bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of type 1 IFN after oral Toxoplasma gondii infection (111.15)
Authors: Han Seong-Ji
Published: January 2023

The protective role of type I interferons (IFN) in host immunity to viral infections has been well characterized. However, the role of type I IFN following parasitic infection is less clear. To investigate this further, we used type I interferon receptor knock out (IFNAR -/-) mice and orally infected them with the protozoan parasite, Toxoplasma gondii (T. gondii). We observed a significant increase in the parasite load in the mesenteric lymph nodes (MLNs) of IFNAR-/- mice with a concomitant drop in survival relative to wild type mice. Furthermore, we observed decreased levels of serum CCL2 that correlates with reduced recruitment of CD11b+Ly6C+ inflammatory monocyte (IM) into the MLNs of IFNAR-/- mice. We observed that IFN-β in wild type mice is produced in two waves in the MLNs after oral infection and we are currently investigating which cells are responsible for type I IFN production at different time points. We present a model for the protective role of type I IFN during T. gondii infection and provide new insight into innate defense against protozoan parasites.
Effect of CCL1 antisense ODN on bacterial translocation in mice irradiated with whole body 137Cs γ-rays: elimination of M2b macrophages in translocation sites (111.19)
Authors: Suzuki Fujio , Patel Niket , Kogiso Mari , Cornforth Michael , Kobayashi Makiko
Published: January 2023

Mice irradiated with 5 Gy whole body 137Cs γ-rays (WBI-mice) are shown to be carriers of M2bMϕ (IL-10+CCL1+LIGHT+IL-12- Mϕ), which are inhibitory on the host antibacterial innate immunities in bacterial translocation. M2bMϕ maintain their suppressor cell properties through the production of CCL1. In this study, therefore, we tried to eliminate M2bMϕ from mesenteric lymph nodes (MLN) of WBI-mice by CCL1 antisense ODN gene therapy. WBI-mice were treated with CCL1 antisense ODN with various doses (2, 10, 50, or 250 μg/mouse), routes (i.p., i.m. or s.c.) and schedules (twice a day for 2 to 7 days, starting 1 to 4 weeks after γ-irradiation). F4/80+ MLN-Mϕ isolated from these mice were identified as M2bMϕ by their abilities to express intracellular IL-10 and LIGHT mRNA. As a control, MLN-Mϕ were isolated from WBI-mice treated with scrambled ODN and subjected to the same test. In the results, the numbers of MLN-M2bMϕ were greatly reduced in WBI-mice treated s.c. with 10 to 50 μg/mouse of CCL1 antisense ODN twice a day for 2 days, 5 days or 7 days, started 1 to 3 weeks after γ-irradiation. However, MLN-M2bMϕ were detected in WBI-mice treated with CCL1 antisense ODN twice a day for 2 days started 4 weeks after the irradiation. These results indicate that M2bMϕ are eliminated from MLN of WBI-mice s.c. treated with CCL1 antisense ODN twice a day for 2 days starting 1 to 3 weeks after γ-irradiation.
Fatal outcome of pandemic H1N1 2009 influenza infection is associated with neutrophil and macrophage lung infiltration but not enhanced viral burden in pregnant mice. (111.3)
Authors: Marcelin Glendie , Aldridge Jerry , Boon Adrianus , Duan Susu , Rehg Jerold , Webby Richard
Published: January 2023

Pandemic H1N1 2009 Influenza A virus (pH1N1) infection in pregnant or postpartum women is quite severe. The immunological mechanisms that affect infection outcome in these populations are not well understood. To address this, pregnant Balb/c and non-pregnant mice were inoculated with a wild-type pH1N1 virus. We determined that early cytokine responses were impaired in pregnant mouse lungs. Moreover, pulmonary infiltration of neutrophils and macrophages strongly correlated with an increase in mortality in pregnant mice. Increased mortality in pregnant animals was not associated with higher virus load because equivalent viral burden (virus titers and immunohistochemical staining) was observed in the nasal cavities and lower respiratory tracts of all mice. Pregnancy altered innate responses despite the presence of similar viral burden. Also, pregnant mice demonstrated equivalent influenza-specific activation of pulmonary CD8+ T lymphocytes however; they displayed elevated T-regulator lymphocyte (Tregs) responses in the lung and mediastinal lymph nodes. Despite these immunologic alterations, pregnant animals mounted equivalent hemagglutinin inhibition and IgG subclass titers in response to virus or immunization with monovalent inactivated pH1N1 vaccine. Therefore, the severity of pH1N1 in pregnant mice is associated with pulmonary neutrophil and macrophage invasion, dysregulated cytokine responses but not increased viral load.
IL-10 antisense oligodeoxynucleotides (ODN) improve the resistance of severely burned mice intradermally infected with MRSA (111.8)
Authors: Asai Akira , Kobayashi Makiko , Herndon David , Suzuki Fujio
Published: January 2023

The effect of IL-10 antisense ODN on the susceptibility of burned mice to intradermal (i.d.) infection of MRSA was studied. IL-10 antisense ODN (6 μg/mouse) was administered to mice twice a day for 3 days beginning 1 day after burn injury (3rd degree, 25% TBSA flame burn). Two days after burn injury, these mice were infected i.d. with 108 CFU/mouse of MRSA. Abscess formation in the infection site and mortality rates of these mice were tested. Also, F4/80+ Mϕ isolated from tissues surrounding the infection sites of these mice were biophysically characterized. In the results, sepsis caused by MRSA i.d. infection did not develop and abscess formation was demonstrated in burned mice treated with IL-10 antisense ODN. However, all burned mice treated with scrambled ODN (control group) died from sepsis, and abscess formation was not shown in these mice. Mϕ isolated from the infection site tissues of burned mice treated with IL-10 antisense ODN were identified as M1Mϕ, while those from control mice were shown to be M2Mϕ. Abscess formation was demonstrated in burned mice inoculated with M1Mϕ (but not M2Mϕ), and these mice resisted a lethal dose of MRSA i.d. infection. These results indicate that burned mice treated with IL-10 antisense ODN are resistant against i.d. infection of MRSA. M1Mϕ appearing in the infection site tissues of burned mice that are treated with IL-10 antisense ODN may play a role on abscess formation and inhibiting sepsis caused by MRSA i.d. infection.
Discrimination of membrane antigen affinity by B cells requires dominance of kinetic proofreading over serial triggering (112.10)
Authors: Tsourkas Philippos , Liu Wanli , Das Somkanya , Pierce Susan , Raychaudhuri Subhadip
Published: January 2023

B cells receptor (BCR) signaling in response to membrane-bound antigen increases with antigen affinity, a process known as affinity discrimination. However, if BCR molecules become signaling-capable immediately upon binding antigen, the decrease in serial engagement as affinity increases results in weaker signaling as affinity increases. We use computational modeling to show that a threshold time (kinetic proofreading) for antigen to stay bound to BCR before the latter becomes signaling-capable is needed to overcome the decrease in serial engagement with increasing antigen affinity, and replicate the monotonic increase in B cell signaling with affinity observed in experiments. This finding matches well with the experimentally observed timescale (~20 seconds) of antigen-mediated conformational changes to BCR that lead to Src-family kinase recruitment. The physical basis of the threshold time of antigen binding may lie in the formation timescale of BCR dimers. The latter decreases with increasing affinity, resulting in shorter threshold antigen binding times as affinity increases. Such an affinity-dependent kinetic proofreading requirement results in affinity discrimination very similar to that observed in experiments. We also develop a measure to characterize affinity discrimination in a quantitative manner. B cell affinity discrimination is critical to the process of affinity maturation, and thus our results have important implications in applications such as vaccine design.
ERK activation is required for PGE2-induced MMP-9 production in bone-morrow derived DCs (112.23)
Authors: Yen Jui-Hung , Kocieda Virginia , Ganea Doina
Published: January 2023

Dendritic Cells (DCs) play an important role in the initiation of the immune response. Immature DCs take up antigen, mature and migrate to the regional lymph nodes where they activate antigen-specific naïve T cells. Prostaglandins such as PGE2 are essential for the migration of DCs to lymph nodes. Matrix metalloproteinases (MMPs) play an essential role in DC migration, through the degradation of extracellular matrix (ECM) and basement membranes. MMP2, and particularly MMP9, are essential for dendritic cell migration in vitro, as well as in vivo to the draining lymph nodes. In this study, we show that PGE2-induced MMP9 expression is mediated through the EP2/EP4-cAMP-PKA/PI3K-ERK signaling pathway. EP2 and EP4 receptors as well as cAMP analog mimic the effect of PGE2 to upregulate MMP-9. In the presence of PKA, PI3K or ERK inhibitors, PGE2-induced MMP-9 production is abolished. Furthermore, PGE2-induced c-Fos expression mediated through ERK activation is required for the binding of AP-1 transcription factor to the MMP-9 promoter. In the presence of ERK inhibitors, PGE2-induced AP-1 binding is abrogated. Our results describe a new molecular mechanism for the effect of PGE on MMP-9 production in DCs, which could lead to a new therapeutic approach by using ERK inhibitors to regulate DC migration.
Mouse CD99 interact with adhesion molecule CD99L2 (112.27)
Authors: Lee Hye-Yeong , Ma Min-jung , Choi Eun Young
Published: January 2023

CD99 and CD99-like-2 protein (CD99L2) are membrane glycoprotein leukocyte antigens that do not belong to any known protein family. They are expressed on leukocytes and at the endothelial cell junction, and participate in leukocyte diapedesis process. Based upon the similarity in expression pattern, we tested whether they would interact each other, and show here that the interaction of the two molecules is mediated via cytoplasmic domain of both molecules. In bimolecular fluorescence complementation (BiFC) assay, co-expression of CD99 and CD99L2 emitted fluorescence suggesting the interaction between the two molecules and heterodimer formation. The interaction was further confirmed by fluorescence resonance energy transfer (FRET) assay. Previously, we have reported that CD99 forms CD99-CD99 homodimer. Unlike CD99, CD99L2 does not form homodimer, rather it binds to CD99 and forms CD99-CD99L2 heterodimer. CD99-CD99 homodimer and CD99-CD99L2 heterodimer exist in a cell without competition. With cytoplasmic domain mutant of CD99, CD99L2 could not form heterodimer, indicating two molecules join through cytoplasmic domain. In reverse case when cytoplasmic domain mutant of CD99L2 was used for BiFC, CD99 could not form heterodimer. The interaction between CD99 and CD99L2 was verified by co-immunoprecipitation using mouse spleen cells. Our results reveal that mouse CD99 interact with CD99L2 and form heterodimer through its cytoplasmic domain.
Autocrine function of CD8+ T cells by modulation of CCR5 expression (112.3)
Authors: Carreno Roberto
Published: January 2023

Although we have shown CCR5+ T cells are known to play a role in the pathogenesis of graft-versus-host disease (GVHD), the detailed biofunction of CCR5 remains uncertain. Recently we have shown that CCR5+CD8+ T cells exhibit an effector function due to the secretion of cytolytic cytokines TNF-α and INF-γ. In addition, CCR5+CD8+ T cells have been shown to proliferate upon DC stimulation and also secrete the proliferative cytokine IL-2. Thus CCR5+CD8+ T cells maintain a polyfunctional state including both effector and memory. Moreover, in effector CD8+ T cells (CD45RA+CD27- [M3]) there is an upregulation of MIP1-β, the ligand for CCR5. These works indicate that CCR5 plays a role in memory/effector phenotype and function. Here we use CCR5 to further define memory and effector CD8+ T cells, and correlate this to their function. Using FACs we characterized central (CD45RA-CD27+ [M1]) and effector (CD45RA-CD27- [M2]) memory CD8+ T cells have high CCR5 expression. We also observed a decline in CCR5 expression in M3 CD8+ T cells. As a result, we propose that CD8+ T cells utilize autocrine signaling by promoting lymphocyte migration and infiltration to sites of inflammation by modulating the expression of CCR5 and its ligands. Further understanding of this autocrine type signal may help understand the role of CCR5 in GVHD pathogenesis.
Association of elevated expression of Toll-like receptor-2 in carotid endarterectomy specimens with the severity of atherosclerotic disease (112.9)
Authors: Tan Xiaoyu , Cherian Greoge , Cherian Rachel , Stechschulte Daniel , Dileepan Kottarappat
Published: January 2023

Significant interest is now focused on the role of Toll-like receptors (TLR) and cyclooxygenases (COX) in vascular disease. In this study we determined whether TLRs and COX gene expression in carotid atherosclerotic lesions is associated with clinical manifestations of the disease. Carotid endarterectomy specimens collected during surgery in nine patients with clinically significant carotid artery occlusive disease were analyzed for the expression of TLR2, TLR4, COX1 and COX2 mRNA by real-time PCR. Immunohistochemical evaluations of the specimens were carried out to quantify the presence of mast cells, lymphocytes, and macrophages using CD117, CD3, and CD68 antibodies. The level of expression of specific genes and density of the inflammatory cell population in the plaques were compared with the characteristics and location of plaques, and the severity and distribution of the clinical disease. The analysis of the expression of TLR2 with the angiographic location of the plaque revealed that the plaques expressing the higher levels of TLR2 were located at or near the carotid bifurcation whereas the plaques with the lowest expression of TLR2 were located distal to the carotid bifurcation. The plaques with the higher levels of TLR2 mRNA also showed relatively increased numbers of mast cells, T lymphocytes, and macrophages. The magnitude of TLR2 expression in the plaque appears to be associated with the severity of the atherosclerotic disease.
CD180 (RP105) intrinsically activates B cells to produce polyclonal Ig and integrates diverse TLR signals to modify cell fate (113.15)
Authors: Chaplin Jay , Kasahara Shinji , Clark Edward , Ledbetter Jeffrey
Published: January 2023

CD180 is homologous to TLR4 and regulates TLR4 signaling, yet its function is disputed. In this work, injection of anti-CD180 mAb into mice induced rapid Ig production of all classes and subclasses except IgA and IgG2b, with up to 50-fold serum increases of IgG1 and IgG3. IgG production after anti-CD180 injection was not due to reactivation of memory B cells and was retained in T cell-deficient (TCR KO) and MyD88 KO mice. Expansion of splenic B cells occurred in WT and TCR KO mice whereas T cell expansion occurred in WT but not in B cell-deficient (μMT) mice, indicating a direct role for B cells but not T cells in CD180 stimulation in vivo. Combinations of anti-CD180 with various TLR ligands biased B cell fate as co-injection diminished Ig production but purified B cells exhibited synergistic proliferation. Anti-CD180 had no effect on cytokine production from B cells but increased IL-6, IL-10, and TNF-α production in combination with LPS or CpG. Thus CD180 stimulation induces intrinsic B cell proliferation and differentiation, causing rapid increases in IgG, and integrates MyD88-dependent TLR signals to regulate proliferation, cytokine production, and differentiation.
Deletion of factors in the Id3 pathway results in a dramatic phenotype where Vγ1Vδ6.3 (γδ NKT) T cells become the dominant T lymphocyte (113.2)
Authors: Alonzo Eric , Sant'Angelo Derek
Published: January 2023

Recent work from our lab and others has shown a significant expansion of γδ NKT cells in mice that lack the HLH-protein Id3. In contrast to WT mice, nearly all Id3-deficient γδ NKT cells express high levels of the innate T cell determinant, PLZF and produce copious amounts of IL-4 upon activation. We have been exploring the transcriptional network that controls the development of γδ NKT cells. In an effort to identify additional factors that play a key role in expansion of γδ NKT cells, we have undertaken a candidate approach to evaluate genes that function in the Id3 pathway. To do this we are studying single and double gene deficient mice to determine the interrelation of different transcription factors. These studies have led to the discovery of a gene combination that results in a dramatic alteration of thymopoiesis. In these mice, while thymic cellularity was not affected, the frequency of γδ T cells versus αβ T cells was reversed. γδ T cells were found to be the dominant lymphocyte, representing ~65% of the cells. Furthermore, nearly all of the γδ T cells were now found to express PLZF. This dramatic phenotype appears to be T cell-specific, as both myeloid and lymphoid lineages are present. Analysis of thymic progenitors revealed major alterations in DN2-DN4 stages of development. Our findings uncover a fundamental regulatory pathway that favors the development of γδ NKT cells, while restricting the maturation of pro-T cells into the conventional T cell lineage.
Acute stress-induced thymic atrophy is mediated by intrathymic inflammatory and wound healing responses (113.25)
Authors: Billard Matthew , Gruver Amanda , Sempowski Gregory
Published: January 2023

Productive thymopoiesis is essential for a robust and healthy immune system. Thymus unfortunately is acutely sensitive to stress resulting in involution and decreased T cell production. Thymic involution is a complication of many clinical settings, including infection, malnutrition, and irradiation or immunosuppressive therapies. Little is known, however, about intrathymic mechanisms that may actively contribute to thymus atrophy or initiate thymic recovery following stress events. In this work, phenotypic, histologic and transcriptome/pathway analysis was performed to identify putative intrathymic mechanisms that drive endotoxemia-induced involution of murine thymic tissue. Thymus atrophy in this murine model was confirmed by down-regulation of genes involved in T cell development, cell activation, and cell cycle progression, correlating with observed phenotypic and histologic thymus involution. Significant gene changes support the hypothesis that multiple key intrathymic pathways are differentially activated during stress-induced thymic involution. These included direct activation of thymus tissue by LPS through TLR signaling, local expression of inflammatory cytokines, inhibition of T cell signaling, and induction of wound healing/tissue remodeling. Taken together, these observations demonstrated that both systemic and direct intrathymic responses to endotoxin challenge concurrently contribute to thymic involution during endotoxemia.
Expansion of Th17 cells and IL-17 are present only in a subset of patients with acute coronary syndrome (114.10)
Authors: Golino Paolo , Borriello Francesco , Barra Giusi , D'Aiuto Elena , Cirillo Plinio , Abbate Gianfranco , Cimmino Giovanni , De Palma Raffaele
Published: January 2023

T cells have a central role in atherosclerosis and Acute Coronary Syndromes (ACS). Th17 cells have a key role in chronic inflammation since they secrete a wide array of cytokines, including IL-17, having pro-inflammatory effects by recruiting other inflammatory cells and acting directly on target cells. So far, Th17 role in atherosclerosis is still a matter of debate. To study the role of Th17 cells in the pathophysiology of ACS, we studied 38 patients (ACS,n=23; Stable Angina,n=15. Cytokine production was measured by Luminex™ technology in sera obtained from the aorta(Ao)and the coronary sinus(CS)during diagnostic coronary angiography. Th17 cells were measured by flow cytometry after staining with MoAb for CD3,CD4,CCR6 and CD161. 5 out of 23 patients with ACS had a high number of Th17 cells (>15% of total T cells) in the coronary sinus and high amounts of IL-17 (>50pg/ml). No correlations were found with the presence of Th1 or Th2 or related cytokines. Only one patient with Stable Angina had a high number of Th17 cells in the coronary sinus. After a 18 months follow-up we found that patients with ACS having high number of Th17 presented a worse disease course, in terms of successive needs for hospitalization, stroke and/ or exitus. Further exploitation of these data could lead to a better understanding of processes leading to atherosclerosis and ACS, and to the identification of new prognostic biomarkers in atherosclerosis and to the set up of new therapeutic strategies.
Enhanced T cell proliferation, Th17 differentiation and unaltered IL-10 production lead to marginally enhanced development of experimental autoimmune encephalomyelitis in EBI3-deficient mice (114.12)
Authors: Bai Xue-Feng , Liu Jin-Qing , Carl, Jr. Joseph , Liu Zhenzhen , Zhang Xuejun , Talebian Fatemeh , Shi Fu-Dong , Whitacre Caroline , Trgovcich Joanne
Published: January 2023

Epstein-Barr virus-induced gene 3 (EBI3) encoded protein can form heterodimers with IL-27P28 and IL-12P35 to form IL-27 and IL-35. IL-27 and IL-35 may influence autoimmunity through inhibiting Th17 differentiation, and facilitating the inhibitory roles of Foxp3+ Treg cells, repectively. In this study we have evaluated the development of experimental autoimmune encephalomyelitis (EAE) in EBI3-deficient mice that lack both IL-27 and IL-35. We found that MOG peptide immunization resulted in marginally enhanced EAE development in EBI3-deficient C57BL6 and 2D2 TCR transgenic mice. EBI3-deficiency resulted in significantly increased proliferation and Th17 differentiation of MOG-specific CD4 T cells in the peripheral lymphoid organs and in the central nervous system (CNS). However, IL-10 production was not affected or even enhanced in the peripheral lymphoid organs or the CNS of EBI3-deficient mice. Treg cells from EBI3-deficient 2D2 mice had reduced capacity of inhibiting proliferation of 2D2 T cells. Taken together, we have found that EBI3-deficiency results in marginally enhanced EAE severity, which is associated with enhanced T cell proliferation and Th17 differentiation. However, the relatively milder EAE enhancement and lack of impact on IL-10 production by EBI3-deficiency suggest that alternative signaling pathways (presumably IL27P28-related) can partially compensate for EBI3-deficiency in EBI3-/- mice.
JAK2 is overexpressed in human multiple sclerosis Th17 cells and stabilizes cell surface expression of IFN-γR2 (114.14)
Authors: Novelli Francesco , De Palma Raffaele , Rolla Simona , Conti Laura , Clerico Marinella , Cintessa Giulia , Silveinnonen Olli , Durelli Luca
Published: January 2023

IFN-β inhibits the expansion of T helper 17 (Th17) cells in active multiple sclerosis (AMS), and this might contribute to improve the clinical symptoms. The effectiveness of this inhibition, however, requires intact IFN-γ signaling in T cells. Here we report that both mRNA and cell surface expression of the signaling chain of the IFN-γ receptor (IFN-γR2) and its cognate tyrosine kinase JAK2 are enhanced in peripheral blood Th17 cells from patients with AMS compared with those with inactive MS (IMS) or healthy subjects (HS). Anti-CD3 mAb stimulation and IFN-γ decreased the frequency of Th17 cells in peripheral blood mononuclear cells (PBMC) from AMS patients. Stimulation of PBMC from HS in Th17-polarizing conditions resulted in enhancement of JAK2 expression and accumulation of cell surface IFN-γR2. The role of JAK2 in the modulation of IFN-γR2 was demonstrated as its transduction prevented rapid internalization and degradation of IFN-γR2 in JAK2-deficient γ2A cells. In conclusion, these data identify JAK2 as a critical factor that stabilizes IFN-γR2 surface expression in Th17 cells from AMS patients, making them sensitive to IFN-γ. These data may have clinical implications for a better use of IFNs in MS and possibly other inflammatory diseases.
Th17 is involved in the pathogenesis of Beçhet’s disease (114.15)
Authors: Yasuoka Hidekata , Chen Zhu , Takeuchi Tsutomu , Kuwana Masataka
Published: January 2023

Behçet’s disease (BD) is an autoinflammatory disease with a unique distribution characterized by uveitis, and mucosal and skin lesions. To examine a role of Th17 response in the pathogenic process of BD, peripheral blood samples from 20 patients with BD and 13 controls were used to evaluate phenotypic and functional properties relevant to the Th17 response. Expression levels of RORC mRNA in peripheral blood mononuclear cells by RT-PCR and proportion of CD4+ cells expressing intracellular IL-17 by flow cytometry were increased in BD patients than in controls (P < 0.05 in both comparisons). Expression of chemokine receptor CCR6 was detected in nearly all IL-17-expressing cells. The proportion of CD4+CCR6+ was higher in BD patients in remission compared those with active disease (P < 0.05), suggesting that these cells are migrated to the lesions at active disease phase. In addition, CD4+ T cells from BD patients had an enhanced migration capacity induced by CCL20, than did those from controls. Finally, plasma CCL20 level was higher in BD patients than in controls (P < 0.05). These results together suggest that Th17 cells are involved in the pathogenesis of BD by migrating into skin and mucosa through the CCL20-CCR6 axis.
The role of Stat4 in IL-23-dependent IL-17 production (114.2)
Authors: Glosson Nicole , Stritesky Gretta , Kaplan Mark
Published: January 2023

Signal transducers and activators of transcription (STAT) proteins are induced upon cytokine and growth factor stimulation and play a pivotal role in immune and inflammatory responses. Stat4 is a critical mediator of inflammatory immunity and is required for all known IL-12 biological responses, including the induction of IFN-γ and development of Th1 cells. IL-23, an IL-12-related cytokine, also induces Stat4 activation, which may be important for optimal IL-17 secretion from CD4+ T cells. We further characterized the requirement for Stat4 in IL-23-mediated cytokine production using cells from Stat4-deficient mice. Stat4-deficient CD4+ T cells display reduced cytokine-induced IL-17 production in comparison to control cells, and this phenotype is most prominent in IL-23-primed memory CD4+ T cells. Furthermore, innate IL-17-producing cells, such as γδ T cells, natural killer T (NKT) cells and natural killer (NK) cells display distinct dependence on Stat4 activation for IL-17 production following antigen receptor or cytokine stimulation. Taken together, these data demonstrate a restricted requirement for Stat4 in the responses of innate and adaptive IL-17-secreting cells and define candidates for further defining the role of Stat4 in inflammatory immunity.
Interleukin-2 independent expansion of Helios-expressing T-cells contributes to autoimmunity in regulatory T-cell deficient mice (115.13)
Authors: Sharma Rahul , Abaya Christian , Ju Chiao-Ying , Ju Shyr-Te
Published: January 2023

The CD4+Foxp3+ T regulatory cells (Treg) are important for peripheral tolerance. Expression of the transcription factor Helios (Hel) was recently identified as a marker for thymus-derived Treg (tTreg), and was not expressed on the induced Treg. A minor population of CD4+Foxp3negHel+ T-cells is present in normal B6 mice. We hypothesized that this population may be related to tTreg in lineage and may contribute to autoimmunity during lack of Foxp3 expression. Indeed, in Treg deficient Il2-/- and Scurfy (Sf) mice, the CD4+Foxp3negHel+ T-cells expanded in the periphery. These cells produced high levels of inflammatory cytokines and were present at high numbers in the inflamed organs. The peripheral maintenance of the CD4+Foxp3negHel+ cells was independent of interleukin (IL)-2 because they expanded in the periphery of Il2-/- and IL-2 deficient Sf mice (Sf.Il2-/- mice). In this respect, the majority of the peripheral Treg in the Il2-/- mice are Hel+, suggesting that they are also relatively IL-2 independent. Importantly, abnormal Foxp3 mRNA could be detected in the Sf CD4+ T-cells that contain high percentage of Hel+ T-cells. The data strongly suggest that abnormal tTreg contribute significantly to the CD4+Foxp3negHel+ T-cell -mediated autoimmunity in Sf mice. In contrast to Treg, the expansion of CD4+Foxp3negHel+ T-cells becomes IL-2 independent
Regulatory T cells (Treg) from B cell deficient (B-/-) mice are more effective at suppressing spontaneous autoimmune thyroiditis than WT Treg (115.15)
Authors: Ellis Jason , Braley-Mullen Helen
Published: January 2023

NOD.H-2h4 mice given NaI in their water develop SAT with chronic inflammation of the thyroid by T and B cells. B-/- mice are resistant to SAT, but develop SAT after transient depletion of Treg by anti-CD25. After Treg depletion, Treg in B-/- mice take longer to repopulate than WT Treg. WT Treg have fewer FoxP3 low cells than B-/- Treg, suggesting they may have different suppressive capacities. B-/- mice have more FoxP3+ Treg, and a more activated phenotype than WT Treg with higher CD25, TGFβ, and CD44 expression, and lower CD62L and TNFR II p75. CD28-/-B-/- NOD.H2-4 mice have very few CD4+CD25+FoxP3+ Treg. We established a transfer model using splenocytes from CD28-/-B-/- mice (effector cells) cultured with or without sorted Treg from FoxP3GFP WT or B-/- mice. After transfer to mice lacking T cells, mice given Treg from B-/- mice developed significantly lower SAT severity than mice given Treg from WT mice. Although few Treg were detected at the end of culture, recipients given either WT or B-/- Treg had comparable numbers of splenic Treg 8 wks after transfer. The data herein suggests that despite being present in similar numbers both after culture and in vivo at the peak of disease, Treg that develop in B-/- mice are more effective at suppressing SAT than those in WT mice. In B-/- mice, a lack of B cells as APC during initiation of SAT may lead to preferential activation of Treg, resulting in reduced effector T cell activation and lack of SAT.
Tregs in autoimmunity to an inducible epidermal antigen (115.19)
Authors: Gratz Iris , Rosenblum Michael , Abbas Abul
Published: January 2023

The skin has unique immunological characteristics and it is known to be the most difficult organ to establish tolerance in a transplant-setting. The regulation of CD4+ T cell responses to epidermal self-antigen are largely undefined. Our goal was to establish a mouse model to study CD4+ T cell responses to epidermal neo-self-antigen. We established a mouse model that features tetracycline-inducible expression of chicken ovalbumin (Ova) in the epidermis under the control of the keratin K5 promoter. We analyzed the response of TCR-transgenic T cells (DO11.10) to Ova. Ova mRNA was inducibly expressed in the skin in a strictly tetracycline-dependent manner. In the thymus Ova was constitutively expressed in a tet-independent manner, leading to partial deletion of endogenous DO11 as well as induction of high percentages of natural Tregs. Interestingly, upon antigen-induction, mice developed an inflammatory dermatitis which was associated with IFN-γ and IL-17 production by DO11 T cells. This disease developed despite the presence of high starting numbers of natural DO11 Tregs, and, in fact, the Tregs proliferated in response to skin-Ag recognition. A remarkable feature of the skin disease was that it resolved spontaneously. This resolution correlated with changes in phenotype and, apparently, functional activity of Foxp3+ DO11 Tregs. We are currently using this inducible Ag-expression model to define the dependence of regulatory and effector T cells on persistent Ag recognition.
Anti inflammatory role of IFN gamma in a viral model of encephalitis (115.21)
Authors: Ramakrishna Chandran , Shen Yuewei , Cantin Edouard
Published: January 2023

HSV1 induced encephalitis in susceptible mice such as 129 and BALB/c mice is characterized by CNS inflammation associated with pathogenic Ly6Chigh macrophages and neutrophils. Mortality in this model correlates with degranulation by these cells rather than virus mediated cytopathology. IVIG, composed of pooled human IgG protects these mice by induction of regulatory CD4 T cells including IL-10 secreting Tregs and Tr1 cells secreting IL-10 and IFNγ. Surprisingly, IFNγ has an anti-inflammatory role in this model as infection of IFNγKO mice results in severe early morbidity and mortality associated with unabated CNS inflammation by neutrophils and Ly6Chigh monocytes suggesting that IFNγ is critical in the control of expansion of these cells. Interestingly, IVIG cannot protect IFNγKO mice despite control of infectious virus but not CNS inflammation typified by the inflammatory Ly6Chigh macrophages, indicating that IFNγ is crucial to IVIG’s anti-inflammatory activities. Adoptive transfer of T cells subsets from either WT or IFNγKO mice into Rag recipients reinforced the importance of IFNγ on CNS inflammation. Furthermore, IFNγ appears to have a role in the IVIG induced expansion of Tregs while in the absence of IFNγ, there was an increase in γδ T cells secreting IL-17, which may contribute to the early unmitigated expansion of the neutrophils and monocytes. IFNγ related effects on CEACAM1a and iNOS on monocytes, on Tregs, and the cell subset that secretes IFNγ will be discussed.
Emergence of polyclonal autoantibody responses to multiple platelet surface glycoproteins in regulatory T cell-deficient mice. (115.28)
Authors: Nishimoto Tetsuya , Satoh Takashi , Simpson Elisa , Ni Heyu , Takeuchi Tsutomu , Kuwana Masataka
Published: January 2023

Naturally occurring CD4+CD25+Foxp3+ regulatory T cells (Tregs) are known to play an important role in preventing pathogenic autoimmune responses. Recently, we have found that approximately one third of mice deficient of CD4+CD25+Foxp3+ Tregs spontaneously develop thrombocytopenia with increased platelet-associated IgG, analogous to patients with immune thrombocytopenia. To identify platelet autoantigens in Treg-deficient mice, plasma samples from thrombocytopenic and non-thrombocytopenic Treg-deficient mice were subjected to immunoblots using platelets as an antigen in reduced and non-reduced conditions. IgG antibodies to platelet surface glycoproteins, such as GPIb, GPIIb, and GPIIIa, were detected specifically in thrombocytopenic mice in various combinations. IgG antibodies to native platelets were further assessed by flow cytometry using platelet eluates and splenocyte culture supernatants. IgG antibodies capable of binding to native platelets were detected in thrombocytopenic, but not in non-thrombocytopenic mice. Antibody reactivity was cancelled in the majority of samples when platelets derived from GPIb-deficient or GPIIIa-deficient mice were used instead of wild-type platelets as an antigen. In summary, Treg deficiency alone leads to emergence of polyclonal IgG autoantibody responses to native and denatured platelet surface glycoproteins, supporting a critical role of CD4+CD25+Foxp3+ Tregs in suppressing induction of autoimmunity.
Dendritic cells initiate early autoimmunity in the absence of functional regulatory T cells (115.7)
Authors: Hoyer Katrina , Isakson Sara , Katzman Shoshana , Abbas Abul
Published: January 2023

BALB/c IL-2-KO mice develop systemic autoimmunity, dying within 4 wks from autoimmune hemolytic anemia. Disease in these mice is Th1-mediated, and IFNγ production is required for early autoimmunity. We asked whether APC are required for IFNγ production by T cells in the IL-2-KO mouse. Our data show that APC:T cell interactions through B7, but not via B cells, are necessary for IFNγ production in the absence of IL-2, suggesting that dendritic cells (DC) mediate this alteration. Disease is marked by DC accumulation, activation and elevated cytokine production, including IL-12 and type1-interferons. The depletion of either conventional (cDC) or plasmacytoid (pDC) significantly augmented the survival of IL-2-KO mice, demonstrating that DC contribute to the progression of autoimmunity. In the absence of IL-12, T cell activation and disease is only moderately altered, thus Th1 differentiation and IFNγ induction as a part of the underlying kinetics of autoimmunity is primarily IL-12-independent. Elimination of type 1 interferon signals in addition to IL-12 further augmented survival, indicating that cytokines derived from both pDC and cDC contribute to disease severity. Enhanced DC function is not dependent on T cell activation as DC in an environment without overt T cell activation are still functionally activated. Overall, our data suggest that DC activation can be an initiating event during the development of disease and thus DC are critical regulators of autoimmune development.
Characterization of antigen-specific CD4+CD25+ regulatory T-cells expanded with IL-2/IL-2mAb complexes in NOD mice (115.9)
Authors: MANIRARORA Jean , Wei Cheng-Hong
Published: January 2023

Type-1 diabetes is an autoimmune disease caused by the destruction of insulin-producing pancreatic beta cells by auto-reactive T-cells. It results from a failure of the immune system to maintain tolerance to self-antigens. CD4+CD25+ regulatory T-cells represent one major mechanism by which auto-reactive T-cells are controlled in the periphery. Deficiency in this cell population number or function could tilt the regulatory cell-pathogenic cell balance and lead to type-1 diabetes development. In this study, we describe a new method to expand antigen-specific CD4+ CD25+ regulatory T-cells from autoimmune-prone non-obese diabetic mice. CD4+ CD25+ regulatory T-cells were expanded in vivo using a combination of IL-2, anti-IL-2 antibody, BDC 2.5 peptide, and rapamycin. The expanded CD4+ CD25+ regulatory T-cells expressed a classical phenotype, displayed suppressive functions in vivo, and preliminary data suggest that these cells can delay diabetes development in NOD mice. This new method of peripheral tolerance induction could be used in humans to promote tolerance to islet cell transplants without relying on immunosuppressive drugs.
BCAP negatively regulates TLR signaling in macrophages (116.13)
Authors: Ni Minjian , MacFarlane Alexander , Campbell Kerry , Lowell Clifford , Hamerman Jessica
Published: January 2023

B cell adaptor for phosphoinositide 3-kinase (BCAP) was originally identified as an adaptor molecule that binds to the p85 subunit of phosphatidylinositol 3-kinase (PI3K). BCAP-deficient mice have decreased numbers of mature B cells and attenuated B cell function due to defects in B cell receptor signaling. BCAP is also expressed in macrophages and activates PI3K downstream of Toll-like receptor (TLR) ligation. Here we investigated the responses of BCAP-deficient mice to stimulation through TLRs. BCAP-deficient macrophages produced higher concentrations of inflammatory cytokines in response to a variety of pathogenic stimuli in vitro. Additionally, BCAP-deficient mice produced more IL-12 in response to LPS in vivo. The TLR mediated Akt activation is impaired in BCAP-deficient macrophages. Furthermore, the PI3K inhibitor wortmannin enhanced TLR mediated proinflammatory cytokine production in wild type but not in BCAP-deficient macrophages. In B cells, YXXM motifs of BCAP are required for BCR induced PI3K activation. Surprisingly, we found that YXXM motifs of BCAP are only partially responsible for the BCAP mediated negative regulation of TLR signaling in macrophages. BCAP is constitutively phosphorylated and associated with PI3K in resting macrophages, and, unlike in B cells, does not require Syk for its phosphorylation. Taken together, our findings show that BCAP negatively regulates TLR signaling in macrophages partly through regulation of TLR-mediated PI3K activation.
Induction of endotoxin tolerance in vivo inhibits LPS-inducible activation of IRAK4, p38 and NF-κB, decreases expression of pro-inflammatory cytokines and induces negative regulators of TLR4 signaling (116.14)
Authors: Xiong Yanbao , Medvedev Andrei
Published: January 2023

Toll-like receptors (TLRs) are conserved sensors of microbial pathogens and environmental or host-derived “danger” molecules. Development of TLR tolerance in septic patients re-programs monocyte responses to LPS, decreasing expression of pro-inflammatory cytokines without inhibiting anti-inflammatory and antimicrobial mediators, and increases a risk of secondary infections. To address the impact of endotoxin tolerization on activation of proximal TLR4 signaling pathways and expression of regulators of TLR4 signaling, we used an in vivo model of endotoxin tolerance. C57BL/6 mice were i.p. injected with LPS or PBC (used as a control), followed by in vitro challenge of isolated macrophages with medium or LPS and analyses of TLR4 signaling outputs and negative regulatory molecules. Compared to the control group, administration of LPS led to decreased TLR4-driven induction of IRAK4 kinase activity, degradation of IκB-α and phosphorylation of p38, and reduced expression of TNF-α, IL-6, and KC mRNA in peritoneal and splenic macrophages upon in vitro LPS challenge. In contrast, real-time PCR analyses revealed increased expression of IRAK-M, Tollip, SHIP, SARM, and SIKE in macrophages obtained from LPS-injected mice. We conclude that induction of endotoxin tolerance in vivo inhibits expression of pro-inflammatory cytokines and chemokines via impaired activation of IRAK4, p38 and NF-κB and up-regulation of negative regulators of MyD88- and TRIF-dependent pathways.
NF-κB activation is critical for bacterial lipoprotein tolerance-enhanced bactericidal activity in macrophages during microbial infection (116.15)
Authors: Liu Jing Hua , Jiang Yong , Redmond H. Paul , Wang Jiang Huai
Published: January 2023

Tolerance to bacterial cell-wall components including gram-positive bacterial lipoprotein (BLP) represents an essential regulatory mechanism during bacterial infection. Our previous work has demonstrated that BLP-induced tolerance, characterised by hyporesponsiveness in producing proinflammatory cytokines and simultaneously enhanced antimicrobial functions, protects against microbial sepsis-related lethality. However, the underlying mechanisms remain unidentified. In this study we reported that upon S. aureus or E. coli challenge, significantly enhanced IκBα phosphorylation and p65 translocation into the nucleus were observed in BLP-tolerised bone marrow-derived macrophages (BMM), indicating that NF-κB is activated in BLP-tolerised macrophages during bacteria infection. To further clarify whether activation of the NF-κB pathway is required for efficient bacterial killing by BLP-tolerised macrophages, we used two NF-κB inhibitors, SN50 and SC-154. Both inhibitors substantially restrained BLP-tolerised macrophage-induced intracellular bacterial killing, and this was closely associated with delayed phagolysosome fusion. Furthermore, the expression of LAMP-1 and Rab5, two membrane-trafficking regulators participated the process of phagosome maturation was significantly downregulated when the NF-κB pathway was blocked. Collectively, our results highlight a novel role of the NF-κB pathway in bactericidal activity displayed by BLP-tolerised macrophages during microbial infection.
A carboxyl terminal loop structure is the client-binding domain of gp96 (Hsp90B1, Grp94) in chaperoning Toll-like receptors and integrins (116.22)
Authors: Wu Shuang , Gewirth Daniel , Li Zihai
Published: January 2023

gp96, known also as grp94 and HSP90b1, is a molecular chaperone for Toll-like receptors (TLRs) and integrins. Unlike its cytosolic counterpart HSP90, the structure-function relationship of gp96 remains obscure. In particular, the client-binding domain (CBD) of gp96 has not been identified, although a C-terminal loop structure of gp96 may fulfill such a function from a recent crystallographic study. Herein, we provide genetic and biochemical evidence to definitively demonstrate that this loop structure is the CBD of gp96 for TLRs and integrins. Deletion of the CBD completely abolishes the chaperoning ability of gp96. Further fine mapping of this region demonstrates critical residues within the CBD for the folding of integrins but not TLRs. Moreover, we demonstrate that the binding of TLR9 to CBD is dependent on the C-terminal dimerization domain but not the N-terminal ATP-binding pocket of gp96. As both TLRs and integrins play important roles in inflammation, sepsis, autoimmunity and cancer, the identification of the CBD of gp96 in their folding may facilitate the design of gp96-selective inhibitors for the treatment of a variety of pathological conditions.
Toll-like receptors and NOD-like receptors in otitis media with effusion (116.24)
Authors: Yeo Seung Geun , Lee So Yoon , Park Dong Choon
Published: January 2023

Objectives; Bacterial infection in a normally sterile environment of the middle ear cavity usually triggers host immune response which innate immune system plays a dominant role as host’s first line of defense mechanism. Here, we evaluated pediatric patients with otitis media with effusion (OME) in order to assess expression levels of Toll-like receptors (TLRs) -2, -4, -5, -9, and nucleotide-binding oligomerization domain-containing proteins (NODs) -1 and -2 which are all related to bacterial infection. Methods; The study population consisted of 46 pediatric patients with OME, of whom all had ventilating tubes inserted. The expression levels of TLR-2, -4, -5, -9, NOD-1 and -2 mRNA in middle ear effusion were assessed by performing real-time PCR. Results; All effusion fluid samples collected from patients with OME showed expressions of TLR-2, -4, -5, -9, NOD-1, and -2 mRNA. However, we found no significant correlation among expression levels of pattern recognition receptors (PRRs) in relation to characteristics of exudates, presence of bacteria, comorbidity with allergic rhinitis, and frequencies of ventilation tube insertion (p>0.05). Conclusion; This finding suggests that expression of PRRs during bacterial infection may possibly be associated with the development of OME in children.
RLR signaling is amplified by reactive oxygen species and dysfunctions in autophagy (116.25)
Authors: Tal Michal , Sasai Miwa , Iwasaki Akiko
Published: January 2023

Infected cells utilize the RIG-I-like receptors (RLRs) to recognize the presence of RNA viruses in the cytosol. RLRs signal through IPS-1, which is localized to the mitochondrial membrane, resulting in the production of pro-inflammatory and antiviral cytokines. We are interested in the functional implications for the cellular localization of RLR signaling as it is clear that this is a point of intersect for important cellular processes and antiviral defense. We show that pro-inflammatory cytokine production in response to viral ligands is increased in several situations in the absence of autophagy due to increased levels of oxidative stress. The process of autophagy maintains the integrity of cellular organelles such as mitochondria and long-lived proteins by transporting them to the lysosomes for degradation. We have examined the role of defective autophagy and aging on cytosolic antiviral signaling using several ssRNA viruses as well as the dsRNA analog Poly I:C. We found that autophagy contributes to homeostatic regulation of innate antiviral defense through the clearance of dysfunctional mitochondria, and revealed that ROS associated with mitochondria play a key role in potentiating RLR signaling. We also present that RLR mediated cytokine production is increased in a human physiological condition that is known to involve decreased autophagy and increased oxidative stress.