bluuebunny/crossref_metadata_2025 huggingface dataset:
CD4+ T regulatory and effector cells display a similar wide range of affinities for self during autoimmune disease (BA13P.129)
Authors: Hood Jennifer , Evavold Brian
Published: January 2023

The affinity of a T cell receptor (TCR) from a regulatory T cell (Treg) for endogenous self peptide MHC complex is unknown. It is thought that thymic derived Tregs (nTregs) are enriched for high affinity, self-reactive TCRs as an alternative to negative selection. During experimental autoimmune encephalomyelitis (EAE), MOG self-antigen pMHC tetramers identified few (<10%) of the infiltrating CD4+ Tregs and Teffs. The micropipette adhesion frequency assay has previously proven more sensitive for assessing TCR specificity, affinity and frequency of antigen specific cells. Using this assay, it was determined that most of the Tregs and Teffs (>80% each) localized in the central nervous system (CNS) at peak EAE are in fact MOG self antigen specific. Tregs and Teffs possessed similar broad 1000 fold ranges in affinities for MOG with geometric means of 1 and 4 X 10-5 μm4. In the CNS, greater than 90% of the Tregs expressed helios which was consistent with frequencies in the thymus (94%), which suggests an enrichment of nTregs in the CNS during peak EAE. Contrary to the hypothesis that nTregs arise as an alternative to negative selection due to a high affinity for self, our data show that TCR affinity of MOG specific Tregs mimics Teffs with no enrichment of high affinity cells.
Prolonged exposure to type-I IFN inhibits IL-10 signaling in memory and regulatory T cells: a new mechanism contributing to the development of autoimmune diseases (BA13P.130)
Authors: Raimondi Giorgio , Nicholson Yawah , Chen Leo
Published: January 2023

Interleukin 10 (IL-10) is a fundamental immunoregulatory cytokine. Mice with memory T cells (Tmem)- or regulatory T cells (Treg)-specific depletion of the IL-10 receptor (IL-10R) develop fatal autoimmune diseases. However, no correlation is known between pathological conditions and altered IL-10 signaling in T cells. We investigated the influence of prolonged exposure to the supernatant of mouse maturing dendritic cells, exposed to LPS, on the integrity of the IL-10 signaling pathway in Tmem and Treg. Following 36-48h incubation, a block in phosphorylation of STAT3 in response to IL-10, but not in response to IL-6, was evident. This IL-10-specific unresponsiveness was not associated with lower IL-10R expression. Additionally, this inhibition was not induced by IL-6 or IL-1. Instead, incubation with IFN-β impaired IL-10 signaling in both Tmem and Treg. This effect was not blocked by AKT inhibition, suggesting a role for Jak/STAT or other IFN-β-initiated signaling pathways. In NOD mice, a model of human type-1 diabetes (T1D), diabetes development is linked to high levels of type-I interferons in pancreatic lymph nodes (PNC-LN). We analyzed the IL-10 response in T cells from spleen, PNC-LN, other LNs, and pancreata of NOD mice and discovered that starting at 4 weeks of age, Tmem and Treg in PNC-LN and pancreata display suppressed responses to IL-10. Overall, these data reveal a new molecular mechanism that controls the development of autoimmunity and the pathogenesis of T1D.
Pathogenicity of autoreactive Th17 cells is dependent on T-bet expression in non-T-helper cells in experimental autoimmune encephalomyelitis (BA13P.117)
Authors: Kwong Brandon , McGavern Dorian , Lazarevic Vanja
Published: January 2023

Experimental autoimmune encephalomyelitis is a murine model for multiple sclerosis in which autoreactive CD4+ T-helper cells infiltrate the central nervous system (CNS) and initiate autoimmune inflammation. Previous studies have shown that EAE induction via the adoptive transfer of CNS antigen-specific Th17 cells requires T-cell-intrinsic expression of the transcription factor T-bet, but the potential pathogenic role of T-bet expression in non-T-helper cells remains unclear. Using T-bet-ZsGreen reporter mice, we confirmed that multiple non-T-helper cell populations express T-bet in the CNS during active EAE. To assess the pathogenic contribution of T-bet in non-Th cells, we transferred 2D2-transgenic (expressing a CNS antigen-specific TCR) T-bet-/- or WT Th17 cells into T-bet-/- or WT recipient mice. Consistent with previous reports, 2D2 T-bet-/- Th17 cells did not induce significant levels of EAE disease in either T-bet-/- or WT recipient mice, supporting the role for T-bet in CD4+ Th cells. Surprisingly, WT Th17 cells, while fully pathogenic in WT hosts, were unable to induce EAE in T-bet-/- hosts. Our preliminary data suggest that 2D2 WT Th17 cells were unable to breach the blood-brain barrier and infiltrate the CNS parenchyma due to ineffective local re-activation of 2D2 WT cells in the CNS of T-bet-/- recipients. Ongoing studies will seek to identify the role of T-bet expression in non-Th cell populations and their interactions with 2D2 Th17 cells in EAE induction.
VAV1 adaptor functions regulate both T cell polarization and susceptibility to immune mediated diseases (BA14P.211)
Authors: Kassem Sahar , Gaud Guillaume , Dejean Anne , Bernard Isabelle , Fournié Gilbert , Malissen Bernard , Colacios Céline , Saoudi Abdelhadi
Published: January 2023

Vav1 is a guanine nucleotide exchange factor (GEF) that is essential for signal transduction of the T cell receptor (TCR) and plays an important role in the development and activation of T cells. Our previous results identified a polymorphism in the Vav1 gene that impacts on its adaptor functions without notable effect on its enzymatic activity. To analyze the impact of this polymorphism on the susceptibility to immune diseases, we generated a knock-in (KI) mouse bearing an arginine (R) to tryptophan (W) substitution in the Vav1 protein (Vav1R63W KI). Using this model, we confirmed that Vav1R63W mutation had no impact on Vav1 enzymatic activity in CD4 T cells but decreased its adaptor function, as revealed by the reduction of TCR induced calcium flux and ERK/AKT activation. Moreover, we showed that Vav1R63W favors Th2 cytokine production by CD4 T cells. We next analyzed the susceptibility of these mice to immune-mediated diseases and showed that KI mice were less susceptible to MOG-induced central nervous system inflammation. This finding likely resulted from a reduced production of effector cytokines (IFN-γ, IL-17 and GM-CSF) by autoreactive CD4 T cells. Finally, we found an increased susceptibility of the KI mice to asthma features, consistent with enhanced Th2 polarization. Together, these data highlight the importance of Vav1 adaptor functions in T cell polarization and susceptibility to immune mediated diseases, with opposite effects on autoimmune and allergic diseases.
Epithelial lymphotoxin beta receptor regulates embryonic medullary thymic epithelial cell development for central tolerance (BA14P.212)
Authors: Wu Weiwei , Shi Yaoyao , Fu Yang-Xin , Zhu Mingzhao
Published: January 2023

Medullary thymic epithelial cells (mTEC) play critical roles in central tolerance. Given multiple cell types involved and complex cellular and molecular crosstalk, the mechanisms underlying mTEC development remain intriguing. Previous studies showed reduced mTEC number in lymphotoxin beta receptor (LTβR) knockout (KO) mice, but exactly how LTβR functions is still unclear. Here, by using conditional knockout (cKO) mice, in which LTβR is specifically depleted from thymic epithelial cells, we found dramatically reduced number of mTECs in LTβR cKO adult mice. Unexpectedly, further experiments did not find any homeostatic proliferation or apoptosis defect of mTEC in postnatal cKO mice at any stages. Further analysis of embryonic thymi interestingly found mTEC number is reduced while the number of progenitor of mTEC (mTECp) is comparable between WT and cKO mice, suggesting differentiation and/or expansion defects of mTEC in cKO mice. To further investigate the biological impact of epithelial LTβR deficiency, thymic expression of peripheral tissue restricted antigens and self-reactivity at periphery were determined in adult mice. Thymic negative selection was also directly addressed using TRAMP and SV40-TCR tg mice. Current data suggest significant impairment of thymic tolerance and autoimmune phenotype at periphery. Collectively, our study has revealed that thymic epithelial LTβR is essential for embryonic mTEC development and establishment of central tolerance in adult mice.
Indirect presentation of antigen-coupled cells mediates specific CD4+ T cell tolerance by PD-L1 for the prevention of type 1 diabetes (BA14P.207)
Authors: Fife Brian , Spanier Justin , Pauken Kristen , Heffernan James , Sahli Nathanael , Mitchell Jason
Published: January 2023

Antigen coupled to the surface of cells using ethylene carbodiimide is a potent inducer of tolerance in multiple models of autoimmunity including Type 1 Diabetes (T1D). Yet, the mechanisms contributing to the induction of T cell tolerance in vivo during T1D are unclear. We used the BDC2.5 T cell receptor transgenic T cell transfer model to study how p31-coupled cells inhibit T cell function in vivo. Antigen-coupled cells that were either deficient in MHC class II or MHC-mismatched induced tolerance similarly to MHC class II-sufficient matched cells, suggesting that indirect presentation of coupled cell antigens is required for tolerance induction in vivo. We also show that BDC2.5 T cells predominantly encounter coupled cell antigen that has been ingested by host antigen presenting cells, and that the inhibitory receptor PD-L1 is required on the host but not the coupled cells to induce tolerance, further highlighting an indirect mechanism of tolerance induction. These data add to our current understanding of how antigen-coupled cells induce tolerance and aid in the translation of this therapeutic into the clinic.
Bacillus infantis NRRL B-14911-induced myocarditis: a novel disease model to study the autoimmune mechanisms of myocardial injuries in the myocarditis-susceptible, A/J mice (BA15P.221)
Authors: Krishnan Bharathi , Massilamany Chandirasegaran , Smith Tim , Loy John , Barletta Raul , Reddy Jay
Published: January 2023

We recently discovered that Bacillus spp., NRRL B-14911, a marine microbe, has the ability to induce myocarditis through molecular mimicry. The bacterium contains a mimicry epitope designated, BAC 25-40, and it induces autoimmune myocarditis by generating cross-reactive T cells for cardiac myosin heavy chain-alpha 334-352 (Myhc). We have analysed the complete genome sequence leading us to identify this microorganism as Bacillus infantis NRRL B-14911, a close relative of the strain that has been isolated in sepsis of newborn infants. Conditions have been optimized for the bacterium to grow in a Zobell marine medium at 37oC in an aerobic environment. Our preliminary studies indicate that A/J mice infected with Bacillus infantis NRRL B-14911 show the generation of CD4 T cells that can react with both BAC 25-40 and Myhc, as determined by major histocompatibility complex class II/IAk tetramer staining. We propose that Bacillus infantis NRRL B-14911-induced myocarditis can serve as a useful disease model to study the immune-mediated mechanisms of bacterial myocarditis as they relate to molecular mimicry and/or epitope spreading.
Gender differences in the development of autoimmune myocarditis induced with cardiac myosin heavy chain-alpha, 334-352 in C57Bl/6 mice (BA15P.222)
Authors: Gangaplara Arunakumar , Massilamany Chandirasegaran , Steffen David , Reddy Jay
Published: January 2023

Various animal models of experimental autoimmune myocarditis (EAM) have been developed to understand the immune pathogenesis of myocardial injuries. Although, genetic susceptibility is one major predisposing factor in the causation of myocarditis, other factors like gender-bias can also influence the disease outcome. For example, A/J mice carrying IAk allele on ‘A’ background are highly susceptible to EAM induced with cardiac myosin heavy chain-alpha (Myhc); whereas C57Bl/6 mice on ‘B’ background possessing IAb allele are resistant. Whether these phenotypic differences are due to inherent variations in the presentation of immunodominant epitopes of cardiac antigens are not known. We addressed this question using Myhc 334-352 as a putative cardiac antigen that induces EAM in A/J mice. Unexpectedly, we discovered that C57Bl/6 mice immunized with Myhc 334-352 showed antigen-specific proliferative responses regardless of gender as determined by T cell proliferation assay and major histocompatibility complex (MHC) class II/IAb dextramer staining. However, in contrast to females, only male mice developed myocarditis which showed higher frequencies of CD4 T cells producing T helper 17 family cytokines namely, interleukin (IL)-17A and IL-22 but not IL-17F. The observation that both IAb and IAk alleles can present single peptide but with varied disease outcomes provides opportunities to dissect the influence of non-MHC genes and also sexual dimorphism on myocarditis-susceptibility.
Pyranopyran-1, 8-dione, an active compound from Viticis Fructus ameliorates cigarette smoke induced lung inflammatory response in a murine model (CAM5P.239)
Authors: Bae Hyunsu , Jung Kyung-Hwa , Kang Geun-Hyung , Kim Won Seob , Kim Hyun Seong , Hong Moochang , Kim Sun Kwang
Published: January 2023

Chronic obstructive pulmonary disease (COPD) is defined as poorly irreversible airflow obstruction due to the inhalation of noxious chemicals and gases, most commonly cigarette smoke (CS). The extract of Viticis Fructus (VF), is widely used as a traditional herbal medicine in various Asian countries for the treatment of migraine pain and allergic diseases. Therefore, we attempt to isolate active compound from VF. After several rounds of activity-guided fractionations, 1H, 8H-Pyrano [3, 4-c] pyran-1, 8-dione (PPY) was isolated. In this study, we hypothesized that PPY, constituent of VF, could suppress CS exposure lung inflammation in mice model. The mice were exposed to 3R4F reference cigarettes for 2 weeks with 6 cigarettes a day. The mice were administered various dose of PPY (1, 2, 10 mg/kg body wt) via an oral administration 2 hr prior to CS exposure. The results of this study indicate cellular profiles and histopathologic analysis demonstrated that peribronchial and perivascular inflammatory cell infiltrates were significantly decreased in the PPY treated groups when compared to the CS exposure group. In summary, the results of this study indicate that PPY has significantly inhibited CS induced lung inflammation. The remarkable anti-inflammatory effects of PPY suggest its therapeutic potential in COPD treatment. Keywords: COPD, 1H, 8H-Pyrano [3, 4-c] pyran-1, 8-dione (PPY), cigarette smoke, IL-6, TNF-α
15d-PGJ2 ameliorates the inflammatory process on rheumatoid arthritis-induced into rats’ temporomandibular joint (CAM5P.240)
Authors: Clemente-Napimoga Juliana , Quinteiro Mariana , Macedo Cristina , Freitas Fabiana , Napimoga Marcelo
Published: January 2023

The aim of this study was to evaluate the peripheral effect of 15d-PGJ2 in Rheumatoid Arthritis-induced inflammation in the temporomandibular joint (TMJ) of rats. Antigen-induced arthritis (AIA) was generated in rats with methylated bovine serum albumin (mBSA) diluted in complete Freund's adjuvant. Pretreatment with an intra-articular injection of 15d-PGJ2 (100 ng/TMJ) before mBSA intra-articular injection (10 µg/TMJ) (challenge) in immunized rats significantly reduced the RA-induced hypernociception (p<0.05; ANOVA, Tukey test). Corroborating these results, 15d-PGJ2 was able to inhibit plasma extravasation, leukocyte migration and the release of inflammatory cytokines TNF-α, IL-1β, IL-6, IL-12, IL-18 and the chemokine KC and CINC-1 (p<0,05: ANOVA, Tukey Test). Moreover, the effect of 15d-PGJ2 to increase the decay accelerating factor (CD55) expression associated to decrease pro-inflammatory cytokines release may explain the inhibition of leukocyte infiltration, through a shift in the balance of the pro- and anti-adhesive properties. To confirm these results histopathological analysis showed that 15d-PGJ2 reduced the inflammatory parameters induced by RA. Thus, 15d-PGJ2 might be used as a potential anti-inflammatory drug to treat RA inflammation of the temporomandibular joint.
Evolutionary origins and structural analysis of phosphatidylserine binding by the TIM gene family (CAM5P.244)
Authors: Jones Jennifer , Brandmaier Andrew , Umetsu Sarah , Bu Xia , Umetsu Dale , DeKruyff Rosemarie , Freeman Gordon
Published: January 2023

The T cell Immunoglobulin and Mucin-like domain (TIM) proteins are a family of receptors that bind phosphatidylserine (PS) and regulate adaptive and innate immune cell functions. We find that two TIM-specific motifs: a G(W/L/I/F)(F/M)ND (GWFNDor) sequence and four conserved cysteine residues in the FG and CC’ segments of the TIM IgV domain form the distinctive PS-binding pocket identified in TIM-PS co-crystal structures. Because BALB/c and C57BL/6 mice have significant polymorphisms in TIM-3 and TIM-1, we determined the haplotypes of immunologically diverse and genetically divergent mice. Despite a high degree of variation in the IgV domain and in the mucin like stalk, the GWFNDor motif is unmodified in all known polymorphisms of TIM-1, -3, and 4. We identified two genomic alterations that cause divergent expression profiles of TIM-1 in commonly studied mouse strains. A major polymorphism in mouse TIM-1 deletes 23 amino acids of the mucin domain, and is caused by a retroviral insertion in the C57BL/6 genome that prevents expression of part of the mucin domain. Genomic and phylogenetic analyses show that a sytenic locus encoding generally 3 TIMs (1, 3, and 4) is conserved from bony fish to primates, and with emergence of the GWFND-4 motif in the most primitive TIM loci in bony fish, and the TIM family occupies a distinct PS-binding branch among the family of V-domain receptors that modulate immune activation via lipid, glycan, or protein binding.
Administration of phospholipase D1 inhibitor attenuates aminoacetophenone-induced acute liver injury (CAM5P.245)
Authors: Kwon Soonil , Lee Sung Kyun , Lee Mingyu , Lee Ha Young , Kim Sang Doo , Bae Yoe-Sik
Published: January 2023

A phospholipase D1 (PLD1)-selective inhibitor, N-[2-[4-(5-chloro-2,3-dihydro-2-oxo-1H-benzimidazol-1-yl)-1-piperidinyl]-1-methylethyl]-2-naphthalenecarboxamide, strongly blocked aminoacetophenone-induced acute liver injury. Administration of 500 mg/kg aminoacetophenone strongly increased alanine transaminase levels in serum, which was markedly decreased by PLD1 inhibitor administration in a dose-dependent manner, showing almost complete inhibition by 4 mg/kg of the inhibitor. During the pathological progress of acute liver injury, GSH levels are decreased, which is recovered by the administration of PLD1 inhibitor. JNK mediates aminoacetophenone-induced oxidative stress and liver injury. PLD1 inhibitor administration strongly blocked JNK phosphorylation in aminoacetophenone-induced acute liver injury model. Several inflammatory cytokines including TNF-α, IFN-γ, CCL2, and IL-1β were strongly increased from liver in aminoacetophenone-induced acute liver injury, which were also markedly decreased by PLD1 inhibitor administration. Taken together, the results indicate that the PLD1 inhibitor strongly show therapeutic effects reverse the progression of acute liver injury, and PLD1 can be regarded as an important target molecule to develop therapeutic agents against acute liver injury.
Copper nanoparticles suppresses nitric oxide production in macrophages (CAM5P.234)
Authors: Arancibia Sergio , Barrientos Andrea
Published: January 2023

Copper and copper oxide nanoparticles (CuNP) have been utilized as anti-microbial agent, metal catalyst, diet supplement, high thermal conductive material, lubricant additive, among others. Although the uses of CuNP are relatively new, there is an increasing interest to know their immunotoxicology. CuNP have shown to induce one of the most potent acute inflammatory reactions among several types of nanoparticles. Recently, in a pulmonary infection model, it was demonstrated that mice, which had been previously exposed to CuNP, presented an impaired bacterial clearance. These effects were due to a reduction in macrophage function; however, the precise molecular mechanism underlying these effects is still unknown. Here, we described the effects of CuNP in the response of peritoneal macrophages against microbial products. Transmission electron microscopy (TEM) data showed that CuNP were rapidly internalized by macrophages. The internalization of the particles induced a significant reduction of cell viability in comparison with other nanoparticles. The challenge with CuNP inhibited LPS-mediated nitric oxide (NO) production in a dose dependent manner. These results showed that CuNP modulate the expression of a key inflammatory mediator that is crucial to eliminate bacterial infection.
Inhibitory effect of 5-Pyridin-2-ylmethylene-6,7-dihydro-5H-benzo[b]thiophen-4-one (PT-2-318) on NF-kB and AP-1 activation in macrophages (CAM5P.235)
Authors: Baek Seung-il , Lee Eung-Seok , Han Sang-Bae , Kim Youngsoo
Published: January 2023

TGF-beta activated kinase1 (TAK1) has been reported to play an essential role in pro-inflammatory cellular signaling pathway. Activated TAK1 phosphorylates IkB kinase β (IKKβ), leading to NF-kB activation. Nuclear factor-kB (NF-kB) is a ubiquitous transcriptional factor that controls the expression of genes involved in immune responses. Here, treatment with 5-Pyridin-2-ylmethylene-6,7-dihydro-5H-benzo[b]thiophen-4-one (PT-2-318) inhibited not only autophosphorylation of TAK1 but also acute liver injury of E.coli LPS/D-galactosamine-challenged mice. As a mechanism, PT-2-318 directly inhibited autophosphorylation of TAK1. So, phosphorylation of both IkB and MAPKs was inhibited. Also, LPS-induced NF-kB and AP-1 promotor activity in lipololysaccharides (LPS)-stimulated macrophages RAW 264.7 was inhibited by blocked TAK1. As TAK1 was blocked, PT-2-318 attenuated LPS-induced mRNA expression of cytokines (IL-1β, TNF-α). On the other hand, PT-2-318 did not inhibit phosphorylation of IRAK4 and degradation of IRAK1. in vivo, intravenous treatment with PT-2-318 protected against LPS/GalN-induced acute liver failure in C57/BL7 mice. These results suggest that PT-2-318 might be considered as a potential agents for the treatment of inflammatory diseases.
A novel population of myeloid Gr-1+ cells expressing FasL and perforin mediate contact hypersensitivity responses in skin (CAM5P.236)
Authors: Kish Danielle , Fairchild Robert
Published: January 2023

Allergic contact hypersensitivity (CHS) is a CD8 T cell mediated response to hapten sensitization and challenge of the skin. Hapten-primed CD8 cell infiltration into the skin challenge site requires prior Gr-1+ cell infiltration into the site. Despite WT levels of CD8 priming, sensitized gld/perforin-/- mice have no CHS to hapten challenge. The response is restored in these mice by transfer of WT bone marrow derived Gr-1+ cells at the time of hapten challenge. The goal of this study was to further characterize these Gr-1+ cells. Gr-1+cells were purified from the bone marrow of sensitized and challenged mice by depletion of CD3+, B220+, NK1+, F4/80+, and Ly6G+ cells. qRT-PCR and Western Blot analyses confirm expression of FasL and perforin by the negatively selected cells that are 90% Gr-1+CXCR2+. Flow cytometry analyses indicate the FasL+ cells within this population express CD11c, CXCR2, CCR2, Ly6C, and CX3CR1. These Gr-1+cells, but not neutrophils, induce apoptosis of cultured endothelial cells. Transfer of these Gr-1+ myeloid cells to sensitized gld/perforin-/- mice during hapten challenge induces expression of the T cell chemoattractants CCL1, CCL2, and CCL5 in the challenged skin. These results describe a novel population of bone marrow derived Gr-1+CXCR2+ myeloid cells expressing FasL and perforin that induce expression of the chemoattractants required to direct hapten-primed T cell infiltration into the challenged skin site to mediate CHS.
Blood monocytes expressing CCR6 and monocyte-derived dendritic cells in dermis and epidermis are critical for IL-23-induced psoriasis-like inflammation in mice (CAM5P.238)
Authors: Singh Tej Pratap , Zhang Howard , Farber Joshua
Published: January 2023

Human studies have implicated IL-23 in psoriasis, and injecting IL-23 in mouse skin produces IL-22-dependent, psoriasis-like inflammation. IL-23 injection leads to accumulation of dendritic cells (DCs), which are also abundant in psoriatic skin, and depleting CD11c+ cells blocked IL-23-induced IL-22 and inflammation. In analyzing myeloid cells, we found monocyte-derived langerin+ cells (mLCs) appearing in the epidermis and more of other monocyte-derived cells, including mDCs, plus non-monocyte derived DCs in the dermis after IL-23 injection. Batf3-/-, diphtheria toxin-treated Bdca2-DTR, and Flt3l-/- mice, which lack CD103+ cDCs, pDCs, and all cDCs and pDCs, respectively, each showed only modest protection against IL-23-induced changes. Although depleting conventional LCs alone had no effect, inflammation was much diminished in langerin-DTR mice also depleted of mLCs. Together, these data suggested that CD11c+ mDCs and mLCs contributed significantly to pathology. We have reported that Ccr6-/- mice are resistant to IL-23-induced pathology, and the skin of Ccr6-/- mice showed no changes in DCs after IL-23 injection. Using Ccr6-EGFP mice, we identified expression of Ccr6 in blood monocytes, and found that Ccr6+/+, but not Ccr6-/- monocytes restored inflammation when injected i.v. into Ccr6-/- mice. Together, our data suggest that blood monocytes are recruited to inflamed skin using CCR6 and give rise to mDCs and mLCs, which are critical for IL-23-induced psoriasis-like pathology.
Direct binding and inhibition of interleukin-2 with a small molecule, HH-236 (CCR5P.259)
Authors: Oh Jin-Gyo , Heo Tae-Hwe
Published: January 2023

Interleukin-2 (IL-2) is a growth factor for regulatory, activated and memory T cells. Thus, IL-2 plays a critical role in stimulation as well as suppression of immune responses. Recently, IL-2/anti-IL-2 antibody (Ab) complexes were found to significantly enhance the activity of IL-2, leading either to expansion of regulatory T cells or that of memory CD8+ T cells and NK cells. In this study, we had tried to discover a small molecule that regulates IL-2 activities and identified HH-236 as a direct IL-2 binder and inhibitor of the biological activity of IL-2. HH-236 competes with anti-mouse IL-2 Ab (S4B6) for IL-2 binding, however, unexpectedly promotes the interaction between IL-2 and IL-2 receptor-alpha, CD25. HH-236 neutralized the proliferative activity of IL-2 on the IL-2 dependent cell line, CTLL-2. In addition, anti-tumor activity of IL-2 in B16F10 melanoma mouse model was blocked by HH-236 co-treatment. Small molecule IL-2 modulators may have a potential to be used as an oral therapeutics for IL-2-related pathological conditions.
Involvement of CGRP in peripheral nerve injury-induced behavioral hypersensitivity and chemokine production (CCR5P.260)
Authors: Malon Jennifer , Cao Ling
Published: January 2023

Previously, we have observed increased expression of Calcitonin Gene Related Peptide (CGRP), a pro-nociceptive neuropeptide, along with selected cytokines and chemokines, in the lumbar spinal cord following spinal nerve L5 transection (L5Tx, a murine model of neuropathic pain) in BALB/c mice. To further study the effects of CGRP in neuropathic pain, we intrathecally (i.t.) injected a CGRP antagonist (CGRP8-37) into BABL/c mice following L5Tx. Mice injected with CGRP8-37 displayed a reduced L5Tx-induced mechanical hypersensitivity, but not heat hypersensitivity. CGRP8-37 also caused significant reduction of the level of lumbar spinal cord CCL5 (RANTES) post-L5Tx when compared to vehicle injected-mice and non-injected mice. i.t. injection of a CCL5 neutralizing antibody significantly inhibited L5Tx-induced mechanical hypersensitivity. To test whether CGRP played its pro-nociceptive role through stimulating glial response, mixed glial cells were treated with various doses of CGRP. CGRP induced a slight increase of CCL5 24 hours post-treatment, which was correlated with the increased expression of MAP Kinase (MAPK) signaling pathway molecules: p38 and JNK. Thus, CGRP may promote MAPK-mediated glial activation and the subsequent production of CCL5, which potentiate behavioral hypersensitivity following L5Tx.
Meprin α mediates CCL2 activity in murine bone marrow cells using a model of breast cancer chemotherapy (CCR5P.262)
Authors: Crisman Jackie , Sena Maria , Ishmael Faoud , Gerwitz Andrew , Marshall Joseph , Ferry Michelle , Vandervoort Brittany , Salamone James
Published: January 2023

Meprins metalloproteases are comprised of hetero or homo-oligomers of α and β subunits. These proteases degrade extracellular matrix proteins and have been linked to cancer cell metastasis and leukocyte transmigration. Meprins are also known to cleave cytokines like interleukin-1 and cytokine receptors like the epidermal growth factor receptor, thereby modifying their activity. Thus the expression of meprins in a tissue can have a profound impact on cytokine activity in tissue microenvironments. Breast cancer metastases to bone marrow often elude the apoptotic effects of chemotherapy. One potentially important chemokine is CCL2, which supports the chemotaxis of cancer cell and monocytes that support metastatic disease. Additionally, patients with elevated levels of CCL2 in tissue microenvironments and serum have a poor prognosis. Herein, data is presented supporting the role of meprin α in the inactivation of chemokines by N-terminal truncation, including CCL2 (monocyte chemoattractant protein-1), CCL5 (RANTES), CCL3 (macrophage inflammatory proteins-1α) and CCL4 (macrophage inflammatory proteins-1β). Additionally, while the abundance of MCP-1 transcripts increased after induction of bone marrow apoptosis, the abundance of meprin α transcripts decreased below detection. These data indicate that apoptosis induced by chemotherapeutic agents can increase CCL2 activity in bone marrow due to a decline in meprin α, thus supporting cancer cell growth in bone marrow.
A novel recombinant chemokine protein TAT-SDF-1/54-KDEL for blocking CXCR4: bioinformatics analysis and prokaryotic expression (CCR5P.263)
Authors: Chen Hongyuan
Published: January 2023

To explore the potential application of SDF-1/54 mutant in Immunology, a prokaryotic vector pTAT-HA was exerted to express a novel recombinant protein ,TAT/SDF-1/54/KDEL, which contains three distinct functional domains: murine SDF-1/54, a 11-peptide TAT (47-57) derived from human immunodeficiency virus tat trans-activator protein and an endoplasmic reticulum retention four-peptide sequence KDEL.Bioinformatics analysis showed that the estimated half-life of the protein in mammalian reticulocytes,yeast,Escherichia coli in vitro are 2.8h, 10h and 2min. Respectively.Endoplasmic reticulum distribution and localization ratio is 13.0%. The data also shows the novel protein on the secondary structure and topology proteins , protein-binding sites, the three-dimensional map and a possible prediction catalytic pocket and the binding sites. Our expression experiment showed that the expected band of the protein appeared in the precipitate by SDS-PAGE electrophoresis. Western blot confirmed the specificity of the immune response of this protein to the rabbit anti-His-probe antibody.These suggest that the bioinformatics analysis for TAT/54R/KDEL might be an effective approach to predict its structure and functional expression. The expression of the protein in pTAT-HA prokaryotic vector is in the form of inclusion bodies. It offered a new method targeting for CXCR4 implicated immunity regulation,such as HIV, WHIM syndrome, cancer and stem cells mobilization and so on.
Leptin induction of aggrecanases-1 and -2 genes expression in human chondrocytes is mediated by p38 mitogen-activated protein kinase pathway (CCR5P.265)
Authors: Hatipoglu Omer , Yaykasli Kursat , Dogan Mustafa , Yaykasli Emine , Kaya Ertugrul , Ozsahin Mustafa , Uslu Mustafa
Published: January 2023

The cloning of leptin initiated to explore new functions of adipose tissue. The studies revealed that adipose tissue like endocrine organ involves in critical roles by secreting adipokines. Among them, leptin is the most studied and characterized adipokine, and involves in several pivotal processes including inflammation. The degradation of articular cartilage components by aggrecanases-1 and -2 (called also a disintegrin and metalloproteinase with thrombospondin motifs -4 and -5) is a key event for arthritic diseases. The catabolic effect of leptin on articular cartilage degradation by increasing aggrecanases was established, but the pathways involved in this upregulation have not been elucidated. It was aimed to investigate putative molecular pathways involving in the upregulation of aggrecanases by leptin. The cultured human articular chondrocytes were incubated with SB203580 (a specific inhibitor of p38) and subsequently recombinant human leptin. At the end of the 48 hours, total RNA was extracted. The mRNA was reverse-transcribed into cDNA and the expression levels of aggrecanases were analyzed using real-time polymerase chain reaction. The leptin induced aggrecanases expressions were suppressed by SB203580. The elucidation of underlying pathways for aggrecanases upregulation may promote the new approaches for the treatment of arthritic diseases. It can be concluded from our result, p38 MAPK might be one of the potential target for cartilage degradation caused by leptin.
Regulation of IL-4-induced IRS-2 activation by serine phosphorylation. (CCR5P.248)
Authors: Warren Kristi , Gowda Nagaraj , Thompson Joshua , Heller Nicola
Published: January 2023

Allergic asthma is characterized by acute inflammation of the airways with alternatively activated (M2) macrophages as key regulators of disease severity. Robust, yet transient tyrosine phosphorylation (pY) of IRS-2 in response to IL-4 leads to enhanced M2 gene activation. Mechanisms for downregulation of IL-4-induced IRS-2 signaling have not been determined although serine phosphorylation (pS) of the IRS proteins terminates insulin-activated IRS signaling. We hypothesize that serine phosphorylation of IRS-2 plays a role in inhibiting IL-4-induced responses. Human monocytic U937 cells were stimulated with IL-4 ± calyculin A, a Ser/Thr phosphatase inhibitor. In separate experiments, U937 cells and bone marrow derived macrophages (BMM) were stimulated with IL-4 and four kinase (JNK, GSK-3, TORC1, and TORC2) inhibitors. The amount of total IRS-2, pY-IRS-2 and pS-IRS-2 was determined. pY-IRS-2 inversely correlated with pS-IRS-2 following IL-4 stimulation. Calyculin A prevented pY-IRS-2, while increasing pS-IRS-2, thus diminishing the IL-4 signal. Rapamycin treatment prolonged pY-IRS-2 in U937 cells and BMM in a similar manner. More specifically, TORC2 inhibition did not prolong IRS-2 activation. These results suggest that inhibiting TORC1 activity prolonged pY-IRS-2 by diminishing pS-IRS-2. Understanding how IRS-2 signaling is regulated is crucial for finding novel targets to suppress the differentiation of M2 macrophages and lower severity of allergic lung inflammation.
IFN-γ controls IL-33 protein level through a STAT1- and LMP2-dependent mechanism (CCR5P.268)
Authors: Kopach Pavel , Lockatell Virginia , Todd Nevins , Luzina Irina , Atamas Sergei
Published: January 2023

IL-33 levels and activity are differentially associated with Th1 and Th2 phenotypes. IL-33 mRNA is rapidly regulated but the fate of synthesized IL-33 protein is unknown. To assess the interplay between IL-33, IFN-γ, and IL-4 proteins, replication-deficient adenoviruses (AdV) were constructed for dual expression of IL-33 & IFN-γ or of IL-33 & IL-4. Combined IL-33 & IFN-γ or IL-33 & IL-4 effects were compared with similar AdV expression of each of these cytokines alone. Co-delivery of IL-33 & IFN-γ led to mutual suppression, and co-delivery of IL-33 & IL-4 led to mutual elevation of these proteins in cell culture and in vivo. Purified IFN-γ also attenuated IL-33 protein but not mRNA driven by the recombinant CMV promoter in AdV-IL-33-infected cells, suggesting that IFN-γ controls IL-33 protein degradation. Pharmacological inhibition, siRNA-mediated silencing, or gene deficiency of STAT1 potently upregulated IL-33 protein expression levels and attenuated the downregulating effect of IFN-γ on IL-33. Inhibition of caspase-1, -3, or -8 had minimal effect on IFN-γ-driven IL-33 protein downregulation. siRNA-mediated silencing of LMP2 proteasome subunit, which is known to be essential for IFN-γ-regulated antigen processing, also abrogated the effect of IFN-γ on IL-33. Thus, IL-33, IFN-γ, and IL-4 are engaged in a complex interplay, part of which involves IFN-γ-activated, caspase-independent, STAT1-mediated degradation of IL-33 protein through non-canonical use of LMP2 proteasome.
C-kit/SCF autocrine loop in human NK cells (CCR5P.254)
Authors: Zakiryanova Gulnur , Karpova Oxana , Khamdiyeva Ozada , Kopytina Dilyara , Urazalieva Nataliya , Kustova Elena , Biyasheva Zarema
Published: January 2023

Expression of c-kit receptor on CD56bright subset of NK cells has been previously reported. Based on the fact that c-kit receptor and its ligand - stem cells factor (SCF), are co-expressed on many cell types, we have tested the hypothesis that c-kit and SCF might be co-expressed on human peripheral blood NK cells using RT-PCR, flow cytometry and DNA sequencing. NK cells were harvested using the immunomagnetic NK isolation kit and their purity was confirmed by flow cytometry. Analysis of RT-PCR data revealed the presence of two DNA fragments (about 330 bp and 420 bp) in NK cells. Identity of PCR products was directly confirmed by sequencing. To determine the specificity of SCF transcript formation only in c-kit-positive cell subset, NK cells were separated on c-kit-positive and c-kit-negative subsets using CD117 microbeads. No SCF mRNA expression was found in c-kit-negative NK cells even after increasing the quantity of total RNA to 2 μg and the number of amplification cycles to 40. Intracellular flow cytometry analysis with specific anti-SCF antibodies confirmed the expression of SCF protein in human NK cells. Thus, our results provide a definite proof of the existence of c-kit/SCF autocrine loop in c-kit-positive subset of human peripheral blood NK cells. The verification of the functional significance of this new phenomenon is in progress in our laboratory.
Lack of exacerbated atherosclerotic lesions in apolipoprotein E-inhibitory FcγRIIb deficient mice is associated with increased anti-inflammatory cytokine response (CCR6P.269)
Authors: Nagarajan Shanmugam , Zhu Xinmei , Ng Hangpong
Published: January 2023

Functionally FcγRs can be classified into the activating (CD64, CD16 and FcγRIV) and inhibitory (FcγRIIb) receptors. Previous finding from our lab and others have shown that deletion of activating FcγR in apoE knockout (apoE KO) mice decreased atherosclerosis. This led to our hypothesis that deficiency of inhibitory FcγRII (CD32b) exacerbates atherosclerosis. We tested this hypothesis using apoE-CD32b double knockout mice congenic to C57BL/6 background (apoE-CD32bB6 dKO). On the contrary to our hypothesis, compared to apoE KO mice apoE-CD32bB6 dKO mice did not show exacerbated atherosclerotic lesions. Our finding is in contrast to previous reports showing exacerbated lesions in apoE-CD32b dKO mice in a mixed background (129s/v and C57BL/6, CD32b129/B6). Molecular mechanisms to understand the difference revealed that CD32b129/B6 mice showed more Th1 and Th17 response, while congenic CD32bB6 showed predominant anti-inflammatory Th2 and Treg responses. Moreover, macrophages from CD32b129/B6 mice showed more IL-1β response in response to LPS or IC, while macrophages from CD32bB6 mice showed more IL-10 response. These findings revealed a novel paradigm that inhibitory CD32b does not directly contribute to atherosclerosis. As CD32b polymorphism has been implicated in lupus susceptibility, the role of CD32b, as pro-atherosclerotic mediators in vivo in lupus-associated cardiovascular disease need to be investigated.
IL-6 deletion attenuates psoriasis-like skin inflammation in K5-IL-17C mice. (CCR6P.270)
Authors: Ward Nicole , Klenotic Philip , Diaconu Doina , McCormick Thomas , Johnston Andrew
Published: January 2023

IL-17C is the most abundant IL-17 family member found in lesional psoriasis skin. IL-17C elicits a 4-fold increase in TNFα (P<0.05) from primary human endothelial cells (ECs) and primary human keratinocytes (KCs) stimulated with IL-17C/TNFα produce synergistic increases in psoriasis signature genes. Mice engineered to overexpress IL-17C in KCs (K5-IL-17C) develop a psoriasiform skin phenotype that improves upon TNFα inhibition. IL-17C also elicits a 3-fold increase in IL-6 from ECs (P<0.05), and IL-6 protein levels significantly increase in uninvolved and involved skin of K5-IL-17C mice (42% and 480%; P<0.05). We hypothesized that IL-6 synergizes with IL-17C to elicit chronic skin inflammation, akin to IL-17C/TNFα. To test this, we mated K5-IL-17C mice with IL-6 KO mice. K5-IL-17C-IL-6 KO mice develop less severe skin disease at 8-10 weeks of age vs K5-IL-17C mice (40% decrease in mouse PASI; n=15, P<0.05) and have significant decreases in serum TNFα (64%; n=4; P<0.035). Histological analyses of involved skin show no differences between K5-IL-17C vs K5-IL-17C-IL-6 KO mice, however analyses of uninvolved skin reveal a significantly thinner epidermis (2-fold decrease; P<0.001), decrease in dermal angiogenesis (27%; P<0.04), and decreases in CD4+ and CD8+ T cells and F4/80+ macrophages (30-51%; P<0.05) in K5-IL-17C-IL-6 KO vs K5-IL-17C mice (n=8/group). These data suggest that IL-6 may contribute to proinflammatory IL-17C signaling critical for sustained psoriasis pathogenesis.
A computational model for mechanistic investigation of negative feedback in Inerleukin-17 receptor signaling (CCR6P.274)
Authors: Sheehan Robert , Garg Abhishek , Gaffen Sarah , Faeder James
Published: January 2023

IL-17 is a pro-inflammatory cytokine that promotes autoimmunity and protects against certain pathogens. Here, we present a computational model of signaling downstream of the IL-17 receptor. To manage the complexity of the system we use a rule-based modeling approach, in which signaling proteins are modeled as structured objects and rules describe their biochemical interactions. The model includes 19 molecules and 46 reaction rules, which expand to a reaction network with 168 distinct chemical species - complexes and phosphoforms of the basic molecules - which are connected through a network of 5,145 unidirectional reactions. The model is defined and simulated using the BioNetGen software. This model encompasses major signaling components downstream of IL-17R, which activate NF-κB and in turn promote the production of pro-inflammatory cytokines. NF-κB also promotes production of A20, a deubiquitinating enzyme that inhibits NF-κB activation in multiple settings. We recently showed that A20 is a negative regulator of the IL-17 signaling pathway. By modeling A20 interactions with TRAF6 and IKK, we are able to recapitulate experimentally-observed oscillations in NF-κB-dependent A20 expression at both the mRNA and protein levels. Further expansion of the model, along with additional experimental work tracking the dynamics of signaling intermediates, will enable us to explore novel hypotheses about the regulatory mechanisms of A20 and other potential regulators of IL-17 signaling.
Noxious pain fibers drive interleukin-23 mediated psoriasiform skin inflammation (CCR6P.275)
Authors: Riol Lorena , von Andrian Ulrich , Ordovas-Montanes Jose
Published: January 2023

To protect against invading pathogens, the skin harbors specialized immune cells, including dermal dendritic cells (dDCs) and T cells, such as IL-17 producing γδT cells (γδT17) whose aberrant activation by IL-23 can provoke psoriasis-like inflammation. The skin is also innervated by peripheral nerves consisting of autonomic and sensory fibers. Interactions between the autonomic nervous system and immune cells in lymphoid organs are known to contribute to systemic immunity, but how peripheral nerves regulate cutaneous immune responses remains unclear. Here, we have exposed the skin of mice to, imiquimod (IMQ), which induces IL-23 dependent psoriasis-like inflammation. We show that a subset of TRPV1+ NaV1.8+ pain fibers is essential to drive this inflammatory response. Imaging of intact skin revealed that a large fraction of dDCs, the principal source of IL-23, is in close contact with pain fibers. Upon selective pharmacological or genetic ablation of nociceptors, dDCs failed to produce IL-23 in IMQ exposed skin. Consequently, the local production of IL-23 dependent inflammatory cytokines by dermal γδT17 cells and the subsequent recruitment of inflammatory cells to the skin were dramatically reduced. Intradermal injection of IL-23 bypassed the requirement for nociceptor communication with dDC and restored the inflammatory response. These findings indicate that nociceptors, by interacting with dDCs, regulate the IL-23/IL-17 pathway and control dermal immune responses.
Critical target cell-specific roles for IL-17 family cytokines/CIKS-mediated signaling in mouse models of skin and lung inflammation (CCR6P.276)
Authors: Siebenlist Ulrich , Claudio Estefania , Ha Hye-Lin
Published: January 2023

The inflammatory cytokine IL-17 has been strongly implicated in human psoriasis and in the imiquimod-induced mouse model of this disease, while the IL-17 family member IL-25 has been associated with allergic asthma in humans and with allergen-induced inflammation in mouse models. Both cytokines signal via the obligatory adaptor protein CIKS (a.k.a. Act1 or Traf3ip2). However what cells are targeted by these cytokines and to what end has not been clearly delineated in these disease contexts. We demonstrate that in imiquimod-induced psoriatic inflammation, IL-17 signals into keratinocytes to dysregulate their growth/differentiation. On the other hand, IL-17 signals into non-keratinocytes to promote cellular infiltration in this psoriasis model and to drive a positive feedback loop, resulting in increased production of IL-17. We further demonstrate that in a chronic house dust mite-induced asthma model, IL-25 uniquely contributes to generation of Th9 cells and to airway remodeling, at least in part by targeting dendritic-like cells. These findings reveal previously unappreciated, cell-type specific functions IL-17 cytokines that contribute to inflammatory diseases at barrier sites.
Chronic activation with a staphylococcal superantigen drives the expansion of CD4, CD8 double negative T cells and promotes multiorgan inflammation mimicking systemic lupus erythematosus in HLA class II transgenic mice. (HUM7P.306)
Authors: Rajagopalan Govindarajan , Tilahun Ashenafi , Chowdhary Vaidehi
Published: January 2023

CD4 and CD8 coreceptors play an important role in the development and maturation of αβ TCR+ T cells. However, T cells lacking CD4 and CD8 (double negative T or DNT cells) are present in the periphery and are significantly expanded in systemic lupus erythematosus (SLE) and Sjogren’s syndrome. The antigens that cause expansion of these DNT cells are unknown. As T cell activation by superantigens does not require coreceptors, we hypothesized that superantigens could activate DNT cells and contribute to autoimmunity. HLA-DQ8 and HLA-DR3 transgenic mice lacking both CD4 and CD8 were generated by intercrossing CD4 and CD8 single knockout mice. The CD4.CD8 double knockout (DKO) mice harbored a diverse repertoire of DNT cells in the periphery, albeit at reduced numbers compared to wild type (WT) mice. DNT cells from DKO mice responded robustly to the superantigen, staphylococcal enterotoxin B, but not to HLA restricted conventional antigens. Implantation of mini-osmotic pumps delivering 10 or 50 μg of SEB continuously over 7 or 28 days induced a lupus-like disease in WT as well as DKO DR3 and DQ8 mice. However, the immunopathology was significantly more pronounced in DKO mice. Immunostaining revealed extensive infiltration of multiple organs including lungs, liver and kidneys with CD3+ T cells in DKO mice. Overall, our study unraveled the role of CD4 and CD8 coreceptors in T cell development/function and the role of superantigens and DNT cells in the pathogenesis of lupus.