bluuebunny/crossref_metadata_2025 huggingface dataset:
CD40 as a T cell biomarker in multiple sclerosis (171.38)
Authors: Wagner David , Waid Dan , Corboy John
Published: January 2023

Multiple sclerosis is a neurodegenerative disease with strong evidence for autoimmune components. Auto-aggressive T cells are implicated in the pathogenesis and maintenance of the disease in both mouse models and human disease. We described a T cell subset, CD4 cells expressing CD40, which prove highly pathogenic in autoimmunity. Using the McDonald criteria, diagnosed relapsing/remitting (RR) MS subjects have a significantly increased percentage of CD4CD40 cells in PBL compared to controls. Comparing to type 1 diabetes (T1D), MS subjects (78 total) had CD4CD40 cell levels equal to or greater than T1D (238 total) subjects. CD4CD40 cells from MS subjects recognize MS associated antigens and produce Th1 cytokines and IL-17, suggesting that CD4CD40 cells bridge Th1 and Th17. CD4CD40 cells produce high levels of IL-6 that shunts regulatory T cells into a Th17, pro-inflammatory, pathway. During RRMS CD4CD40 cell expansions occur regardless of age or gender or disease duration. In many MS subjects a clonal expansion of TRAV 8.1.3+ cells within the CD4CD40 population was evident. During pregnancy CD4CD40 cell levels dropped dramatically corresponding with the remission phase of disease, and returned to pathogenic levels post partum. In the mouse EAE model of MS CD4CD40 cells are expanded in percentage and adoptively transfer EAE to non-sick hosts; thus demonstrating pathogenicity. This suggests that controlling CD40 relative to T cells will prove therapeutic in MS.
A case for microbial involvement in the activation of lupus-antigen reactive T cells (171.39)
Authors: Szymula Agnieszka , Szczerba Barbara , Bagavant Harini , Fu Shu Man , Deshmukh Umesh
Published: January 2023

Although events responsible for the activation of lupus-antigen reactive T cells are not known, the strong environmental influence on SLE is suggestive of microbial involvement. Microbes can influence T cell activation through molecular mimicry. To test this hypothesis, we selected Ro60 as the candidate antigen and HLA-DR3 transgenic mice as the experimental mouse model system. Autoantibodies against Ro60 are present in lupus patients and HLA-DR3 has the strongest genetic linkage to lupus. Using a panel of T cell hybridomas, 3 T cell epitopes on Ro60 were characterized. Amino acids critical for T cell activation were identified and pattern search analysis was carried out. This strategy identified hundreds of mimicry peptides. Several mimicry peptides originated from microbes residing in the human oral cavity, gut and skin, and were able to activate the Ro60 reactive T cell hybridomas. In addition, immunization of HLA-DR3 transgenic mice with some of the mimicry peptides induced autoantibody responses. Our data suggests that T cells reactive against Ro60 can be activated by exposure to multiple peptides originating from different microbes that are present in the human microbiome. Thus, we would like to suggest that repeated exposure to different microbes shapes the self-reactive T cell repertoire in SLE and a dysregulated immune response against normal microbiota can initiate autoimmune responses in lupus.
Alteration of the germinal center checkpoint by the lupus associated NZM2410/NZW SLAM family genes in the Sle1b sub-locus (171.40)
Authors: Wong Eric , Khan Tahsin , Rahman Ziaur
Published: January 2023

C57BL/6 (B6) mice carrying the Sle1b sub-locus that harbors the lupus-associated NZM2410/NZW SLAM family genes produce anti-nuclear antibodies (ANA). However, the role and mechanisms of alteration in the germinal center (GC) checkpoint in the development of autoantibodies in these mice (named B6.Sle1b mice) is not clear. Here we show significantly higher spontaneously formed GC (Spt-GC) response in aged B6.Sle1b mice compared to B6 controls. The increased Spt-GC response in B6.Sle1b mice did not result from a generalized defect in the response of B cells expressing Sle1b as no difference was observed between two groups of mice in the anti-sheep red blood cell (sRBC) GC responses. We found a significant increase in CD4+CXCR5hiPD-1hi follicular helper T cells (TFH) within Spt-GC and anti-sRBC GCs in B6.Sle1b mice. The increase in Spt-GC and TFH cell responses in B6.Sle1b mice was not due to reduced CD4+CD25+Foxp3+ T effector (iTreg) and TFH regulatory (TFR) cells in GCs. Sle1b GCs compared to controls had significantly higher proliferating B cells. This elevated Spt-GC response in B6.Sle1b mice was associated with increased DNA and nucleosome-specific plasma cells (PCs), memory B cells and ANAs. These data indicate that the mechanisms that control the formation of Spt-GCs and generation of autoreactive B and TFH cells in GCs are altered by the NZM2410/NZW SLAM family genes located in Sle1b.
Altered CD4+ T cell differentiation with preferential expansion of Th1 cells in TLR7-driven autoimmunity (171.45)
Authors: Horton Christopher , Pan Zijian , Lawrence Christina , Bolland Silvia , Farris Darise
Published: January 2023

TLR7 is a single-stranded RNA-recognizing innate immune receptor with roles in anti-viral immunity and systemic lupus erythematosus (SLE) in humans and SLE-like autoimmunity in mice. TLR7 gene duplication drives inflammation and autoimmunity in the BXSB mouse model and other Y autoimmune accelerator (Yaa)-derived models of murine lupus. Additionally, TLR7 SNPs associate with SLE in Eastern Asian populations. In this study, we investigated effects of TLR7 overexpression on CD4+ T cell subsets. In unmanipulated, 5-6 month old TLR7 BAC transgenic mice, we observed amplified activated:naïve T cell ratios and preferential expansion of the Th1 subset compared to wild type controls. We also evaluated the phenotype of TCR transgenic T cells specific for the RNA-binding autoantigen, human La, following adoptive transfer into TLR7 BAC transgenic mice. These human La-specific T cells demonstrated enhanced differentiation to Th1 and PSGL1lowCXCR4+ extrafollicular helper phenotypes after recovery from TLR7-overexpressing human La transgenic mice compared to wild type human La transgenic controls seven days after transfer. Moreover, TLR7 overexpression in recipient mice resulted in significantly reduced expansion of the CD4+ Foxp3+ phenotype among transferred, human La-specific cells. These data indicate that TLR7 overexpression alters CD4+ T cell populations in a manner that potentially contributes to autoinflammatory disease in these animals.
Role of RIG-1 signaling in the Pathogenesis of Alzheimer’s Disease (172.1)
Authors: de Rivero Vaccari Juan Pablo , Brand III Frank , Sedaghat Christina , Mash Deborah , Dietrich W. Dalton , Keane Robert
Published: January 2023

Recent evidence has accumulated that Alzheimer’s disease (AD) is associated with a central nervous system (CNS) inflammatory response. Neuroinflammation is a complex response against harmful effects of diverse stimuli and plays a critical role in the pathogenesis of AD. An important arm of the inflammatory response is the innate immune response. The innate immune response is regulated by pattern recognition receptors (PRRs) and their signaling pathways. Here we report that the expression of the innate immune response proteins, retinoic acid-inducible gene-I (RIG-1), virus-induced signaling adaptor (VISA), and the NACHT leucine-rich domain and pyrin-containing protein, NLRP12 are significantly elevated in neurons of the temporal cortex, the cerebrospinal fluid and plasma in presymptomatic AD patients, post-mortem diagnosis. Increased expression of RIG-1 and VISA were not found in pathologically confirmed cases of amyotrophic lateral sclerosis, Diffuse Lewy Body disease or Huntington’s disease, suggesting that activation of RIG-1 signaling may be a specific marker for early AD pathogenesis. Moreover, in vitro results show that stimulation of RIG-1 signaling induces expression of amyloid precursor protein and β-amyloid, supporting the idea that activation of the RIG-1 signaling system occurs in presymptomatic AD.
Modulation of the unfolded protein response via XBP1 splicing: a novel mechanism that regulates Natural Killer cell effector function. (172.10)
Authors: Mujaj Sally , Gandhi Maher , Vari Frank , Nourse Jamie
Published: January 2023

The transcriptional factor X-box-binding protein 1 (XBP1), a critical gene in the unfolded protein response (UPR), has recently been demonstrated to be essential for the development, survival and function of B cells, T cells and Dendritic Cells. However, the involvement of the UPR in Natural Killer (NK) cell function has yet to be investigated. To address this we examined XBP1 during NK cell killing. Following activation of the UPR, XBP1 mRNA is converted to its active form (XBP1s) through splicing mediated by inositol-requiring enzyme-1 (IRE1). Splicing of XBP1 mRNA was observed within 1 hour following initiation of NK cell direct-cytotoxicity or antibody-dependent cell-mediated cytotoxicity (ADCC) towards leukaemia or lymphoma target cells. This indicates that the IRE1/XBP1 arm of the UPR is rapidly induced during killing of target cells. To determine the impact of induction of the IRE1/XBP1 pathway on NK cell cytotoxicity, IRE1 mediated splicing of XBP1 was pharmacologically inhibited. This significantly impaired both NK cell mediated direct-cytotoxicity and ADCC. Together these results indicate that induction of the UPR and splicing of XBP1s is essential for optimal NK cell cytotoxicity and demonstrates a novel mechanism to modulate NK cell effector function.
Identification of hemopexin as an anti-inflammatory factor that inhibits hemoglobin synergy with HMGB1 in sterile and infectious inflammation (172.11)
Authors: Lin Tian , Sammy Fatima , Yang Huan , Thundivalappil Sujatha , Tracey Kevin J. , Warren H. Shaw
Published: January 2023

In many clinical settings such as trauma, infections and other hemorrhagic diseases, multiple endogenous substances are released into the extracellular space, including hemoglobin (Hb) from degrading RBC and HMGB1, an intracellular DNA-binding protein that has been shown to be pro-inflammatory, from injured or immune cells. We studied the effects of Hb and HMGB1 on the activation of macrophages. In the supernatant of cultured bone marrow-derived macrophages from C57BL/6 or C3H/HeN mice, significantly increased levels of pro-inflammatory cytokines (TNF and IL-6) were detected when Hb was added with HMGB1 compared to the culture with HMGB1 alone ( P < 0.01), while Hb itself did not induce detectable TNF and IL-6. This synergistic effect was also present in the culture of TLR2 knockout and TLR4 deficient (C3H/HeJ) macrophages. Addition of hemopexin (Hx), an endogenous heme-binding plasma protein, significantly decreased TNF and IL-6 induced by Hb and HMGB1 (P < 0.01). Co-incubation of microbial ligands LPS or Pam3Cys with Hb and HMGB1 in the culture resulted in further dramatic increases of TNF and IL-6 that were also significantly decreased by Hx (P < 0.01). These findings suggest that Hb may play an important role in amplifying sterile as well as infectious inflammation, and that endogenous Hx may play a role in controlling it. Administration of Hx could be a beneficial strategy in clinical settings where extracellular Hb and HMGB1 are both present.
Mice deficient in Complement Receptor 1 produce normal to enhanced quantities of antigen specific immunoglobulin in response to naïve immune challenge (172.14)
Authors: Donius Luke , Handy Jennifer , Weis Janis , Weis John
Published: January 2023

Complement receptors (Cr)1 and 2 have been established to have important roles in control of complement activation in the immune microenvironment and B cell responses. Previous work investigating the effects of these receptors in vivo has relied on a double knockout mouse line in which each of these receptors was deleted. However, two drawbacks of this mouse must be acknowledged: 1) that both receptors are deleted and roles of each must be inferred, and 2) that the essential B cell signaling protein CD19 is expressed at a significantly higher level than WT cells. We have created a Cr1 deficient line of mice (expressing Cr2) by homologous gene targeting that expresses normal levels of Cr2 gene products on B cells. This animal has normal ratios of bone marrow, B2 cell, and B1 B cell populations. Cr2 negative marrow B cells express the same amount of surface CD19 as WT but mature Cr2-expressing B cells express less. These Cr2-only mice generate normal to slightly enhanced antibody titers to T-dependent and T-independent (both 1 and 2 antigens.) In conclusion Cr1 is not critical for development and maintenance of B cell populations nor for the generation of antibodies to naïve antigens. This mouse, along with the Cr1/2KO, will be a critical tool in delineating Cr1 specific immune response effects.
Lamprey variable lymphocyte receptors mediate complement-dependent cytotoxicity (172.20)
Authors: Yu Xiaoli , Wu Fenfang , Chen Liyong , Liu Xin , Wang Huaying , Su Peng , Han Yinglun , Li Mo , Feng Bo , Qiao Xu , Zhao Jing , Ma Ning , Liang Meng , Liu Huijie , Zheng Zhen , Li Qingwei
Published: January 2023

An alternative adaptive immune system is present in the most basal vertebrates, lampreys and hagfish, the only surviving jawless vertebrates. These eel-like fish use leucine-rich-repeat (LRR)-based receptors, called variable lymphocyte receptors (VLRs), for antigen recognition instead of the immunoglobulin (Ig)-based receptors used in jawed vertebrates. We showed that in Japanese lamprey (Lampetra japonica), antigen-specific VLRB antibodies interact with C1q-like and C3 proteins to mediate complement-dependent cytotoxicity for bacteria and tumor cells. The immune based lysis involved deposition of VLRB and C1q-like protein complex on the surface of target cells, activation of C3, and ultimate disruption of cell wall integrity. The demonstration of functional interaction between VLRB and complement components in lamprey provides evidence for the emergence of cooperative innate and adaptive immune responses at a pivotal point in vertebrate evolution, before or in parallel with the evolution of Ig-based antibodies and the classical complement activation pathway.
Complement mediates membrane rearrangements and lipogenesis (172.21)
Authors: Fattakhova Gulnar , Cascalho Marilia , Platt Jeffrey
Published: January 2023

The complement is a major effector part in innate immunity. Its main biological function is to recognize foreign particles and macromolecules, and to promote their elimination either by opsonization or lysis. In order to avoid potential risk of host cells lysis, an efficient system of regulatory proteins is involved. Additional protective mechanisms include removal of membrane attacking complex by both vesicle shedding and internalization followed by exocytosis. The aim of research is to investigate alterations in membrane biogenesis and lipid biosynthesis in cells targeted by complement. Treatment of mouse NIH 3T3 fibroblasts with complement induces early and late phase responses. The early phase response is characterized by increase in intracellular Ca2+, alterations in phospholipid packing order and plasma membrane asymmetry, as well as translocation of lysosomal vesicles to the cell surface followed by activation of acid sphingomyelinase and ceramide production. We demonstrated that prestimulation of cells with sublityc complement doses followed by treatment with lytic concentrations substantially reduces levels of both plasma membrane-associated acid sphingomyelinase and ceramide, and protects cells from lysis. During late phase response we observed alterations in mRNA levels of several SREBP-targeted genes and lipid biosynthesis. Taken together, our data link regulated lipid dynamics and metabolism to acquired cell resistance to complement attack (accommodation).
Chaperone like activity of HMGB1 reduces the formation of polyglutamine aggregates (172.24)
Authors: Min Hyun Jin , Shin Jeon-Soo
Published: January 2023

High mobility group box protein 1 (HMGB1), which functions as a DNA chaperone, has recently been shown to play intracytoplasmic roles as an autophagy regulator and a nucleic acid sensing molecule. In order to identify a new role for HMGB1 in cytoplasm, we studied the chaperone-like activity of HMGB1, since HMGB1 is a ubiquitous protein. Using light scattering after heat and chemical stress, we found that HMGB1 inhibited the chemically-induced aggregation of insulin and lysozyme as well as the heat-induced aggregation of citrate synthase, suggesting that HMGB1 has a chaperone-like activity in vitro. Also, HMGB1 restored the heat-induced suppression of luciferase activity as a reporter protein in O23 hamster fibroblast cells after co-transfection of firefly luciferase and HMGB1. Next, we demonstrated that HMGB1 inhibits the aggregation and therefore toxicity of elongated polyglutamine (polyQ), one of the main causes of Huntington’s disease. HMGB1 proved to be co-localized with polyQ in the co-transfection study, while over-expression of HMGB1 or exogenous treatment of recombinant HMGB1 protein remarkably reduced polyQ-induced aggregates in SHSY5Y neuroblastoma cells, upon filter trap and immunofluorescence assay. Taken together, we show that HMGB1 demonstrates chaperone-like activity and inhibits the cytotoxicity of polyQ-mediated disease.
Macrophage chitin binding proteins in phagocytosis and M1 activation in response to chitin microparticles (172.27)
Authors: Shibata Yoshimi , Kogiso Mari , Huang Chun-Jung , Nouri-Shirazi Mahyar , Mizoguchi Emiko , Dorey C Kathleen
Published: January 2023

Unlike soluble or non-phagocytosable 40 - 100 µm chitin, chitin microparticles (1 - 10 μm, CMP) induce MAPK activation within 20 min in a manner dependent on TLR2, phagocytosis and a cellular cholesterol-mediated mechanism, followed by a typical expression of M1 phenotypes. Based on the previous studies, we hypothesized that trans-membrane signal through surface TLR2 ligation is insufficient and additional chitin binding proteins (CBP) involved in the early CMP phagocytosis are obligatory for M1 activation; CBP are distinct between M1/M2 macrophages (MΦ). Peritoneal M1, M2 and control MΦ were isolated from mice 1 - 2 days after ip administration of CMP, 40 - 100 µm chitin, and saline, respectively. These MΦ were cultured with CMP; CMP with CBP were isolated shortly after phagocytosis, stained by specific antibodies and analyzed by FACS. Several CBP on CMP, including TLR2, CD206, CD62L, CHI3L1, and LAMP-1, exhibited distinct patterns between M1/M2 activations. For example, TLR2, fully engaged at the early phagocytosis, was increased in M1 and reduced in M2 relative to unstimulated MΦ. Our results suggest that, although exact mechanisms of CBP-mediated M1 activation still remain to be elucidated, the profiles of selected CBP that bind to CMP during phagocytosis are associated with the polarization of MΦ. These profiles offer a potential tool as diagnostic/prognostic biomarkers to monitor chronic inflammatory diseases and the efficacy of immunotherapy.
Direct regulation of Interleukin-6 expression by Notch signaling in macrophages (172.36)
Authors: Wongchana Wipawee , Osborne Barbara , Palaga Tanapat
Published: January 2023

Notch signaling is required for immune cell development. The intracellular domain of Notch (NIC) induces expression of target genes by binding to CSL, a DNA-binding protein. In macrophages, Notch signaling regulates inflammatory cytokines TNFα and IL-6. IL-6 is secreted by various cell types, including activated macrophages and functions in immune regulation and inflammation. The consensus CSL binding site in the promoter region of IL-6 was shown to negatively regulate IL-6 mRNA, but the role of Notch signaling in IL-6 expression in macrophages remained unclear. We investigated the role of Notch signaling in the regulation of the IL-6 transcript in macrophages. Our results showed that bone marrow derived macrophages (BMMΦ) and RAW264.7 cell lines activated with lipopolysaccharide (LPS) and interferon gamma (IFNγ) induced IL-6 mRNA expression and upregulation of Notch1. Inhibition of Notch signaling in BMMΦ using a gamma secretase inhibitor partially decreases the level of IL-6 mRNA and protein. In contrast, RAW264.7, transiently transfected with constitutively active Notch1, displayed a dramatic increase in IL-6 mRNA. Inhibition of NF-κB completely abrogated IL-6 mRNA expression induced by the over expression of NIC. Chromatin immunoprecipitation assay demonstrated that Notch1 binds to the promoter of IL-6 in activated RAW264.7 but not in unstimulated cells. These results strongly suggest that Notch1 positively regulates IL-6 expression via NF-κB in activated macrophages.
TGF-b/TGF-bRII-SOCS3 interactions signifies the tolerogenic pathway(s) for down-regulating CD11c+dendritic cells-mediated osteoclastogenesis & bone loss (172.38)
Authors: Teng Andy Yen-Tung , Liu Yen Chun
Published: January 2023

Inflammation-induced bone-loss is linked to elevated osteoclasts(OC). Dendritic cells are antigen-presenting-cells involved in T-cell-immunity and can act as OC-precursors. We reported the development of functional-OC from CD11c+DC in-response-to RANKL plus microbes(Aa)-stimuli [DDOC]. Aims: to: i) identify molecules for regulating DDOC; ii) study the signals of modulating tolerogenic pathway that down-regulates DDOC-mediated osteoclastogenesis. Methods: DDOC are generated using co-cultures of CD11c+DC with 100ng/ml-RANKL (or-CD4+T/Foxp3+T-reg) & 10g/ml-Aa-sonicate. The results showed that: i) micro-arrays & functional analyses have identified key regulator(s) involved in DDOC-development, with a positive correlation to TGF-bRII. Neutralization of TGF-b abolishes DDOC development, based on TRAP & resorptive-pits (p<0.02); ii) transfection of dominant-negative SOCS3-cDNA into CD11c+DDOC via lenti-viral-vector and adoptive-transfer yield a significant inhibition of osteoclatogenesis & bone-resorption (p<0.05); iii) addition of Foxp3+CD4+T-reg into CD11c+DC plus Aa-Ag co-cultures results in reduced SOCS3-&-TRAP expressions & lowered-osteoclastogenesis in bone-resorption assays & NOD/SCID-mice (p<0.05). Conclusion: i) TGF-b/TGF-bR-SOCS3 interaction is involved in modulating the tolerogenic pathway(s) of down-regulating DDOC activity, ii) Foxp3+T-reg may be an integral player underlying inflammation-induced osteoclastogenesis and bone-loss through DC-T-cell interactions.
Endothelial Cells Bind Immune Complexes in the Kidney (172.39)
Authors: Wenderfer Scott
Published: January 2023

Inflammatory kidney diseases often involve the deposition of antibodies (Ab) or antibody-antigen immune complexes (IC). The glomerulus is a highly evolved vascular system which filters blood to produce urine. Ab-mediated glomerular injury can lead to bleeding into the urine, protein wasting, hypertension, decreased urine output, edema, and kidney failure. However, the mechanisms by which IC deposit and the means by which they are cleared remain unclear. In order to model endothelium-Ab interactions, kidney endothelial cells (KEnCs) were isolated from C57BL/6 mice and cultured in vitro. Immuno-phenotyping was performed to assess expression of CD31, VEGF receptors, von Willebrand factor (vWF), and alpha smooth muscle actin. CD31+ vWF+ VEGF-R2+ KEnCs displayed a phenotype in culture similar to other microvascular cells. Monomeric and heat aggregated Ig (HA-IgG) and peroxidase-antiperoxidase IC (PAP-IC) were used for binding and stimulation assays. Effects of stimulation of KEnCs were identified using PCR arrays and multiplex cytokine analysis. KEnCs stained brightly with HA-IgG and PAP-IC, but only dimly with monomeric IgG. KEnCs responded to IC with robust chemokine and cytokine responses in a dose dependent manner. Competitive binding studies mapped the interaction to the Fc-portion of IgG, but suggest that the KEnCs bind independently of classical Fc-receptors. Results support an important role for KEnCs in the pathogenesis of IC kidney diseases.
MiR-106b family regulates autophagy via targeting ATG16L1 (172.41)
Authors: Lu Changming , Xu Zhi-xiang
Published: January 2023

Autophagy is a lysosomal degradation pathway that serves an adaptive role to protect organisms against diverse pathologies. Dysfunction of autophagy is implicated in the development of numerous human diseases. However, regulation of autophagy at the molecular level remains largely unknown. In this study, we demonstrated that microRNA (miRNA) 106b family members were a critical regulator of autophagosome formation as well as autophagy-mediated functions. We found that during the differentiation of human monocytes into dendritic cells, expression of autophagy-related genes (ATGs) and autophagosome were substantially induced, whereas a number of miRNAs were down-regulated. Silencing of dicer resulted in the increased expression of ATGs and autophagosome formation. Bioinformatic search uncovered that miR-106 family members were highly potential regulators for ATG proteins. Indeed, our experimental investigations demonstrated that miR-106b and miRNA-93 mimics remarkably decreased autophagosome induction under physiological and stress conditions. In contrast, administration of antagonists of miR-106b and miRNA-93 sensitized autophagy formation. Immunoblot and luciferase report assays demonstrated that ATG16L1 was a major component targeting by miRNA-106b family members. Over-expression of ATG16L1 partially rescued miRNA-106b transduction-induced autophagy suppression. Previous reports indicated that ATG16L1 suppression and autophagy deficit are potential factors that lead to inefficient elimination of bacteria in colon epithelial cells during the development of Crohn’s disease. Consistent with these reports and our aforementioned findings, miRNA-106b mimic dramatically reduced the clearance of LF82 bacteria in HeLa epithelial cells. Thus, we have identified a family of miRNA members (miR-106b) as a main inhibitor of autophagy, which may play a role in the eradication of intracellular bacteria and development of Crohn’s disease.
Potential contribution of the interaction between spinal cord-infiltrating Th1 cells and astrocytes to the development of nerve injury induced neuropathic pain (173.10)
Authors: Cao Ling , Draleau Kyle , Maddula Swathi
Published: January 2023

We previously demonstrated that CD4+ T cells contribute to the maintenance of L5 spinal nerve transection (L5Tx)-induced neuropathic pain in BALB/c mice, and T-Bet+ Th1 cells were found to be the dominant subtype of CD4+ T cells within the lumbar spinal cord following L5Tx. To investigate the specific downstream responses mediated by these infiltrating Th1 cells, we examined the cytokine expression of the spinal cord-infiltrating CD4+ T cells via flow cytometry. At day 7 post-L5Tx, the peak time for detecting the lumbar infiltrating CD4+ T cells, there were trends indicating increases in the numbers of IFNγ+ and TNFα+ lumbar spinal cord-infiltrating CD4+ T cells in the L5Tx group compared to the sham group. Further, we examined the expression of glial fibrillary acidic protein (GFAP), an indication of the level of astrocytic activation, in the L5 spinal cord via immunohistochemistry. As expected, in wild type BALB/c mice, L5Tx induced an increase in GFAP expression in the ipsilateral side of the spinal cord compared to sham-operated mice 7 days post-surgery. This increase appeared to be reduced in CD4 knockout mice post-L5Tx. Altogether our data suggest the involvement of the Th1 cytokines IFNγ and TNFα in the maintenance of L5Tx-induced neuropathic pain, which may have potential role in regulating spinal cord astrocyte activation.
Microfluidic Assay for Neutrophil Chemotaxis Towards Chemoattractant Gradients (173.13)
Authors: Jones Caroline
Published: January 2023

Chemoattractants represent an essential group of molecular guidance signals that regulate the transit of leukocytes out of the mainstream of blood and into tissues at sites of inflammation. In this project, we developed a microfluidic device that is simple to operate and allows for precise and robust measurements of neutrophil chemotaxis speed and directionality towards precisely defined chemoattractant gradients at single-cell resolution. These devices consist of non-planar, enclosed microenvironments of increasing complexity, from straight channels, to channels with posts and simple mazes. The cross-section of the channels (3 × 6 μm) is smaller than the human neutrophils, so that cells are mechanically confined and allowed to move only along the axis of the channels toward a gradient of chemoattractactant. This assay is currently being applied in a clinical setting to characterize the immune function in burn patients during treatment. Comparing samples from healthy donors to burn patients revealed impaired directional migration speed starting as early as 24 hours after burn injury, reaching a minimum at 72 hours, correlated to the size of the burn injury and potentially serving as an early indicator for concurrent infections. Further characterization of neutrophil chemotaxis using this new assay may have important diagnostic implications not only for burn patients, but also for patients afflicted by other diseases that compromise neutrophil functions.
Dynamin 2 regulates S1P1 signalling and T cell migration (173.15)
Authors: Willinger Tim , Ferguson Shawn , De Camilli Pietro , Flavell Richard
Published: January 2023

Sphingosine 1-phosphate (S1P) is essential for T cell egress from lymphoid tissues by acting on its receptor S1P1. S1P agonism on S1P receptors is now used to treat autoimmune diseases by sequestering lymphocytes in lymphoid organs. However, not much is known about molecules that mediate T cell egress downstream of S1P1. Dynamin 2 is a large GTPase that is required for vesicle scission during endocytosis. Mice with a T cell-specific deletion of dynamin 2 had profound lymphopenia and an accumulation of mature thymocytes. Intrathymic dye injection revealed that thymic egress was inhibited in the absence of dynamin 2. Dynamin 2 regulated T cell migration downstream of S1P-S1P1 since dynamin 2-deficient thymocytes did not migrate in response to S1P in vitro. This was not due to a general defect in cell motility because T cells lacking dynamin 2 had a normal migratory response to CCL21. Dynamin 2 was required for ligand-induced S1P1 downmodulation, consistent with its known function in receptor-mediated endocytosis. In addition, dynamin 2 knockout thymocytes co-expressed S1P1 and CD69 on the cell surface demonstrating impaired S1P1 signalling in vivo. Surprisingly, the critical role of dynamin 2 in S1P1 signalling was independent of receptor internalization. Instead, dynamin 2-deficient T cells showed faster desensitization of S1P1 after agonist stimulation in vitro. Our data reveal an unexpected role for dynamin 2 in promoting T cell egress by regulating S1P1 signalling.
KLF2 and S1Pr1: Aiding in memory T cell trafficking and retention in non-lymphoid tissue (173.16)
Authors: Skon Cara , Lee June-Yong , Jameson Stephen
Published: January 2023

Memory T cells, unlike naïve T cells, have the capacity to traffic and be retained in non-lymphoid tissue (NLT). Because most infections originate in NLT and early detection of pathogens can lead to a more effective immune response, identifying factors contributing to memory T cell residence in NLT is an important aspect of memory T cell potential. The transcription factor kruppel-like factor 2 (KLF2) controls naive T cell trafficking (primarily through inducing sphingosine 1 phosphate receptor 1 (S1Pr1)), yet KLF2 expression in memory T cells has not been extensively addressed. Using eGFP-KLF2 reporter mice we report that almost all CD8+ memory T cells in lymphoid tissue express KLF2, yet the vast majority of CD8+ memory T cells in NLT (including small intestine, salivary glands, kidney and brain) are KLF2lo. Forced-expression of the KLF2 target gene S1Pr1, using retroviral transduction, caused a decrease in memory CD8+ T cells in NLT versus spleen, confirming a functional consequence of S1Pr1 down-regulation. We also showed that inflammatory cytokine milieu causes a decrease in KLF2 protein in the absence of antigen. Our data suggest that local cues from the non-lymphoid environment, including cytokines, cause a decrease in KLF2 and S1Pr1 leading to a cessation in CD8+ memory T cell trafficking that aids in memory T cell retention in NLT.
Tissue exit - a novel control point in the accumulation of antigen-specific CD8 T cells in the influenza A virus-infected lung (173.20)
Authors: Jennrich Silke , Lee Michael , Lynn Rachel , Dewberry Kristofer , Debes Gudrun
Published: January 2023

Memory/effector T cells efficiently migrate into extralymphoid tissues and sites of infection, providing immunosurveillance and a first line of defense against invading pathogens. Even though it is a potential means to regulate the size, quality, and duration of a tissue infiltrate, T cell egress from infected tissues is poorly understood. Using a mouse model of influenza A virus infection, we found that CD8 effector T cells egressed from the infected lung in a CCR7-dependent manner. In contrast, following antigen-recognition, effector CD8 T cell egress decreased and CCR7 function was reduced in vivo and in vitro, indicating that the exit of CD8 T cells from infected tissues is tightly regulated. Our data suggest that the regulation of T cell egress is a mechanism to retain antigen-specific effectors at the site of infection to promote viral clearance, while decreasing the numbers of bystander T cells and preventing overt inflammation.
A cyclic AMP signal participates in T cell tissue retention by selectively blocking tissue egress. (173.21)
Authors: Lees Jason , Li Xin
Published: January 2023

Accumulation of effector T cells within target tissue(s) is a critical step in the induction of tissue directed immunity. Cyclic AMP (cAMP) is a known regulator of T cell activity and function. In this study we examined the effects of cAMP on T cell trafficking and retention. We found that increasing levels of cellular cAMP, either through direct uptake of a cAMP analogue or induced by treatment with nonanedioic acid, decreased the ability of T cells in vitro to traffic to CXCL12 and to suboptimal, but not optimal, doses of CCL21. Levels of cAMP that decreased chemotaxis had no effect on T cell viability, activation, proliferation, or cytokine production. To determine the role of cAMP on T cell retention, we examined the capacity of transferred T cells to traffic from the foot pad to the draining lymph node. Increasing T cell cAMP levels significantly decreased T cell movement to the lymph node but not the capacity of cells to enter lymph nodes via high endothelial venules. We suggest that cAMP can act to increase T cell tissue retention by blocking responses to suboptimal levels of chemokines.
High endothelial venules at the nexus of an IL-6/gp130/STAT3 axis controlling immune surveillance during fever (173.22)
Authors: Vardam Trupti , Appenheimer Michelle , Zhou Lei , Chen Qing , Wang Wan-Chao , Ernst Matthias , Rose-John Stefan , Baumann Heinz , Evans Sharon
Published: January 2023

Fever remains the least understood component of acute inflammation although it confers a survival benefit in endothermic and ectothermic vertebrates. Here we show that LPS- or turpentine-induced fever enhances immune surveillance by augmenting lymphocyte trafficking via high endothelial venules (HEV) in lymph nodes. We identified an autocrine feed-forward loop during the effector phase of fever in which IL-6 produced by HEV acts via the gp130 signal transducing subunit of the IL-6 receptor to enhance ICAM-1-dependent lymphocyte trafficking. Comparative analysis of the thermal response in gp130Y757F and gp130ΔSTAT mutant mice implicated STAT3/1 in the pathway downstream of fever that controls lymphocyte trafficking. An obligate role for STAT3 signaling was further delineated using Stat1-/- mice. While gp130-dependent MEK1-ERK1/2 signaling was not required for enhanced lymphocyte trafficking, ERK1/2 activation was necessary for the metalloproteinase-dependent shedding mechanism that restores endothelial ICAM-1 to homeostatic levels during the resolution phase of inflammation. Finally, thermally-induced lymphocyte trafficking across HEV lowered the threshold for antigen-driven priming of naïve T cells in lymph nodes. Taken together, these studies provide insight into a novel IL-6/gp130/STAT3 axis that acts as a highly integrated thermally-sensitive alert system to heighten immune surveillance during febrile responses.
The E3 ubiquitin ligase TRIM21 forms a complex with p62 and TRAF6 and promotes degradation of TRAF6 (P1176)
Authors: Yoshimi Ryusuke , Watanabe Reikou , Tsukahara Toshinori , Hama Maasa , Ihata Atsushi , Ueda Atsuhisa , Takeno Mitsuhiro , Morse III Herbert , Ozato Keiko , Ishigatsubo Yoshiaki
Published: January 2023

NF-κB signaling pathway plays a pivotal role in inflammatory response, and its excessive activation causes systemic inflammatory disorder, such as systemic lupus erythematosus (SLE). TRIM21, one of the tripartite motif (TRIM) family members, is an E3 ubiquitin ligase that functions in both innate and acquired immunity. It is also an autoantigen known as Ro52/SS-A, which is observed in patients with SLE. Recent studies, including Trim21 gene disruption studies, suggest that TRIM21 is a negative regulator for NF-κB-dependent proinflammatory cytokine production. However, the molecular mechanism still remains to be clarified. Here we report that TRIM21 interacted with tumor necrosis factor-associated factor 6 (TRAF6), an essential adaptor protein for Toll-like receptor/interleukin 1 receptor (TLR/IL-1R)-mediated activation of NF-κB signaling pathway. The TRIM21-TRAF6 interaction was indirect and mediated by a scaffold protein, p62/sequestosome 1. TRIM21-overexpressed fibroblast cell line showed decreased expression of TRAF6 as compared to the control cells. TRIM21 promoted not only K63-linked ubiquitylation but also K48-linked ubiquitylation of TRAF6 in the presence of p62. Furthermore proteasome inhibitor MG132 increased the ubiquitylated TRAF6 proteins. These data suggest that the function of TRIM21 as a negative regulator for NF-κB signaling is attributed to the proteasome-mediated degradation of TRAF6.
Induction of expression of LAT1, an essential amino acid transporter, in activated T cells (P1182)
Authors: Hayashi Keitaro , Endou Hitoshi , Anzai Naohiko
Published: January 2023

Activation of T cell accompanies remarkable enhancement of metabolism. Sufficient and continuous supply of nutrient such as amino acid is thus considerable importance to support immune reaction in T cell. However, the molecular mechanism of efficient incorporation of amino acid into activated T cell has not been determined. We characterized LAT1 as an essential amino acid transporter which is highly expressed in activated T cell. TCR stimulation and co-stimulation of human T cell triggered the dramatic induction of LAT1, although neither TCR stimulation nor co-stimulation alone did not affect the LAT1 expression. The promotion of LAT1 expression in activated T cell was prohibited by inhibitor of NF-kB and AP-1. These results indicate that LAT1 expression is induced by full activation of T cell and LAT1 could work as a potential transporter for essential amino acid in activated T cell. Our result that NF-kB and AP-1 are essential for LAT1 expression suggests that nutrient supply system and immune reaction system are included in one program and those systems are activated by the same molecular machinery in T cell.
Defining the roles of calcium mobilization in the initial decondensation of chromatin during T cell activation (P1162)
Authors: Rawlings Jason , Bingham Kellie , Lee Megan , Wooten Sam
Published: January 2023

Condensation of chromatin within the nucleus of T lymphocytes occurs during thymocyte development and is a requirement for proper T cell development and maintenance of the naïve (quiescent) state. T cell activation via stimulation of the T cell receptor (TCR) is a necessary step in an immune response, allowing T cells to gain the ability to proliferate in response to cytokines, such as IL-2. During this activation process, the chromatin within T cells decondenses, allowing access to the previously inaccessible DNA, permitting the transcription of genes required for proliferation. We demonstrate that stimulation of either the IP¬3 (inositol triphosphate) or DAG (diacylglycerol) pathways downstream of the TCR leads to chromatin decondensation, but with different kinetics. Within the IP3 pathway, we show that extracellular calcium is not required for the initial decondensation of chromatin. Furthermore, we show that the release of calcium from intracellular stores is sufficient and required for the initial decondensation of chromatin following TCR engagement. Interestingly, while calcium mobilization results in a decondensation of chromatin, it is not sufficient for STAT5 target gene expression. However, chromatin decondensation via the DAG pathway is capable of making T cells competent to respond to IL-2. These findings suggest that there may be at least two distinct domains of condensed chromatin in naïve T cells.
IL17A and IFNγ jointly control rapidly fatal eosinophilic myocarditis in mice (P4120)
Authors: Barin Jobert , Baldeviano G. , Talor Monica , Wu Lei , Ong SuFey , Fairweather DeLisa , Bedja Djahida , Stickel Natalie , LeGault Jillian , Cardamone Ashley , Zheng Dongfeng , Gabrielson Kathleen , Rose Noel , Cihakova Daniela
Published: January 2023

Deviation to autoaggressive T helper 17 (Th17) responses has been reported to account for the protective effect of interferon-gamma (IFNγ) in certain animal models of autoimmune disease. Building on our previous findings that interleukin-17 (IL17) signaling is critical for the progression of experimental autoimmune myocarditis (EAM) to dilative heart failure, we sought to address the question of whether IL17 mediated the severe form of EAM observed in IFNγ-deficient mice. To our surprise, we found that IFNγ-/-IL17A-/- double-knockout (DKO) mice had greater mortality compared to IFNγ-/- mice, starting at day 14 and reaching 50% by day 21 of EAM. DKO hearts were severely infiltrated, indicating acute inflammatory heart failure as the mode of death in these mice. We found pronounced eosinophilic infiltration in DKO mice, implicating eosinophils as mediators of the enhanced morbidity. Cardiac production of CCL11/eotaxin was increased, alongside Th2 deviation among heart-infiltrating CD4+ T cells. Intriguingly, blockade of IL4 in IFNγ-/-IL17A-/-DKO mice inhibited Th2 deviation, but failed to resolve eosinophilia or morbidity. However, developmental ablation of eosinophils partly rescued the survival of DKO mice. These data point to a novel collaboration between IFNγ and IL17A in controlling eosinophil recruitment.
Pre-conditioning with high-mobility group box 1 (HMGB1) attenuates excessive TLR-mediated inflammation. (INM9P.456)
Authors: Lim A Ram , Kim Sol , Park Su Jung , Shin Jeon soo
Published: January 2023

Activation of innate immunity via TLRs plays a pivotal role in host defense against pathogens through the recognition of PAMPs. However, since uncontrolled immune response leads to various kinds of autoimmune and inflammatory diseases, TLR signaling should be tightly regulated. Recent studies have shown that HMGB1 could be critical for the control of TLR signaling. In this study, we demonstrated that HMGB1 mediated negative regulating signals of TLR activation. Our data indicated that HMGB1 induced NF-κB activation via non-canonical pathway by processing of p100 to p52 and translocation of p52 into the nucleus. Also, HMGB1 induced expression of TRIM30α which is a regulator of TLR signaling. Preconditioned HMGB1 attenuated TLR activation by LPS in murine, human macrophage cell lines and mouse BMDM. Treatment of HMGB1 followed by LPS stimulation decreased TNF-α and IL-6 level in mouse BMDM when compared with LPS stimulation alone. Furthermore, HMGB1 pretreatment in the presence of polymyxin B, an endotoxin inactivator, induced HMGB1-mediated tolerance. Pretreatment of HMGB1 promoted rapid termination of signaling in macrophage cells through transcription factor NF-κB by augmenting negative regulators of TLRs such as TRIM30α and SHIP-1. Knockdown experiments using TRIM30α-specific small interfering RNA impaired the HMGB1-mediated TLR tolerance. Collectively, our results demonstrate that HMGB1 mediated the mechanism for prevention of excessive TLR-mediated inflammation.
Characterizing the role of Blimp-1 in regulating encephalitogenic T cells in CNS autoimmunity (IRC8P.483)
Authors: Nuro-Gyina Patrick , Liu Yue , Pei Wei , Larson Michelle , Lovett-Racke Amy , Racke Michael , Yang Yuhong
Published: January 2023

Effector and recall responses of T cells play an important role in the pathogenesis of Multiple Sclerosis and EAE. B lymphocyte induced maturation protein-1 (Blimp-1) is a transcription factor that plays important roles in regulating immunity. However, its role in regulating encephalitogenic T cells in EAE has not been characterized. Using YFP/Blimp-1 reporter mice, we analyzed Blimp-1 expression in myelin-specific CD4 T cells in vitro and in vivo. Blimp-1 expression correlates with the encephalitogenicity of myelin-specific CD4 T cells, which is highly expressed in encephalitogenic Th1 and Th17 cells, but not in non-encephalitogenic Th17 cells. Moreover, Blimp-1 is highly expressed in myelin-specific CD4 T cells in the draining lymph nodes, spleen and CNS during EAE development, specifically in activated CD44+IL7Rα+CD4 T cells. As IL-23 significantly enhances the encephalitogenicity of myelin-specific CD4 T cells, we determined if IL-23 upregulates Blimp-1 expression. Our data showed that exogenous IL-23 significantly enhances Blimp-1 expression in myelin-specific CD4 T cells and Blimp-1 is highly expressed in IFNγ+, IL-17+ and IFNγ+ and IL-17 double positive cells in draining LN cells, splenocytes and CNS infiltrating cells. In addition, we determined the role of Blimp-1 in regulating T cell migration and EAE development. Together, our findings suggest that Blimp-1 might play an important role in regulating autoreactive T cells and the development of CNS autoimmunity.
Heme oxygenase-1/CO protect against post-traumatic suppression of adaptive immune responses (IRC8P.475)
Authors: Darwiche Sophie , Wang Yanting , Brumfield John , Stratimirovic Sladjana , Vreeland Kathryn , Botero Ana , Martinez Sylvia , Escobar Daniel , Luciano Jason , Kautza Ben , Zuckerbraun Brian
Published: January 2023

Traumatic injury induces a systemic dysregulated immuno-inflammatory response that contributes to mortality. This response includes an upregulation of pro-inflammatory cytokines by innate cells and an independent suppression of adaptive immunity characterized by reduced Th1 responses. Heme oxygenase-1 (HO-1) and its byproduct, carbon monoxide (CO), are protective against injury-induced inflammation and end-organ damage, but their beneficial role in the dysregulation of adaptive responses following trauma is unknown. To investigate this, B6 mice received multiple-trauma or no manipulation, and were given either vehicle or a HO-1 inducer (Cobalt protoporphyrin, CoPP; 5mg/kg) prior to injury. Another cohort received either room air or CO (250ppm) for 4 hours directly post-injury. At 48hr post-trauma, Tcell functions were assessed by ex-vivo mitogen-induced Tcell proliferation and Th1 cytokine release (IL-2 and IFN-γ). As expected after injury, a near 50% reduction in Tcell proliferation was seen in vehicle- or air-treated trauma mice, compared to uninjured controls (p=0.007). A parallel reduction in Tcell IL-2 and IFN-γ release was observed. In clear contrast, CoPP or CO treatment prevented trauma-induced reductions in Tcell proliferation and cytokine release (fold difference 0.90±0.10 and 1.21±0.16 to uninjured controls, respectively). Overall, HO-1 represents an endogenous protective pathway that can be exploited following trauma to preserve normal immune functions & survival.