bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and characterization of a novel splicing variant of the MHC class I-related neonatal Fc receptor (FcRn) for IgG (93.9)
Authors: Ye Lilin , Tuo Wenbin , Liu Xindong , Zhu Xiaoping
Published: January 2023

The neonatal Fc receptor for IgG (FcRn), a MHC class I-related molecule, functions to transport maternal IgG into the fetus or newborn and protects IgG from catabolism. The mRNAs of several MHC class I-related molecules have multiple splicing variants. By cloning porcine FcRn cDNA from the intestinal epithelial cell line IPEC, two cDNAs were found: a long form of 1071 bp (pFcRn-L) and a short form of 795 bp (pFcRn-S). These mRNAs were also detected in porcine kidney epithelial cell line LLC-PK1, peripheral blood mononuclear cells, as well as a variety of porcine tissues by RT-PCR and Northern blot. Sequence analysis showed that pFcRn-L cDNA encodes the full-length pFcRn, whereas pFcRn-S cDNA represents a splicing variant of pFcRn mRNA, coding for a truncated pFcRn with deletion of 92 amino acids matching to the alpha2 domain of pFcRn-L. Recombinant pFcRn-L bound IgG at pH 6.0, but not at pH7.5. However, recombinant pFcRn-S bound IgG at both pH 5.0–6.0 and pH 7.5. Furthermore, the intracellular trafficking patterns of pFcRn-L and pFcRn-S exhibited remarkable differences. The pFcRn-L was expressed on the cell surface and mainly localized in the early endosomal compartment, whereas pFcRn-S was primarily found in late endosome/lysosome. In contrast to pFcRn-L, pFcRn-S trafficking to the lysosomal compartment is independent of porcine beta2m expression as assessed in a beta2m-deficient cell line FO-1. Therefore, the pattern of FcRn gene expression which is characterized by the complexity of RNA splicing further implies the functional complication of FcRn.
Properties of a SOCS-1 small molecule antagonist (94.1)
Authors: Waiboci Lilian W , Johnson Howard M , Martin James P , Ahmed Chulbul M
Published: January 2023

Suppressor of cytokine signaling 1 (SOCS-1) is a member of a small family of protein molecules that are regulators of cytokines that signal through JAK2 tyrosine kinase and STAT transcription pathways. SOCS-1 binds to the autophosphorylation site of JAK2 tyrosine kinase and blocks its ability to activate substrates such as STAT1-alpha both directly and by initiation of proteosomal degradation. Thus, SOCS-1 is an attenuator of positive immune regulators such as gamma interferon. We reasoned that a peptide corresponding to the autophosphorylation site of JAK2 in the phosphorylated state, pJAK2(1001–1013), would bind to induced SOCS-1 in cells and block its inhibitory effects on mediators of the immune response. We therefore synthesized pJAK2(1001–1013) with an attached palmitate residue for cell penetration and showed that it enhanced suboptimal IFN-gamma, blocked SOCS-1 induced inhibition of STAT3 phosphorylation in IL-6 treated cells, enhanced IFN-gamma activation site (GAS) promoter activity, and enhanced antigen specific proliferation. In a mouse model of lethal vaccinia virus infection, pJAK2(1001–1013) completely protected mice against an overwhelming lethal dose (106 pfu intranasal) of vaccinia virus. The small molecule SOCS-1 antagonist should be quite useful in enhancing the immune response against infectious agents and cancer.

Supported by NIH grants AI 056152 and NS051245 to HMJ.
Glucocorticoids Augment CXCR4-Mediated Signaling and Function Via the Activation of the Src Kinase, Lck (94.10)
Authors: Ghosh Manik C. , Collins Gary , Carter Arnell , Taub Dennis D
Published: January 2023

Dexamethasone is a synthetic member of the glucocorticoid (GC) class of hormones that possesses anti-inflammatory and immunosuppressant activity and is commonly utilized to treat chronic inflammatory disorders, severe allergies and other disease states. While glucocorticoids are known to mediate well-defined transcriptional effects via GC receptors, there is increasing evidence that GCs also initiate rapid non-genomic signaling events in a variety of cell types. Here, we report that dexamethasone appears to induce the phosphorylation of Lck and the activation of other down stream mediators in resting human T cells including p59Fyn, Zap70, Rac1, Cdc42 and Vav1. DM treatment also appeared to augment CXCL12-mediated signaling in resting T cells through its cell surface receptor, CXCR4, resulting in the enhanced actin polymerization and cell migration upon ligand exposure. Lck was found to be a critical intermediate in these DM-induced signaling activities. Moreover, DM-mediated Lck phosphorylation in T cells was dependent on the presence of both the glucocorticoid receptor and the CD45 molecule. Overall, these results elucidate additional non-genomic effects of DM on resting human T cells, inducing Lck activation and augmenting chemokine signaling and function.
Glucocorticoids Suppress Activation of Mast Cells through induction of Inhibitory Regulators of Signaling Pathways (94.13)
Authors: Peng Ze , Hiragun Takaaki , Qiao Huihong , Beaven Michael A
Published: January 2023

Glucocorticoids suppress production of inflammatory cytokines via the glucocorticoid receptor (GR) by inhibiting activity of relevant transcription factors, a process known as transrepression. Here we provide evidence that dexamethasone and other glucocorticoids also suppress activation of mast cells through induction of synthesis of inhibitory regulators of signaling pathways by a process referred to as transactivation. The glucocorticoid-inducible regulators identified so far include Src-like adaptor protein (SLAP), downstream of tyrosine kinase (Dok)-1, MAP kinase phosphatase-1 (MPK-1) (Mol. Pharmacol. 67:598, 2005; J. Immunology 177:2047, 2006),. Suppressor of cytokine signaling 1 (Socs1), and cytokine inducible SH2-containing protein (Cish) (unpublished data). We have found that the induction of these inhibitory regulators account for the inhibition of key antigen-mediated signaling events such as the activation of the tyrosine kinase Syk, MAP kinase pathways, and downstream transcription factors. The effects of overexpressed wild type GR, and a dimerization-negative mutant GR with minimal transactivation activity, further suggest that the inhibitory actions of glucocorticoids are dependent on gene transactivation as well as cytokine-gene transrepression. These findings have important implications in the design and use of glucocorticoids that exhibit selectivity towards transrepression.
Signaling motifs in the IL-17 receptor (94.2)
Authors: Gaffen Sarah L , Shen Fang , Maitra Amarnath , Hanel Walter , Mossman Karen
Published: January 2023

IL-17 and its receptor IL-17RA have received much attention as the defining cytokine of a new class of T helper cells termed Th17. Mounting evidence suggests that IL-17 causes pathology in autoimmunity, but surprisingly little is known about mechanisms of IL-17 signal transduction. Although produced by T cells, IL-17 activates genes typical of innate immune cytokines such as TNFα and IL-1β, despite little sequence similarity in their respective receptors. A prior bioinformatics study predicted a subdomain in IL-17-family receptors with homology to a Toll/IL-1R (TIR) domain, termed “SEFIR.” However, the SEFIR domain lacks motifs critical for bona fide TIR domains, and its functionality has never been verified. Here, we used a reconstitution system in IL-17RA-null fibroblasts to map functional domains within IL-17RA. We demonstrate for the first time that the SEFIR domain mediates signaling, independently of TIR adaptors such as MyD88 and TRIF. Moreover, we identified a novel “TIR-like loop” (TILL) also required for activation of NF-κB, NF-κB-dependent genes and upregulation of C/EBPβ and C/EBPδ. We further identified a distinct domain required for activation of C/EBPβ and induction of a subset IL-17 target genes. This is the first structure-function analysis of any IL-17R superfamily receptor, and reveals unique structural and functional properties in IL-17 signaling.
Regulation of p53 expression in PBMC by beta Interferon in Multiple Sclerosis (94.21)
Authors: Zhang Fanglin , Sriram Subramaniam
Published: January 2023

Background: Beta Interferon (IFN) is a first line treatment in Multiple Sclerosis (MS); however its mechanism of action remains unclear. P53 is a key player in regulating apoptosis through the transcriptional activation of target genes. Previous studies showed an integration of beta IFN signaling with stabilization of p53 in murine cells. We examined the activation and stabilization of p53 in PBMC following in vitro culture with beta IFN, and in patients receiving beta IFN treatment.

Methods: PBMC were isolated from 6 heath controls (HC) and 6 MS patients and then cultured with beta IFN. We also examined p53 expression in 7 MS patients receiving beta IFN-1b treatment. P53 expression in vitro and in vivo experiments was determined by real time RT-PCR, Western blotting and flow cytometry.

Results: P53 expression increased in a dose and time dependant manner following culture with beta IFN. Flow cytometry studies showed increased stabilization of p53 in CD3+ T cells and monocytes. PBMC isolated from MS patients receiving beta IFN in vivo also showed the increase in p53 stabilization. However, the stabilization varied between patients receiving beta IFN: two patients showed no increase in p53; in the remaining five patients, the kinetics of p53 induction varied.

Conclusion: Our studies show that p53 is vigorously induced in PBMC in vitro and following injection of drug in vivo. P53 expression induced was variable between patients receiving beta IFN. We suggest that p53 can potentially regulate the expansion of autoreactive cells that might contribute to the development and progression of MS.

The study was supported by funds from Serono Inc
Nuclear Factor of Activated T Cells (NFAT) is Suppressed by the Plant-Derived Cannabinoid, Cannabidiol. (94.23)
Authors: Kaplan Barbara L. F. , Kaminski Norbert E.
Published: January 2023

Marijuana is derived from the Cannabis Sativa plant. Although there are over 60 cannabinoid compounds in the plant, Δ9-tetrahydrocannabinol (THC) is the primary psychoactive congener of marijuana and binds to the two known cannabinoid receptors, CB1 and CB2. THC has exhibited immunosuppressive actions both in vivo and in vitro. Interestingly, another predominant cannabinoid from the plant, cannabidiol (CBD), possesses low affinity for both cannabinoid receptors, yet also exhibits immunosuppressive actions. A major target of immune suppression by CBD is interleukin-2 (IL-2) as demonstrated in PMA/Io-stimulated mouse splenocytes. The focus of these studies was to determine the mechanism by which CBD suppressed IL-2. Using purified T cells from mouse splenocytes, CBD suppressed PMA/Io-stimulated IL-2 production, similar to the transplant drug cyclosporin A. Consistent with the observation that CBD does not possess high affinity for CB1 or CB2, CBD also suppressed PMA/Io-stimulated IL-2 from purified T cells derived from CB1/CB2 null mice. IL-2 transcription is regulated by several transcription factors, including NFAT, activator protein-1 (AP-1), and nuclear factor κB (NF-κB). Using the human Jurkat T cell line, in which CBD suppressed PMA/Io-stimulated IL-2 production, NFAT- and AP-1-luciferase reporter constructs were transiently transfected and induced with PMA/Io in order to examine the effect of CBD on transcriptional activation. CBD suppressed both PMA/Io-induced NFAT- and AP-1-luciferase in a concentration-dependent manner. These results are consistent with the mechanism by which other plant-derived, synthetic and endogenous cannabinoids suppress IL-2 and other functional immune endpoints.

(Supported in part by NIH DA007908).
Protein Phosphatase 2A destabilizes TNF alpha mRNA by dephosphorylating Tristetraprolin (94.8)
Authors: Sun Lei , Stoecklin Georg , Van Way Susan , Hinkovska-Galcheva Vania , Guo Ren-Feng , Anderson Paul , Shanley Thomas
Published: January 2023

Tumor necrosis factor (TNF)-α is a major cytokine produced by alveolar macrophages in response to pathogen associated molecular patterns such as LPS. TNF-α secretion is regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation occurs by modulation of TNF-α mRNA stability via the binding of tristetraprolin (TTP) to the AU (adenosine/uridine)-rich elements (AREs) found in the 3’-untranslated region (3’-UTR) of TNF-α transcript. Phosphorylation plays important roles in modulating mRNA stability. Our results show that inhibition of PP2A by okadaic acid or siRNA significantly enhanced the stability of TNF-α mRNA. This result was associated with increased phosphorylation of p38 MAPK and MK-2. PP2A inhibition increased TTP phosphorylation and enhanced complex formation with chaperone protein 14-3-3. A functional consequence of TTP/14-3-3 complex formation appeared to be protection of TTP from dephosphorylation by inhibition of the binding of the catalytic unit of PP2A to phosphorylated TTP. Our data indicates that PP2A regulates TNF-α mRNA stability by modulating the phosphorylation state of members of the p38/MK-2/TTP pathway.

This work was supported by grants from the National Institute of Health (7R01GM066839-03, to TPS).
Herpes Simplex Virus I Obstucts the Type II Interferon Pathway in Keratinocytes by Induction of Suppressor of Cytokine Synthesis-1 (94.9)
Authors: Frey Kenneth Gene , Johnson Howard M , Bigley Nancy J
Published: January 2023

Herpesviruses are masters at evading the immune system. HSV-1 employs a variety of strategies to blunt the immune response, for example, downregulation of expression of class I MHC molecules. Studies using mouse L929 fibroblasts and HEL-30 keratinocytes revealed a differential response to treatment with interferon-γ (IFN-γ). L929 cells were protected from HSV-1-induced lysis by pretreatment with IFN-γ while HEL-30 cells were not. Interestingly, treatment of HEL-30 cell with IFN-γ followed by HSV-1 infection led to a 14-fold increase in SOCS-1 transcripts while treatment with IFN-γ alone increased SOCS-1 by 7-fold, while in comparison L929 cells showed much less increase in SOCS-1. Recent evidence indicates that HSV-1 inhibits the interferon pathway. SOCS-1 is the primary negative regulator of signaling by IFN-γ via the JAK/STAT pathway. Here we establish that infection of HEL-30 cells with HSV-1 (syn 17+) leads to a rapid induction of SOCS-1 at both transcript and protein levels. This induction is evident as early as 2 hours p.i., and is stable up to 8 hours p.i. Use of siRNA against SOCS-1 indicates that knockdown of SOCS-1 prior to treatment with IFN-γ results in inhibition of HSV-1 replication. These results suggest a possible therapeutic strategy to herpes infection by treatment with SOCS-1 siRNA and IFN-γ.
Follicular CD4+ T helper cells induce human B cells to undergo Ig isotype switching and differentiation to Ig-secreting cells through the production of IL-21 (95.1)
Authors: Tangye Stuart Graham , Avery Danielle T , Bryant Vanessa L , Ma Cindy S
Published: January 2023

The primary function of B cells is to produce high-affinity Ag-specific Ig by differentiating into plasma cells. Naïve B cells can also alter the effector function of their Ig molecule by class switch recombination (CSR). CSR and plasma cell differentiation occur within germinal centres (GC) and are believed to be largely regulated by signals delivered by T follicular helper (TFH) cells. TFH cells reside within the GC and can be resolved from other T-cell populations by the expression of elevated levels of CXCR5. The mechanism by which TFH cells induce B-cell differentiation is unknown. We examined the effects of IL-21, a product of TFH cells, as well as these cells themselves, on the fate of human naïve B cells. IL-21 induced CD40L-stimulated naïve B cells to undergo CSR to acquire expression of IgG and IgA, and secrete large amounts of IgG, IgA and IgE. Isotype switching induced in CD40L-stimulated naïve B cells by IL-21 was linked to cell division, as revealed by analysis of CFSE-labelled B cells. The addition of IL-4 to cultures of CD40L/IL-21-stimulated naïve B cells had divergent and isotype-specific effects with IL-4 enhancing IL-21-induced switching to IgG and IgE whilst inhibiting switching to IgA. When naïve B cells were co-cultured with autologous tonsillar TFH cells, but not CD4+CXCR5− T cells, they produced high levels of IgM, IgG and IgA. Strikingly, differentiation of autologous naïve B cells by TFH cells was abrogated by neutralising endogenous IL-21. These findings demonstrate the critical role of IL-21 in TFH-cell dependent B-cell differentiation and suggest that diminished or excessive production of IL-21 may contribute to the development of immunodeficiencies or autoimmune diseases, respectively.
Neuroalternative Enhancement of Development of Th17 Cells by Signals from the Type 1 Receptor for Vasoactive Intestinal Peptide (VPAC1) (95.11)
Authors: Yadav Mahesh , Goetzl Edward
Published: January 2023

VIP (vasoactive intestinal peptide) has diverse immunological effects transduced by class II G-protein-coupled receptors VIP receptor type 1 (VPAC1) and type 2 (VPAC2). The VIP-VPAC1/2 axes in T cells promote hypersensitivity and inflammation in many settings, but also may be anti-inflammatory at high VIP concentrations. The Th17 subset of CD4 T cells, that mediates local tissue and systemic inflammation through IL-17, develops under the control of several cytokines of which IL-23 has a distinctive role. To determine if the immuno-inflammatory effects of VIP are attributable to recruitment of Th17 cells, mouse splenic CD4 T cells from VPAC2 knockout mice that express only VPAC1 were cultured with anti-CD3 + anti-CD28 Ab, without and with IL-23 or VIP. Over 5 days, 0.1 μM and 1 μM VIP enhanced the development of Th17 cells significantly and with the same time-course as IL-23. VIP also augmented IL-17 production by human T lymphocytes of the HUT102 line, which expresses only VPAC1. Our results suggest that the VIP-VPAC1 axis is an alternative to IL-23 in enhancing Th17 cells development normally and in autoimmunity. Drugs that increase or inhibit VPAC1 signals may be therapeutic in infectious and autoimmune diseases.

This work was funded through NIH grant AI029912
Bcl-2 Does Not Rescue T Lymphopoiesis in Interleukin-7 Receptor Transgenic Mice (95.15)
Authors: Van De Wiele C. Justin , Tan Chibing , Teague T. Kent
Published: January 2023

We have previously demonstrated that murine thymocytes down-regulate IL-7 sensitivity between the β-selection and positive selection checkpoints. To assess the consequences of enforced IL-7Rα expression during this developmental phase, we evaluated thymocyte IL-7 responsiveness in transgenic mice expressing the IL-7Rα gene under control of the lck proximal promoter. This model demonstrated that induction of ectopic high-level IL-7Rα expression during thymopoiesis increases the percentage of thymocytes responsive to IL-7 but results in an overall decrease in total thymic cellularity in transgenic mice. Transgenic thymocytes initiated IL-7Rα expression beginning in the DN2 compartment which was maintained through development to naïve peripheral subsets. Interference with thymocyte development became apparent shortly after initiation of transgene expression as there was an ~65% reduction in DN3 thymocytes in transgenic mice. This finding was reminiscent of phenotypes observed in IL-7 signaling-deficient mice insofar as there was a significant loss of pre-T cells, which correlated with a reduction in Bcl-2 expression. Indeed, lower Bcl-2 levels were observed in transgenic DN2 and DN3 thymocytes when assessed directly ex vivo. Additionally, and unlike the IL-7 signaling-deficient models, transgenic restoration of Bcl-2 expression did not correct the thymic hypocellularity induced by IL-7Rα overexpression. These data demonstrate that ectopic overexpression of the IL-7Rα past the pro-T stage interferes with DN3 thymocyte development and inappropriately maintains IL-7 responsiveness in β-selected thymocytes.
IL-23 acts in a novel fashion by antagonizing IL-12 induced IFN-γ secretion (95.16)
Authors: Sieve Amy N. , Meeks Karen D. , Balch Joshua B. , Berg Rance E.
Published: January 2023

Most studies have concluded that interleukin-23 (IL-23) plays a unique role in promoting IL-17 secreting T cells. While some reports have also characterized IL-23 as having redundant pro-inflammatory effects with IL-12, we have instead found that IL-23 can regulate the innate immune response in a novel manner, by antagonizing IL-12 induced secretion of interferon-γ (IFN-γ). Our recent data documents the rapid, innate production of IFN-γ from memory CD8 T cells, as well as NK cells, stimulated with IL-12 and IL-18. When splenocytes are cultured with IL-23, IFN-γ secretion in response to IL-12 is dramatically reduced, as measured by ELISA and intracellular cytokine staining. The impact of IL-23 is most prominent in CD8 T cells, but can also be seen in NK, NK-T, and CD4 T cells. We also show that IL-23 can regulate the induction of IFN-γ by endogenously produced IL-12 from Listeria monocytogenes infected macrophages. Furthermore, IL-23 appears to act directly on purified CD8 T cells to negatively regulate IFN-γ induced by IL-12. We are currently investigating the mechanism by which IL-23 inhibits IFN-γ secretion. In addition, experiments to determine if IL-23 can inhibit other effector functions are ongoing. Our data suggest that IL-23 may be a key player in determining the responsiveness of lymphocytes to IL-12 and their subsequent secretion of IFN-γ.

This research is supported by NIH AI064592.
Characterization of IL-2-stimulatory factor produced from dendritic cells stimulated with a sesquiterpene compound TG651 (95.19)
Authors: RAN LEE YOUNG , KI-HYANG KIM , YOUNG-HEE LEE , HYUNJIN KIM , SUN-A IM , CHONG-KIL LEE
Published: January 2023

Dendritic cells (DCs) have been known to produce cytokines such as IL-2, IL-6, IL-10, IL-12, IFN-α and IFN-γ. None of those cytokines has been shown to activate T cells to produce IL-2 by itself. Here, we show that DCs stimulated with TG651, a sesquiterpene compound, produce a cytokine that induces IL-2 production from T cells. The cytokine, termed as IL-2SF, activated both CD4 and CD8 T cells to produce IL-2. IL-2SF was produced from DCs as a membrane bound form as well as a soluble form. Both form of IL-2SF was produced from DCs within 5 min after TG651 stimulation, and did not require new protein synthesis, although presence of approximately 10% fetal bovine serum was required for optimal production. Soluble form of IL-2SF appeared to be stable at neutral pH, but destroyed readily at alkaline pH when treated at 56°C for 30 min. The molecular weight of IL-2SF was estimated to be 60 KD - 70 KD by gel filtration chromatography. Although the molecular entity of IL-2SF remains to be elucidated, the present study demonstrates that DCs produce a cytokine that induces IL-2 production from T cells.
Differential effects of berberine, baicalein, and triptolide on cytokine production from IL-1β-activated mast cells (95.28)
Authors: Hsieh Chia-Jung , Hall Kenton , Krishnaswamy Guha , Chi David S.
Published: January 2023

Human mast cells are multifunctional cells capable of a wide variety of inflammatory responses. Berberine (BER), baicalein (BAI), and triptolide (TRI) are compounds isolated from traditional Chinese medicinal herbs and have been shown to have anti-inflammatory effects. We examined their effects on the production of inflammatory cytokines from IL-1β-activated human mast cells (HMC-1). Two ml of HMC-1 at 1 x 106 cells / mL were cultured with BER, BAI, and TRI (at predetermined non-toxic concentrations) in the presence or absence of IL-1β (10 ng / mL) for 24 hrs. The supernatants were harvested and assayed for IL-6, IL-8, and MCP-1 by ELISA. The compounds alone did not induce cytokine production from HMC-1. However, BAI (15 and 30 μM) significantly inhibited the production of IL-6, IL-8 and MCP-1 (all p < 0.005) from IL-1β-activated HMC-1 in a dose dependent fashion. TRI (10 and 20 nM) significantly inhibited IL-8 and MCP-1 (p < 0.001 and < 0.05, respectively), but not IL-6 production from IL-1β-activated HMC-1. BER (25 and 50 μM) inhibited the production of MCP-1 (p = 0.02 and = 0.005, respectively), had no effect on IL-6 production, and enhanced the production of IL-8 at 25 μM (p = 0.01) from IL-1β-activated HMC-1. The results show that BER, BAI, and TRI had differential effects on the production of inflammatory cytokines. Since BAI has the most potent inhibitory effect, it may lead to new anti-inflammatory therapy.

(Supported by the Chair of Excellence in Medicine, the Ruth R. Harris Endowment, and RDC of ETSU)
Thymic IL-2R expression is sufficient for production of CD4+CD25+ Foxp3+ T regulatory cells (95.5)
Authors: Bayer Allison L , Yu Aixin , Malek Thomas R
Published: January 2023

CD4+CD25+ FoxP3+ Treg cells critically depend on IL-2, but the contribution of IL-2 to development of Treg cells in the thymus versus controlling their homeostasis in the periphery is unresolved. We addressed this issue using models where IL-2R activity was blocked in the thymus and periphery or only in the periphery. We found that IL-2/IL-2R interaction is active and essential in the thymus by promoting Treg cell expansion and upregulation of Foxp3 and CD25 to normal levels. This thymic signal gave rise to peripheral Treg cells with impaired IL-2-dependent STAT5 activation, yet they persisted in the periphery and suppressed autoimmunity without continual thymic output. Thus, thymic IL-2R function represents one important aspect for Treg cell development. Peripheral Treg cells with impaired IL-2R signaling exhibited slower growth and death rates as assessed by BrdU uptake in vivo and their suppressive activity was slightly lower in vitro. Importantly, by following donor Treg cells in mixed thymic and bone marrow chimeric mice, we found that WT Treg cells dominated peripheral immune tissue compared to Treg cells with impaired peripheral IL-2 signaling. Thus, IL-2 is a dominant mechanism controlling the number of thymic and peripheral Treg cells, although in a non-competitive setting, peripheral Treg cells with minimal IL-2R signaling persist and control autoimmunity.

(Supported by the NIH)
TH17 Augmentation in OTII TCR plus T Cell-Selective Type 1 Sphingosine 1-Phosphate Receptor Double Transgenic Mice (95.9)
Authors: Huang Mei-Chuan , Watson Susan R , Liao Jia-Jun , Goetzl Edward J
Published: January 2023

Sphingosine 1-phosphate (S1P) in blood and lymph controls lymphoid traffic and tissue migration of T cells through signals from the type 1 S1P receptor (S1P1), but less is known of effects of the S1P-S1P1 axis on non-migration functions of T cells. CD4 T cells from a double transgenic (DTG) mouse express OTII TCRs specific for ovalbumin peptide 323–339 (OVA) and a high level of TG S1P1, resistant to suppression by T cell activation. Activated DTG CD4 T cells respond as expected to S1P by chemotactic migration and reduction of OVA-evoked secretion of IFN-gamma. In addition, DTG CD4 T cells stimulated by OVA secrete a mean of 2.5-fold more IL-17 than those from OTII single TG mice with concomitantly higher levels of mRNA encoding IL-17 by real-time PCR and intracellular IL-17 detected by ELISpot assays. OVA challenge of subcutaneous air-pockets elicited influx of more OTII-TCR-positive T cells producing a higher level of IL-17 in DTG mice than OTII control mice. Augmentation of the number and activity of Th17 cells by the S1P-S1P1 axis may enhance host defense against microbes and in other settings increase host susceptibility to autoimmune diseases.

(supported by NIH HL31809)
Complex roles of the CXCR3 chemokines in immune-mediated neurological disease (96.1)
Authors: Campbell Iain L. , Mueller Marcus , Carter Sally , Getts Daniel R. , King Nicholas J.C.
Published: January 2023

The chemokine receptor CXCR3 binds CXCL9, CXCL10 and CXCL11 and promotes the trafficking of activated T-cells. Although these chemokines are highly elevated in the CNS in MS and the animal model, experimental autoimmune encephalomyelitis (EAE), their role is unknown. Here we show that while IFN-γ is the principal mediator of CXCR3 ligand gene expression in the CNS in EAE, the cellular localization of the individual chemokine genes is divergent suggesting functional specialisation of the CXCR3 ligand chemokines. Transgenic mice (GF-) were generated with astrocyte-production of CXCL9, CXCL10 or CXCL11. These mice do not develop neurological disease. In GF-CXCL10 and -CXCL11 mice, modest non-reactive leukocytic infiltrates were seen in the CNS. Thus, these chemokines when produced chronically are a poor stimulus for the CNS recruitment and activation of T-cells. The role of CXCR3 signaling in MOG-induced EAE was examined in CXCR3 KO mice and in GF-CXCL9, -CXCL10 or -CXCL11 mice. CXCR3 KO, GF-CXCL9 and -CXCL11 mice had more severe EAE with widely disseminated demyelinating lesions throughout the CNS. In CXCR3 KO mice with EAE the number of suppressor Treg cells in the brain was reduced. Thus, in EAE, CXCR3 chemokine ligands:have non-redundant functions,are not involved in the initial recruitment of effector T-cells to the CNS, andretain T-cells to the perivascular space and foster Treg cell interactions thereby limiting lesion spread and tissue destruction.

Support: NIH NS044905.
A hemoglobin based oxygen carrier (HBOC-201) has similar effects to conventional resuscitation fluids on cytokine responses in an acute trauma-hemorrhage model (96.12)
Authors: Hutchings Anne , Rodriguez Celina , George Richard , Moon-Massat Paula , Rue Loring , Kerby Jeffrey
Published: January 2023

Although traumatic hemorrhagic shock may be acutely fatal, alterations in the immune response of survivors in the post injury setting may lead to infections, sepsis, multiple organ failure and death. Various fluids, including HBOC-201, have been evaluated as resuscitative fluids for hemorrhagic shock. This study investigated the effects of HBOC-201 on cytokine responses following trauma-hemorrhage. Anesthetized mice (n=8/group) received a 2 cm abdominal incision, and then were hemorrhaged over 30 min through a femoral artery catheter to a mean arterial pressure (MAP) of 25±5 mm Hg. After an additional 1h of sustained hypotension, animals were resuscitated with lactated Ringer’s (LR), shed blood, or HBOC-201 and allowed to recover for 24h. Peripheral blood and spleens were collected for analysis. Survival rates were equal. HBOC-201 significantly increased post-resuscitation MAP compared to LR, but had little effect on serum IL6, IL10, IL12, IFNγ, TNFα or MIP1α levels compared to other groups at 24h post-resuscitation. There were no significant differences between groups in cytokine production by splenocytes stimulated for 24h in vitro with LPS. These results suggest that low volume resuscitation with HBOC-201 is superior to LR for maintenance of blood pressure and has no obvious effects on peripheral blood or splenocyte cell function with regard to cytokine production.

Supported by Biopure Corporation, Cambridge, MA.
IL-17 receptor-deficient mice have increased resistance to otherwise lethal influenza infection (96.14)
Authors: Crowe Christopher , Kolls Jay
Published: January 2023

IL-17 is a CD4+ T cell-derived cytokine that has been previously been shown to play a role in the immune response to several different lung infections. Specifically, it has been shown to induce neutrophil recruitment and inflammation in response to a variety of pathogens, including Pseudomonas aeruginosa, Klebsiella pneumoniae, and Mycoplasma pneumoniae. Additionally, it has been shown that IL-17 is upregulated in respiratory syncitial virus (RSV) infection, but its role in other viral pulmonary infections remains poorly described. In order to investigate the role of IL-17 in influenza infections, BALB/c mice lacking the IL-17 receptor were challenged intranasally with a lethal dose of influenza A/Puerto Rico/8/34 (H1N1). The mice were monitored for weight loss and signs of clinical illness on a daily basis. When compared to BALB/c controls, the IL-17 receptor knockout mice exhibited delayed weight loss and had higher survival rates. Additionally, the control mice exhibited higher levels of protein and lactate dehydrogenase in the BAL fluid, markers of acute lung injury, during the course of infection. These data suggest that the effects of IL-17R signaling contributes to lung injury and the pathology of a lethal influenza infection in a murine model.
Neuronal Nitric Oxide Synthase alters inflammatory response after endotoxin challenge (96.16)
Authors: Khaibullina Alfia , Besch Virginia , Middleton LaShon , Quezado Zenaide
Published: January 2023

We have recently shown that mice deficient in the neuronal isoform of NOS (nNOS−/−, KO) have higher mortality compared to wild type (nNOS +/+, WT) in a polymicrobial sepsis model. We sought to determine what effect the genetic deficiency of nNOS had on LPS-induced expression of cytokines. KO and WT mice were injected with LPS (i.p.) and sacrificed 6 or 24 h later. Peritoneal lavage cells (PLCs) were counted and profiled for cell type. RNA from spleens and PLCs was isolated and the relative expression of cytokines was determined by real-time PCR. After LPS challenge, KO mice had higher mortality (100% vs. 40% at 40 mg/kg), higher number of PLCs at 6 and 24h, higher proportion of macrophages at 6h and higher neutrophils counts at 24h compared to WT. In splenocytes, relative expression of proinflammatory cytokines at 6 hours was lower in KO (TNFα=0.18, IL1β=0.05) compared to WT mice (TNFα= 12.1, IL1β =2.9, p ≤ 0.002). Conversely in PLCs, KO mice had significantly higher IL-1β mRNA levels compared to WT (251 vs. 0.64, p ≤ 0.008). These results suggest that KO mice have altered neutrophil trafficking to peritoneum, do not mount a global inflammatory immune response, and exhibit aberrant local immune response after LPS challenge. These differences may explain increased mortality in knockout mice after LPS challenge and polymicrobial sepsis and indicate that nNOS has an important role in immune response during sepsis.
Manipulaion of TNFalpha delays loss of CD28 expression in human CD8 T cells (96.19)
Authors: Parish Stanley T , Fauce Stanley R , Effros Rita B
Published: January 2023

The CD28 costimulatory receptor is essential for T cell activation, glucose metabolism, and optimal telomerase activity. Increased proportions of CD8+CD28 T cells, seen in HIV infection and aging, correlate with decreased vaccine responsiveness and early mortality. CD28 expression is also lost in chronically stimulated cultures of CD8 T cells as they approach replicative senescence, a state of irreversible cell cycle arrest, associated with shortened telomeres and increased levels of TNFα. Based on the link between TNFα and the CD28 gene promoter, we sought to investigate the effects of manipulating TNFα on replicative senescence. Human T cells were repeatedly stimulated with an allogeneic cell line in the presence of either a neutralizing antibody (anti-TNFα) or a TNFα receptor 1 inhibitor (rhuTNFR:Fc). CD28 expression and gene transcription were monitored by flow cytometry and RT-PCR, respectively. Chronic exposure of antigen-stimulated CD8 T cells to rhuTNFR:Fc or anti-TNFα delayed the loss of CD28 expression. Even after completing 17 population doublings, the treated cultures contained >20% more CD28+ cells; CD28 gene transcription was similarly elevated. Finally, telomerase activity was increased by as much as 6-fold in the treated cultures. Manipulation of TNFα, therefore, may be a useful clinical approach to delay accumulation of senescent CD8 T cell in vivo.

Supported by NIH AI060362 and AG023720
CCL25-CCR9 axis role in ovarian cancer progression (96.2)
Authors: Johnson Erica , Johnson-Holiday Crystal , Singh Shailesh , Singh Udai , Partridge Edward , Lillard James
Published: January 2023

Ovarian cancer (OvCa) is the most lethal among gynecologic malignancies. Recently, chemokines have been shown to function in non-random tumor-cell metastasis to distant organs in a fashion that is similar to chemokine-directed lymphocyte migration. Also, the interactions between CCR9 and its ligand, CCL25, have been implicated in leukocyte trafficking to the small intestines, a frequent metastatic site for OvCa. Previously, our laboratory has shown that ovarian cancer cells strongly express CCR9. We have also shown that CCL25 induces the chemotaxis of OvCa cell lines and increases the invasive potential of OvCa cells. Recent studies in T cells, have shown that CCR9-CCL25 interaction can induce anti-apoptotic pathways, namely through Akt/protein kinase B activation, which is PI3K- and G?i protein-dependent. Activated Akt phosphorylates multiple down stream targets that are involved in cell survival signals, which include glycogen synthase kinase-3? and forkhead transcriptional factor. Cisplatin is a commonly used anticancer drug for the treatment of OvCa. It has been shown that the anti-apoptotic signals of the PI3K-Akt survival pathways are involved in tumor cell cisplatin resistance. Here we characterize the molecular mechanisms of CCL25-CCR9 cell-signaling cascades involved in ovarian cancer survival and show that CCL25-CCR9 interactions mediate cisplatin survival through the activation of anti-apoptosis pathway. These studies will provide novel information regarding the cellular and molecular mechanisms, following CCL25-CCR9 interaction, that modulate ovarian cell metastasis and cisplatin resistance.
Abnormal innate immune response and Th1/Th17 induction in murine and human inflammatory bowel diseases (96.22)
Authors: Kobayashi Taku , Kamada Nobuhiko , Okamoto Susumu , Hisamatsu Tadakazu , Inoue Nagamu , Hibi Toshifumi
Published: January 2023

Recently, a lot of papers concerning IL-23/17 axis in some inflammatory conditions have been reported. IL-12p70/IL-23 are considered to regulate Th1/Th17 reciprocally in vitro, however, in vivo, both IFN-γ and IL-17 were reported to be upregulated in murine and human inflammatory bowel diseases (IBD). We previously reported the excess production of IL-12 and IL-23 by innate immune system in IL-10 deficient mice (Kamada N et al. J Immunol 2005; 175(10)) and in the present study, we have tried to unravel the role of innate immune system in induction of Th1/Th17 in murine colitis and human IBD. Colonic lamina propria CD4+ T cells of colitis mice were examined by intracellular cytokine staining and ELISA, and showed the upregulated production of both IFN- γ and IL-17. Similarly, these cytokines from CD4+ cells isolated from inflamed mucosa of human IBD are also increased in relation to upregulated IL-12/23. As expected, both macrophages and dendritic cells from IL-10 deficient mice produced larger amount of IL-12 and IL-23 than WT mice. Furthermore, macrophages and dendritic cells from IL-10 deficient mice could effectively stimulate both IFN- γ and IL-17 production in vitro. These results indicate that abnormal innate immune response stimulates both Th1 and Th17 simultaneously in murine and human inflammatory bowel disease.
IL1 is Required for CD8 T Cell Stimulation of Endothelial Cells to Produce KC in Contact Hypersensitivity (96.27)
Authors: Kish Danielle D. , Fairchild Robert L
Published: January 2023

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to cutaneous sensitization and challenge with hapten. Our recent studies indicate that hapten-primed CD8 T cells induce CXCL1/KC production in response to hapten challenge, which directs subsequent neutrophil infiltration into the challenge site and these infiltrating neutrophils then recruit effector CD8 T cells into site to mediate the swelling response. Keratinocyte-hapten contact induces IL1 production, which in turn also stimulates CXCL1/KC production. This study investigated the role of IL1 in the elicitation of CHS. IL1R−/− mice had decreased priming of effector CD8 T cells and CHS swelling responses to DNFB sensitization and challenge than wild-type mice. Production of CXCL1/KC following challenge of sensitized IL1R−/− mice was near background levels. Flow cytometry analyses of cell suspensions prepared from the challenged skin revealed decreased neutrophil infiltration in IL1R−/− vs. wild-type mice. Transfer of wild-type hapten-primed CD8 T cells to naïve wild-type recipients induced CXCL1/KC production in response to hapten challenge whereas transfer to naive IL1R−/− mice did not. Transfer of CD8 T cells from sensitized IL1R−/− mice did not induce CXCL1/KC production in wild-type mice. Injection of LPS i.v. stimulated endothelial cells in the skin of the IL1R−/− mice to produce KC indicating the ability of the IL1R−/− endothelial cells to produce the chemokine. We hypothesize that IL1 signaling is required for optimal CD8 T cell priming to hapten as well as for hapten-primed CD8 T cell interaction with endothelial cells in the challenge site to induce CXCL1/KC production and the CHS response.
Antibody microarray analysis of inflammatory mediator release by human leukemia T cells and human non-small cell lung cancer cells (96.28)
Authors: Hommema Eric , Garcia Bradley , Hargrave Aubrey , Kilmer Greg , Morgan Aric , Narahari Janaki , Webb Brian , Wiese Rick
Published: January 2023

In order to expedite the analysis of samples for multiple cytokines/chemokines, we have developed slide-based ExcelArray™ antibody sandwich microarrays. Each slide consists of 16 subarrays (wells) printed with 12 specific antibodies in triplicate and positive and negative control elements. This 16-well format allows for the analysis of 10 test samples using a six-point standard curve. The array architecture is based on the “sandwich” ELISA, in which an analyte protein is “sandwiched” between an immobilized capture antibody and a biotinylated detection antibody, using streptavidin-linked DyLight™ 649 dye for quantitation. We used the Jurkat cell line as a model for human T cell leukemia and the A549 cell line as a model for human lung cancer. In order to evoke a cytokine/chemokine response, cells were stimulated with Tumor Necrosis Factor alpha (TNFα), Phorbol-12-myristate-13-acetate (PMA, TPA) and Phytohemagglutinin (PHA). Cell supernatants derived from both untreated and stimulated cells were analyzed on 41 unique analytes. Stimulated cells showed an increase in the expression level of many of the test analytes, including IL-8, TNF-α, and MIP-1α, compared to the non-treated controls. Our experiments clearly demonstrate the utility of antibody microarray analysis of cell culture supernatants for the profiling of cellular inflammatory mediator release.
Respiratory Syncytial Virus Vaccine-Enhanced Pulmonary Eosinophilia Requires IL-13, IL-4Rα, and STAT6 (96.6)
Authors: Castilow Elaine , Varga Steven M.
Published: January 2023

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in children. A formalin-inactivated RSV vaccine administered to children in the 1960′s led to enhanced morbidity and mortality upon natural RSV infection. Histological analysis of the lungs upon autopsy revealed pulmonary eosinophilia. BALB/c mice primed with a recombinant vaccinia virus expressing the attachment (G) protein of RSV (vacG) develop pulmonary eosinophilia and vaccine-enhanced disease mimicking the response of the vaccinated children. Although the G protein elicits a mixed Th1 and Th2 response in these mice, the Th2 cells are known to be necessary for the induction of pulmonary eosinophilia. Here we have investigated role of individual Th2 cytokines in mediating RSV vaccine-enhanced disease. Our work shows that IL-13, the IL-4 receptor α chain (IL-4Rα), and STAT6, are required for the development of pulmonary eosinophilia in vacG-primed mice challenged with RSV whereas IL-4 is not required. We have found that IL-4, IL-13, IL-4Rα, and STAT6 do not contribute to weight loss or clinical illness whereas STAT4 is necessary. Lung histology and viral titers have also been examined in IL-13-, IL-4-, IL-4/13-, and IL-4Rα-deficient mice. Our results demonstrate a critical role for IL-13 in driving RSV vaccine-induced pulmonary eosinophilia.
Critical role of IL-17R signaling in granulocyte recruitment and airway hyperreactivity in an Aspergillus model of asthma (96.7)
Authors: Kapadia Shernaaz B , McKinley Laura , Henry Adam , Ray Anuradha , Kolls Jay K
Published: January 2023

Rationale IL-17 is a proinflammatory cytokine that enhances neutrophil recruitment in response to allergen in murine lung. In an Aspergillus fumigatus (Af)-induced asthma model, wild-type C57BL/6 mice develop bronchial inflammation and hyperreactivity. We hypothesized that, compared to C57BL/6, Af sensitized IL-17R KO mice would have reduced neutrophil recruitment to the lung, airway resistance and hyperreactivity.

Methods C57BL/6 and IL-17R KO male mice, 6–8 wk old, were sensitized intraperitoneally with 40 ug Af extract and 2 mg Al(OH)3 on days 0 and 7, and intratracheally with 25 ug Af on days 21, 22, and 23. Bronchoalveolar lavage (BAL) differential cell counts and cytokine analysis were done at 24 and 72 hr. Flexivent was performed at 24 hr to assess airway physiology.

Results WT mice had significant influx of both neutrophils and eosinophils in response to Af at 24 hr. In contrast, IL-17R KO mice showed reduced neutrophil and eosinophil emigration to the lung which was associated with lower KC and eotaxin levels in BAL fluid. Furthermore, Flexivent data showed lower airway resistance and bronchial hyperreactivity in IL-17R KO mice.

Conclusion Our findings in Af treated IL-17R KO mice of decreased pulmonary neutrophil and eosinophil recruitment at 24 hr suggest that IL-17 promotes granulocyte recruitment as well as airway hyperreactivity.

Funding: NHLBI
A Th2 response to chronic intermittent antigen exposure induces pulmonary vascular muscularization (96.8)
Authors: Grunig Gabriele , Daley Eleen , Emson Claire , Taraseviciene-Stewart Laima , Kurup Viswanath P. , de Waal-Malefyt Rene , Hogaboam Cory , Grunig Ekkehard , Voelkel Norbert F.
Published: January 2023

Pulmonary arterial hypertension is associated with pulmonary vascular remodeling (PVRm) and its incidence is increased in many autoimmune diseases and chronic inflammatory lung diseases. Currently it is not known whether inflammatory and immune responses are cause or consequence of PVRm. In this study immunized mice were intermittently exposed to inhaled soluble antigen over a period of several weeks. The antigen exposure induced dramatic PVRm in a CD4 T cell dependent manner. PVRm was characterized by hyperplasia of smooth muscle cells. To identify pathogenic mediators, IL-4 deficient mice and wild type mice transiently given an IL-13 blocker were examined. In both cases, the mice developed significantly less PVRm relative to immunized, antigen challenged, control wild type mice. Mice given recombinant IL-13 intranasally did not develop severe vascular muscularization indicating that IL-13 and, most likely IL-4 promote PVRm indirectly. In conclusion, our data are the first to provide proof of concept that the adaptive immune response to inhaled antigen can cause PVRm.

Supported by: Flight Attendant Medical Research Institute, Stony Wold Herbert Fund, American Lung Association of the City of New York