infected IL-10-/- mice, where production of inflammatory chemokines, infiltration of IFN-γ producing cells, and additional production of inflammatory cytokines result in arthritis. This mechanism of arthritis is in contrast to that seen in C3H mice, where arthritis development is linked to transient production of Type I interferon, and develops independently of IFN-γ. Due to the sustained interferon response driven by NK cells and T cells, we propose the IL-10-/- mouse as a potential model to study the prolonged symptoms observed in some human Lyme disease patients.
Salmonella enterica Typhimurium evasion of caspase-1 dependent flagellin recognition promotes mucosal injury and systemic dissemination (169.4)
Authors: Lai Marvin , Quarles Ellen , Lopez-Yglesias Americo , Zhao Xiaodan , Smith Kelly
Published: January 2023

Salmonella enterica Typhimurium is a flagellated bacterium and one of the leading causes of gastroenteritis in humans. Bacterial flagellin is critical for motility and also a prime target of the innate immune system. Innate immune recognition of flagellin is mediated by two independent innate immune pathways, TLR5 and Naip5-Naip6/NlrC4/Caspase-1. The functional significance of each of the two independent flagellin recognition systems in innate immunity to Salmonella infection is modest, and we hypothesized that this was due to efficient modulation of flagellin expression in vivo to evade innate immune detection. Salmonella deficient in the anti-sigma factor flgM overexpress flagellin, are attenuated in vivo, and this attenuation is dependent on flagellin expression. In this study, we used flgM- Salmonella to determine if flagellin recognition by the innate immune system was responsible for the attenuation of flgM- S. typhimurium, and to dissect the contribution of each flagellin recognition pathway to bacterial clearance, inflammation and infection. Our results indicate that Salmonella evasion of caspase-1 dependent flagellin recognition is critical for establishing systemic infection and inducing intestinal inflammation and injury during intestinal infection. In contrast, TLR5 paradoxically promotes bacterial colonization in the cecum, but does not influence systemic infection, and plays a modest role in intestinal inflammation.
Inflammation-resolving mediator resolvin E1 inhibits osteoclast fusion. (169.6)
Authors: Zhu Min , Van Dyke Thomas , Gyurko Robert
Published: January 2023

Resolution of inflammation is an active, highly coordinated process leading to the return of tissues to homeostasis. The ω-3 polyunsaturated fatty acid derivative RvE1 is a proresolving receptor agonist that has been demonstrated to protect from inflammatory bone loss by inhibiting osteoclast formation and bone resorption. It is unclear whether the actions of RvE1 in inflammatory bone disease are indirect through modulation of inflammation or the direct action of RvE1 on bone cells. Investigation of the direct impact of RvE1 treatment on the specific stages of osteoclast maturation in vitro revealed that RvE1 targets late stages of osteoclast maturation. Time-lapse vital microscopy revealed that RvE1 inhibits migration and fusion of osteoclast precursors. Western blot analysis revealed that RvE1 specifically down-regulates the pivotal osteoclast fusion protein DC-STAMP (dendritic cell specific transmembrane protein) through the leukotriene receptor BLT-1. RvE1 does not change NFATc1 induction or nuclear translocation; however, NFATc1 binding to the DC-STAMP promoter was inhibited by RvE1 treatment. The data suggest that RvE1 inhibits the binding of the transcription factor NFATc1 to the promoter of osteoclast fusion protein DC-STAMP, leading to DC-STAMP down-regulation and attenuation of osteoclast fusion. Our observations establish a dual role for inflammation resolution in innate immunity as well as bone preservation through the direct regulation of bone cell function.
Memory Treg cells regulate the immune response to influenza virus infection (170.10)
Authors: Brincks Erik , Roberts Alan , Cookenham Tres , Sell Stewart , Blackman Marcia , Kohlmeier Jacob , Woodland David
Published: January 2023

Regulatory CD4+FoxP3+ T cells (Treg) are key regulators of inflammation and control the magnitude of cellular immune responses to viral infections. However, little is known about Treg regulation of recall responses to previously-encountered pathogens. Here we investigated the influence of Treg on recall responses to influenza virus infection. Utilizing influenza NP311-325/I-Ab Class II tetramers, we tracked the antigen (Ag)-specific Treg during primary and secondary influenza virus infections in C57BL/6 mice. Ag-specific Treg accumulated in the lung-draining lymph node and lung parenchyma during secondary virus infection with accelerated kinetics relative to primary influenza infection, suggesting the existence of a “memory” Treg pool. These Treg contributed to the control of in vivo responses to secondary influenza infection by limiting the magnitude of the Ag-specific memory CD8+ T cell recall response and by contributing to the control of pulmonary inflammation as well as airway chemokine/cytokine expression. In vitro studies verified that memory Treg cells can effectively control the proliferation of memory CD8+ T cells in an Ag-specific fashion, and that this regulatory function was MHC class II dependent. Together, these data demonstrate a previously unappreciated role for Treg in shaping the cellular immune response to secondary influenza virus challenges and need to be considered during vaccine development.
Spred-2 negatively regulates influenza A virus (H1N1)-induced pneumonia (170.11)
Authors: Ito Toshihiro , Yoshimura Akihiko , Matsukawa Akihiro
Published: January 2023

Influenza A (H1N1) viral infections have been identified as the etiologic agents for historic pandemics, and contribute to the annual mortality associated with acute viral pneumonia. Many studies have elucidated the functions of H1N1-viral proteins of this negative-strand RNA virus have been well elucidated. However, the virus-induced intracellular signaling events remain unknown. The Raf/MEK/ERK cascade is the prototype of mitogen-activated protein (MAP) kinase cascades and has an important role in cell growth, differentiation, and survival. The Sprouty-related Ena/VASP homology 1-domain-containing protein (Spred) has recently been identified as a negative regulator of Raf-dependent ERK activation. Here, we demonstrate that Spred-2-knockout (KO) mice led to higher mortality with greater virus load compared with wild-type (WT) mice during influenza A (H1N1) viral infection. In addition, Spred-2 KO mice showed more severe pneumonia with higher mRNA levels of Type-I IFNs (IFN-α and IFN-β) and pro-inflammatory cytokines (TNF-α and IL-6). Moreover, administration of MEK-inhibitor, U0126, improved mortality with lower viral load with lower mRNA levels of Type-I IFNs and pro-inflammatory cytokines. Together, the results of this study show that Spred-2 negatively regulates anti-viral immunity during H1N1 infection. We propose that Spred-2 might be a novel therapeutic target for controlling the immune response against influenza H1N1 virus infection.
Respiratory Syncytial Virus infection in mice reduces spatial learning capacity and long term potentiation. (170.16)
Authors: Espinoza Janyra , Cortes Claudia , Valenzuela Javier , Sandoval Rodrigo , Gómez Roberto , Céspedes Pablo , Pancetti Floria , Riedel Claudia , Kalergis Alexis
Published: January 2023

Respiratory syncytial virus (RSV) is the major cause of bronchiolitis and pneumonia in children, the elderly and immune-compromised individuals causing significant health burden worldwide. Clinical reports have shown that infants infected with RSV suffer CNS disorders like seizures; central apnea, lethargy, feeding or swallowing difficulties, abnormalities of tone or strabismus, abnormalities of the cerebrospinal fluid (CSF) and encephalopathy . Whether these CNS disorders result from either RSV replication or immune system reactivity at nervous tissue is still unknown. In this context, clarifying RSV entry, localization and spread on CNS will seed insights on RSV neuropathology. Preliminary evidences have shown the presence of viral RNA and anti-RSV antibodies in the cerebrospinal fluid of infected host. We show by Immunofluorescence analysis the presence of RSV proteins and by RT-PCR the nucleocapsid transcripts in mice infected with RSV. After one month that the RSV infection has cleared out, these mice showed reduced capacity for spatial learning and long-term potentiation in response to theta-burst stimulation to the Schaffer collateral pathway in the area of CA1 of the hippocampus Ammon’s horn stratum radiatum. These are the first evidences that support that RSV infection can alter brain function.
Galectin-3 promotes HIV-1 budding through association with Alix and Gag-p6 (170.25)
Authors: Chen Huan-Yuan , Wang Sheng-Fan , Tsao Ching-Han , Hsu Daniel , Liu Fu-Tong
Published: January 2023

Galectin-3 (Gal3), a β-galactoside-binding lectin, has been reported to regulate the functions of a number of immune cell types. We demonstrated that Gal3 is translocated to the immunological synapse in T cells upon T cell receptors engagement and associated with ALG-2-interacting protein X (Alix). Alix is known to coordinate with endosomal sorting complex required for transport (ESCRT) to promote HIV-1 virion release through binding to the HIV-1 Gag-P6 protein. We hypothesized that Gal3 plays a role in HIV-1 viral budding. Co-transfection of Gal3 and HIV-1 plasmids in HEK293T cells indicate that endogenous Gal3 facilitates HIV-1 budding. This effect was inhibited by knocking down Gal3 expression by shRNA. Immunoblotting of trypsin-treated virions and immuno-electron microscopy indicate that Gal3 is mainly located inside the HIV-1 virions. Immunofluorescent staining and coimmunoprecipitation (Co-IP) suggest that Gal3, Alix, and Gag-p6 are colocalized in HIV-1-infected cells. Notably, Gal3 expression was found to promote the association between Alix and Gag-p6 as demonstrated by Co-IP. Co-transfection of Gal3 and HIV-1 plasmids in Alix knocked-down cells indicate that promotion of HIV-1 budding by Gal3 is mediated through Alix. Finally, knocking down Gal3 expression in primary CD4+ T cells resulted in a significant decrease of HIV-1 titer. Our results indicate that endogenous Gal3 facilitates the HIV-1 virus budding through stabilizing the association between Alix and Gag-p6.
HLA-G expressing Tregs inhibit bystander immune activation in HIV-1 infection (170.27)
Authors: Li Chun , Pereyra Florencia , Rychert Jenna , Rosenberg Eric , Lichterfeld Mathias , Yu Xu
Published: January 2023

T cells expressing the HLA class Ib molecule, HLA-G, represent an emerging non-classical population of regulatory T cells (Tregs). However, the functional role of these cells during HIV-1 infection remains unclear. Here, we performed a comprehensive investigation of HLA-G- expressing Tregs in patients with different rates of HIV-1 disease progression. Using flow cytometry, we found the proportion of HLA-G-expressing CD4 and CD8 T cells were significantly reduced in individuals with progressive HIV-1 infection (p<0.001 for both CD4 and CD8 T cells). Moreover, HLA-G expressing Tregs were inversely correlated with phenotypic markers of immune activation, CD38 and HLA-DR (p=0.026 for CD4 and p<0.005 for CD8 T cells), and positively correlated with CD4 T cell counts. Functional co-culture assays indicated that HLA-G-expressing CD4 and CD8 T cells were able to potently inhibit TCR-independent bystander activation, but only weakly inhibited TCR-dependent cytokine-secretion and proliferative properties of HIV-1-specific T cells. Furthermore, in vitro infection assay suggested HLA-G expressing Treg were more susceptible to HIV-1 infection (p=0.0039). These data indicate an important role of HLA-G+ Tregs for balancing bystander immune activation and anti-viral immune activity in HIV-1 infection, and suggest that the loss of these cells during advanced HIV-1 infection may contribute to immune dysregulation and HIV-1 disease progression.
The in vivo antiviral state is dependent on IFNγ (170.5)
Authors: Premenko-Lanier Mary
Published: January 2023

Since the discovery of interferons 50 years ago their potential therapeutic use has been a source of active research. Thus far, the most successful interferon therapy has been use of IFNα2 in combination with Ribavirin to treat Hepatitis C (HCV). The success rate for this therapy is only about a 50%. A recent case study showed that adding IFNγ to the standard IFNα/Ribavirin therapy regimen led to clearance of HCV in a patient that had failed the standard therapy. The idea that IFNγ is important for successful therapy against HCV is supported by several in vitro and in vivo studies. Using the well-established mouse model of chronic viral infection, LCMV, we have found that the natural in vivo antiviral state is dependent on interferon gamma (IFNγ). In our whole-organism model early production of IFNγ by CD8+ cells within days of a primary LCMV infection creates an antiviral environment capable of preventing secondary viral infections, whereas IFN-I signaling is surprisingly dispensable. The clearance of the secondary infection is not antigen specific since an acquired immune response to the second infection is not induced. Further, this block is transient and the mice eventually become susceptible to the secondary infection. Finally, the IFNγ induced antiviral state is effective regardless of the route of secondary viral entry. Therefore, a naturally induced sustained antiviral state is dependent on IFNγ production.
Renal Mast Cell recruitment in MRL/lpr mice (173.28)
Authors: Hernandez Sanabria Mayra , Wenderfer Scott , Ke Baozhen , Braun Michael
Published: January 2023

Mast cells (MC) are key effectors cells of the immune system. MC infiltration has been reported in a variety of renal diseases including lupus nephritis. Recently it has been argued that MCs are key contributors to a common pathway of progressive renal injury, however, the chemotaxic mechanism of renal MC recruitment are largely unknown. We observed a progressive age-depended MC infiltrate in kidneys of MRL/lpr mice. MRL/lpr bone marrow-derived MC (BMMC) had a high chemotatic capacity in response to MRL/lpr serum. In order to characterize the factors involved in MCs recruitment in MRL/lpr mice we performed expression array analysis in stimulated and un-stimulated MRL/lpr MCs. Serum stimulation increased BMMC expression of the integrin alpha E and chemokine receptors CCR5 and CXCR4. Kidney immunofluorescence staining confirmed these finding in situ. BMMC chemotaxis in vitro was enhanced in response to CCR5 ligands (MIP-1α and RANTES) and CXCR4 ligand (SDF-1) and was blocked by using a non-peptide chemokine receptor antagonist TAK-779. In vivo, TAK-779 significantly blocked the accumulation of renal MC in MRL/lpr mice. Our study indicate that the chemokine receptors CCR5 and CXCR4 and the integrin alpha E are important mediators of renal MC recruitment.
The role of lipid phosphate phosphatase 1 in lymphocyte egress from lymphoid organs (173.39)
Authors: Ramos-Perez Willy , Escalante-Alcalde Diana , Lynch Kevin , Schwab Susan
Published: January 2023

The signaling lipid sphingosine 1-phosphate (S1P) regulates vascular permeability, lymphocyte trafficking, and inflammation. The concentration of S1P is high in blood and lymph compared to lymphoid organs, and this difference guides lymphocyte exit from lymphoid organs into circulation. However, little is known about how S1P distribution is controlled in vivo, and it is not clear how a ubiquitously made lipid functions as a signal that requires precise spatial and temporal control. Lipid phosphate phosphatase 3 (LPP3) is essential to maintain low S1P in the thymus, and we asked whether its homolog LPP1 cooperates with LPP3 to regulate lymphoid S1P. Measurements of Lpp1 messenger RNA indicate that, like Lpp3, Lpp1 is highly expressed by thymic endothelial and epithelial cells, and lymph node (LN) lymphatic endothelial, blood endothelial, and fibroblastic reticular cells. Using mice deficient in LPP1, we have found that LPP1 is not required to maintain low S1P in thymus or LN or to enable efficient lymphocyte egress. Mice doubly deficient in LPP1 and LPP3 preliminarily have a small accumulation of mature thymocytes and minor reduction in the number of T cells in lymph compared to mice deficient in LPP3 alone, suggestive of an egress defect. Further work will test the hypothesis that LPP1 and LPP3 work together to maintain low lymphoid organ S1P and efficient lymphocyte circulation.
IL1 receptor signaling is required at multiple stages during hapten sensitization and challenge to elicit contact hypersensitivity responses (173.4)
Authors: Kish Danielle , Gorbachev Anton , Fairchild Robert
Published: January 2023

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten skin sensitization and challenge. Elicitation of CHS is initiated when hapten-primed CD8 T cells producing IFN-γ and IL-17 stimulate endothelial cells (EC) in the challenge site to produce CXCL1, which directs required neutrophil recruitment into the challenge site. This study investigated the role of IL1 receptor signaling during hapten-specific CD8 T cell priming and in effector CD8 T cell trafficking to the challenge site to initiate the response. CHS responses were markedly lower following hapten challenge of sensitized IL1R-/- vs. wild-type (WT) mice and were associated with low CXLC1 production and neutrophil infiltration into the skin challenge site. Sensitized IL1R/- mice had marked decreases in hapten-primed CD8+ T cells producing IFN-γ or IL-17 vs. WT mice. Transferred primed WT CD8 T cells failed to induce CXCL1 to hapten skin challenge of naïve IL1R-/- mice, indicating an IL-1R requirement for CD8 T cell activation at the challenge site. Sensitized IL1R/- mice also had marked decreases in migration of hapten-presenting Langerhans cells into the skin-draining lymph nodes and transferred hapten-presenting dendritic cells from WT donors into the skin of IL-1R-/- mice failed to migrate to skin-draining lymph nodes and restore the absence of hapten-specific CD8 T cell priming. These results indicate IL-1R signaling is required during both hapten sensitization and elicitation of CHS.
‘Spin 90’ affects the Lymphocyte Recruitment and Homing to the Peritoneal Cavity (173.42)
Authors: Kim Woo-Seok , Kausar Sadia , Kang Jung-A , Lee Cho-Rong , Song Woo-Keun , Park Sung Gyoo
Published: January 2023

To determine the role of Spin90 in lymphocyte trafficking into the peritoneal cavity, we checked changes of the peritoneal cell population in Spin90 deficient mouse compared to the population in wild-type littermate control mouse. As a result, we found that B220+ cell population is increased in peritoneal cavity of Spin90 deficient mouse. In addition, our results showed that B220+CD23low, B220+IgMhighIgDlow B1 cells were high in Spin 90 deficient mouse as compared to wild-type littermate control. However, we found that the difference was not caused by lymphocyte developments because there were no significant differences observed in the lymphocyte population of bone marrow, thymus and peripheral lymphoid organs i.e. Spleen Lymph nodes. Our data suggest that Spin 90 could be responsible for B1 B lymphocyte migration into peritoneal cavity or into periphery from the cavity.
In vitro studies of interferon-alpha neurotoxicity (174.14)
Authors: Fritz-French Cari , Welshhans Kristy , Zhang Lynn , Bassell Gary , William Tyor
Published: January 2023

Elevated levels of interferon-alpha (IFNα) in the CNS is linked to cognitive dysfunction in patients with inflammatory CNS diseases such as HIV-associated dementia. Previous studies showed that IFNα treatment of neuronal cultures caused a dose dependent decrease in dendritic branching and length, mostly prevented after pre-treatment with IFNα neutralizing antibodies. To begin to evaluate the mechanism of decreased dendritic arborization, microtubule associate protein 2 (MAP2), cytoskeleton protein required for dendritic growth and stability in neurons, was looked at. IFNα was found to cause a decrease in total MAP2 protein levels in vitro as soon as 2 hrs after treatment that remained decreased through 48 hrs.. To begin to determine the signaling cascade leading up to effects on MAP2, the cell signaling pathway involving IFNα receptor (IFNAR) was evaluated to show STAT1 phosphorylation, an indicator for JAK-STAT cascade activation, peaks 2 hrs after IFNα stimulation. Both the MAPK/ERK and CREB pathways are being looked at due to their involvement in neuronal growth and STAT1 interaction. IFNα treatment appears to cause an increase ERK activity. The significance of these changese in transcription factor activity is yet to be determined. Our preliminary studies suggest that IFNα is in part acting through its receptor to reduce MAP2 expression in neurons. Determining the mechanism of IFNα neurotoxicity could lead to therapies for cognitive dysfunction during neuroinflammation.
TNF-alpha Converting Enzyme Degradates Interferon-Gamma. (174.15)
Authors: Kanzaki Hiroyuki , Chiba Mirei , Suzuki Maiko , Taubman Martin
Published: January 2023

Infection induced immune responses cause extensive bone destruction in periodontitis. We previously reported that TNF-alpha converting enzyme (TACE) play a role in soluble RANKL and soluble TNF-alpha cleavage from activated lymphocytes in periodontitis, and these soluble osteoclastogenic cytokines induce osteoclast activation from distant site. Interferon-gamma is known as anti-osteoclastogenic cytokine produced from activated T cells, but little is known about the post-transcriptive modification, especially in inflamed tissue. We hypothesized that TACE might modify interferon-gamma and attenuate anti-osteoclastogenic activity. Recombinant interferon-gamma is incubated with intact or heat-inactivated recombinant TACE, electrophoresed in SDS-PAGE gel, and silver stain was done. In addition, the interferon-gamma concentrations of above samples are measured with ELISA. Recombinant TACE is added into the supernatant of activated mouse splenocyte, and measured interferon-gamma concentration. Silver stain revealed that not heat-inactivated but intact recombinant TACE reduced the 16 kDa band of recombinant interferon-gamma. ELISA also showed the reduction of interferon-gamma concentration by the incubation with recombinant TACE. These data suggest that, TACE might degradate interferon-gamma and attenuate anti-osteoclastogenic activity of interferon-gamma produced from activated T cells in periodontitis.
Tyk2 signaling pathway and Mycobacterium tuberculosis (Mtb) infection (174.19)
Authors: Ray Aurelie , Fountain Jeffrey , Cooper Andrea
Published: January 2023

IL-12 is required for the optimal IFNg T-cell response, which is required for control of Mtb growth. IL-12R signaling is mediated by Tyrosine kinase 2 (Tyk2) which belongs to the janus kinase (JAK) family. Tyk2 phosphorylates STAT4 and STAT3, which subsequently induce transcription of target genes. We wanted to define the role of Tyk2 in IL-12R signaling during control of Mtb infection. In order to do this, we infected Tyk2 deficient mice via the aerosol route with Mtb and followed progression of the disease. We found that absence of Tyk2 resulted in a modest increase in bacterial burden associated with slightly reduced cytokine secretion (IL-17, IFNg) and T cell maturation. These data suggest Tyk2 plays a minor role in the expression of the protective response during Mtb infection. It is likely therefore that the other components of IL-12R signaling (JAK1,2 and 3) are the principal mediators of IL-12 activity in this model. These data may also reflect a role of Tyk2 in other signaling pathways such as those initiated by IL-10 and type I IFNs.
HMGB1 producing by viable cardiac fibroblasts led to myocardial contractile dysfunction via PKC-β/Erk1/2/CBP Signal Pathway (174.21)
Authors: Su Zhaoliang , Yin Jingping , Liu Yanfang , Yuan Hongyan , Zhu Haitao , Xu Huaxi
Published: January 2023

HMGB1 is a nuclear protein actively secreted by immune cells, cardiomyocyte, hepatocytes, pituicyte and passively released by necrotic cells. HMGB1 has been implicated in both lethality associated with endotoxemia/sepsis and cardiac contractile dysfunction. Therefore, the present work was to assess whether viable cardiac fibroblasts could release HMGB1 and whether HMGB1 could affect myocardial function. In vitro, cardiac fibroblasts were isolated and treated by LPS. HMGB1 mRNA showed no significant change during 0-12 h after stimulation with LPS, but increased significantly after 24h. Furthermore, the western blotting and immunofluorescence were also showed the same data. We found that HMGB1 active secretion might be PKC-β, Erk1/2, and CBP signal pathway. Meanwhile, we also found that collagen type I, III and osteopontin expression were increased after LPS stimulation. No evidence of inflammatory cells infiltration were noted in vivo. The type-B ultrasonic also showed that actively secreted HMGB1 by cardiac-fibroblast contributed to myocardial contractile dysfunction in sepsis. The findings of current study indicate that 1) LPS can induce HMGB1 secretion by viable cardiac fibroblasts may through PKC-β, Erk1/2, CBP signaling pathway, and 2) HMGB1 plays a role in LPS-induce myocardial contractile dysfunction. The results of the current study also have broader implications that might be more viable cells could secreted HMGB1.
IL-7-neutralizing antibody enhances in vivo IL-7 activity by interfering with normal IL-7 clearance (174.4)
Authors: Martin Chris , Surh Charles
Published: January 2023

Cytokine treatments are becoming increasingly popular clinical options. However, current methods of administration lack specificity and require massive doses to achieve biological effect. For IL-7 treatment, addition of a cytokine-neutralizing antibody paradoxically augments cytokine activity. This phenomenon was assumed to be due to the antibody endowing the cytokine with a longer in vivo half-life. Here we show that the durability of the antibody is indeed essential to, but not solely responsible for optimal activity of the IL-7/antibody complexes. IL-7 clearance is mediated by IL-7 receptor expressed on hematopoietic cells. Our results indicate that the neutralizing antibody perturbs receptor-mediated IL-7 degradation, thus increasing the availability of IL-7 for viable signaling events. This model is potentially applicable to many cytokines and suggests that pharmaceutical efforts intended to increase activity of a given ligand should also include measures that partially obscure the ligand from its receptor.
Chemokine-mediated Treg trafficking is required to suppress allergic airway inflammation (175.12)
Authors: Afshar Roshi , Strassner James , Seung Edward , Medoff Benjamin , Luster Andrew
Published: January 2023

Research has implicated a role for regulatory T cells (Tregs) in preventing asthma; however, where Tregs exert their regulatory function and the mechanisms that localize Tregs to their functional location have not been addressed. We hypothesized that chemokine receptors expressed on Tregs play an important role in directing these cells to the allergic lung and/or the reactive lymph node. We developed two murine models of allergic airway to address this hypothesis. In the sensitization model, naïve mice are injected with antigen-specific Tregs prior to immunization and aerosol challenge. In the effector model, antigen-specific Tregs are injected into mice after immunization but prior to aerosol antigen challenge. Transferred wildtype Tregs are capable of suppressing allergic airway inflammation in both models. We have found that CCR7 is required in the sensitization model and CCR4 is required in the effector model, suggesting Treg trafficking into lymph nodes is required to suppress allergic airway inflammation during the allergen sensitization phase while Treg trafficking into the lung is required to suppress allergic airway inflammation during the recall response to allergen. Using a segmental allergen challenge model in human allergic asthma subjects we further show an important role for CCR4+ Tregs in human allergic asthma. These results support our mouse data and demonstrate that CCR4 may play an important role in Treg recruitment into the allergic airways of humans.
Alternatively activated macrophage (AAM) phenotype induced in murine lungs by cockroach allergen (CRA) exposure (175.19)
Authors: Beal Dominic , Stepien David , Bouchard Jacqueline , Kim Jiyoun , Remick Daniel
Published: January 2023

Human studies of asthma have shown that a subset of AAM is required for disease progression. These cells differ from classically activated macrophages (mϕ) in that they produce/induce production of Th2 cytokines and are comparably inefficient in antigen presentation. In this study a murine model of CRA-induced asthma was used to study mϕ phenotypes. To induce disease, mice were immunized intra-tracheally with CRA and challenged twice 14 and 21 days later. Mice were sacrificed after the 3rd challenge and compared to naïve controls. Enzymatic digests of whole lungs were analyzed by flow cytometry, and cultured to assess cytokine production. 24 hours after the final challenge, mϕ co-stimulatory molecule expression (CD80, CD86) was reduced, as compared to both pre-challenge and age-matched naïve control mice (~40% vs 60% CD86+ cells). This effect was limited to mϕ, as CD28 and CTLA4 on T lymphocytes were unaffected. ELISA performed on supernatants of cells cultured with CRA showed enhanced levels of IL4, 5, 10, and 13 (~60, 500, 350, and 1000pg/ml respectively, vs undetectable in controls) and reduced MIP1a(~2 vs 12 ng/ml), MIP2 (~10 vs 24ng/ml), and KC (~5 vs 9ng/ml). Mϕ isolated from the spleen and peritoneal cavity showed no significant differences in surface molecule expression, indicating these effects of CRA are limited to the lungs. These data show that CRA exposure results in the generation of an AAM-like phenotype in the lung. All p values for quoted data <0.05.
Tlr4-/- mice have reduced proportions of CD4+Foxp3+ Tregs in the colonic lamina propria and increased susceptibility to allergic responses to food (175.20)
Authors: Tripathi Prabhanshu , Stefka Andrew , Feehley Taylor , Pei Zhiheng , Nagler Cathryn
Published: January 2023

Previous work has shown that allergic responses to food are enhanced in Tlr4-/- mice. We hypothesized that allergy susceptible mice lack populations of Tregs, induced by the commensal microbiota, which are present in wild type (WT) mice. We now report that CD4+Foxp3+Tregs are significantly reduced in the colonic lamina propria (LP), but not the spleen or MLN, of Tlr4-/- mice when compared to C57Bl/6 WT mice or Tlr4+/- littermate controls. CD4+Foxp3+Tregs are also reduced in the colonic LP of mice deficient in the MyD88 or TRIF TLR4 adaptor proteins. In addition, CD4+Foxp3+Tregs isolated from Tlr4-/- mice exhibit functional deficits; their ability to produce IL-10 in response to TCR stimulation in vitro is impaired. Cluster analysis of pyrosequence derived data from fecal samples from Tlr4-/- and Tlr4+/- littermates did not segregate by genotype, suggesting that TLR4 deficiency does not itself alter the composition of the microbiome. When maintained under SPF conditions, allergen specific IgE and IgG1 elicited in response to intragastric sensitization is enhanced in Tlr4-/- mice compared to WT or Tlr4+/- controls. WT and Tlr4-/- mice that have been rederived germ free lack bacteria induced Tregs in the colonic LP; under germ free conditions both WT and Tlr4-/- mice respond robustly to sensitization with allergen. Our data suggests that increased susceptibility to allergy in Tlr4-/- mice correlates with numeric and functional impairment of bacteria-induced Foxp3+ Tregs.
Blockage of high-mobility group box 1 inhibited airway inflammation and remodeling in a murine model of chronic asthma. (175.7)
Authors: Lai Yu-ting , Lee Chen-Chen , Chang Hao-Teng
Published: January 2023

Damage-associated molecular patterns (DAMPs) comprise intracellular molecules characterized by the ability to reach the extracellular environment, and they involvein inflammation and cell repair. The high-mobility group box 1 (HMGB1) is a prototypic DAMP. It has two roles in microenvironment, one is DNA-binding protein and the other is cytokine. HMGB1 release during cell necrosis or apoptosis, and it’s also release by activated immune cell. Increasing HMGB1 level has been found in acute and chronic lung inflammatory conditions characterized by tissue damage and remodeling. In our study, we focus on the role of HMGB1 in chronic asthma. In a animal model of chronic asthma, mice were intraperitoneal injected with ovalbumin on day 0, 10, 20 and exposured with 5% ovalbumin five times a week for 4 weeks. After treated HMGB1 neutralization antibody 30μg/mouse by intratracheal injection three times a week for four weeks in OVA-immunized mice, HMGB1 treated mice showed decreased OVA-induced lung neutrophilia, mucus formation and collegen deposition in lung tissues by histology study and decreased lung HMGB1, HMGB1 receptors-RAGE, TLR-2 and TLR-4, collagen, MMP-9, MMP-2, pro-inflammatory cytokines- IL-17A, IL-17F, and INF-γ mRNA expression by quantitative PCR. In the future, we will investigate the further molecular mechanisms which affected by HMGB1 in lungin a chronic asthma animal model.
Intestinal epithelial cells regulate airway allergic inflammation through induction of IgA Abs and and IL-17A (175.9)
Authors: Bonnegarde-Bernard Astrid , Jee Junbae , Davis Ian , Boyaka Prosper
Published: January 2023

IKKβ-NF-κB signaling controls a large number of biological processes via tissue-specific regulation of inflammatory and anti-inflammatory responses. Using a model of oral antigen sensitization and mice with cell-specific deletion of IKKβ, we addressed the contribution of intestinal epithelial cells (IECs) to regulatory crosstalk between distant mucosal sites of the gastrointestinal tract and the airways. Mice with localized alteration of IKKβ-NF-κB signaling in IECs (IKKβΔIEC) exhibited unaltered IgE, but enhanced Th1-associated IgG2a Abs and IgA responses after oral antigen sensitization in the presence of cholera toxin. Interestingly, these mice showed reduced lung inflammation and mucus production after nasal allergen challenge. This protection required oral sensitization and was associated with the presence of IgA in bronchoalveolar fluids and increased IL-17A in the lungs. When orally sensitized with the same regimen, mice with a generalized IKKβ deficiency in cells of the myeloid lineage (IKKβΔMye) also were protected against nasal allergen challenge. Distinct mechanisms contributed to protection in IKKβΔIEC and IKKβΔMye mice since airway hyper-responsiveness and lung inflammation were completely abolished in IKKβΔMye mice, which also showed no IgA in bronchoalveolar fluid and lower IL-17A in the lung than IKKβΔIEC mice. In summary, IKKβ in IECs is sufficient for shaping immune responses to ingested antigens, and subsequent responses to antigen exposure via the airways.
PDL1 is not needed for peritoneal macrophage suppression of T cell activation (176.1)
Authors: Riggs James , Werda Amy , Cua Jackelyn , Walker Amanda , Bucknum Amanda
Published: January 2023

The myeloid suppression found in tumors can be modeled by studying peritoneal cavity (PerC) macrophages (Mϕs). The high % of Mϕs in the PerC inhibit the proliferation of C57BL/6J (WT) T cells by iNOS catabolism of arginine. PerC T cells from IFNγR-/- mice proliferate indicating that IFNγ drives suppression. IFNγ increases expression of molecules that can promote (B7) or inhibit (B7H1/PDL1) T cell activation. PDL1 was of particular interest as it is upregulated on Mϕs & has been reported to restrict T cell activation in tumors. B7-/-, PDL1-/-, & IFNγR-/- PerC cells were compared to those from WT mice. B7-/- mice had fewer CD4 & CD8 PerC T cells than WT. IFNγR-/- mice had fewer CD4 cells & more CD8 T cells than WT. PDL1-/- mice had fewer CD44hi & CD25+ CD4 T cells & more CD8 T cells, particularly CD44hi & GITR+ cells. B7-/- PerC T cells, like those of IFNγR-/- mice, were less suppressed & responded with PGE2 or IL-10 neutralization. However, they differed in that IL4, via arginase, regulated T cell proliferation in IFNγR-/- mice. Surprisingly like WT mice, PDL1-/- PerC T cells were suppressed by IFNγ-triggered iNOS. Although PDL1 costimulates IL10 production the absence of PDL1 did not increase IFNγ production. These results reveal a challenge for strategies intent on recovering immunity by blocking PDL1 in Mϕ-rich tumor microenvironments.
CD40 induced TCR revision promotes tolerance to self-antigen in Type I Diabetes pathogenic CD4+CD40+ T cells. (176.11)
Authors: Vaitaitis Gisela , Wagner David
Published: January 2023

Biomarkers for effector cells have been difficult to find, but we described a pathogenic CD4 T cell subset expressing CD40. We demonstrated that peripheral CD4+CD40+ T cells are necessary and sufficient to transfer autoimmune disease. Here we demonstrate that CD40 engagement of peripheral CD4+CD40+ T cells in-vitro renders those cells unable to transfer disease. We have shown that CD40 signals primary peripheral CD4+CD40+ T cells to access the recombination machinery that causes revision of TCR Vα. We demonstrate here that CD40 signals also causes revision of TCR Vβ. While this constitutes a mechanism that could result in the generation of autoaggressive T cells in the periphery, we demonstrate that this mechanism can be exploited for tolerizing autoaggressive T cells. We show that CD40 interacts directly with recombination machinery proteins RAG1, RAG2 and Ku-70 in CD4+CD40+ T cells and that CD40 becomes translocated to the nucleus upon CD40 stimulation. We also show that while CD40 can induce RAG1 and RAG2 expression, Fas signals act as a silencing mechanism. It will be important to further understand the involvement of CD40 in TCR revision in order to effectively target and tolerize autoaggressive T cells in autoimmune disease.