bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-17 elicits a neutrophil-attractant response to Neisseria gonorrhoeae infection (38.25)
Authors: Feinen Brandon , Gaffen Sarah L , Jerse Ann E , Russell Michael W
Published: January 2023

Infection of the genital tract with Neisseria gonorrhoeae is typically associated with an influx of neutrophils, but the mechanisms underlying this influx are not well understood. The importance of the newly defined lineage of Th17 cells in inflammatory immune responses to infection, including the recruitment of neutrophils, has become increasingly recognized. We have previously shown that N. gonorrhoeae elicits production of IL-17 and other Th17-associated cytokines in vitro and hypothesized that IL-17 plays a role in the induction of chemokines and cytokines which lead to the recruitment of neutrophils to the site of infection. We tested this using a mouse vaginal gonococcal infection model to compare neutrophil recruitment in wild type and IL-17 receptor (IL-17R) knockout mice. The IL-17RKO mice were severely deficient in recruitment of neutrophils to the genital tract. Mouse genital explants were incubated ex vivo with either N. gonorrhoeae or a control antigen and assayed for production of cytokines and chemokines. Genital explants from IL-17RKO mice incubated with N. gonorrhoeae showed decreased production of IL-6 and the neutrophil-attractant chemokines LIX and MIP2α compared to those from wild type mice. These results indicate that N. gonorrhoeae elicits a Th17 response which drives the production of neutrophil-attractant chemokines, resulting in the influx of neutrophils into the genital tract.
Neither the Peyer’s patches, mesenteric lymph nodes, nor the spleen are required for induction of chronic colitis in mice (166.16)
Authors: Koboziev Iurii , Takebayashi Koichi , Ostanin Dmitry , Gray Laura , Karlsson Fridrik , Jackson Sherry , Kosloski-Davidson Melissa , Burrows-Dooley Angela , Zhang Songlin , Grisham Matthew
Published: January 2023

The objective of this study was to evaluate the role of PPs, MLNs and spleen in the pathogenesis of chronic colitis induced in mice. For the first series of studies, we transferred sorted, naïve (CD4+CD45RBhigh) T-cells (5x105 cells) into sham-operated RAG-1-/- mice or into RAG-1-/- mice that had their MLNs, spleen or both tissues surgically removed. We found that at 8 wks following T-cell transfer, all four of the surgically-manipulated RAG-1-/- groups developed moderate-severe colitis that was not different among the four groups. Flow cytometric analysis revealed large and similar numbers of IFN-γ producing T-cells within the colonic lamina propria (LP) of all four groups. For the second series of studies we intercrossed lymphotoxinβ-deficient (LTβ-/-) mice with RAG-1-/- animals to generate LTβ-/-xRAG-/- offspring that were devoid of Peyer’s patches and most peripheral lymph nodes. Surprisingly, and in contrast to what has been reported for LTβ-/- mice, we found no evidence of MLNs in the LTβ-/-xRAG-/- mice suggesting that lymphocytes may be required for MLN development in LTβ-/- mice. Adoptive transfer of CD45RBhigh T-cells into splenectomized LTβ-/-xRAG-/- mice or their littermate controls (LTβ+/+x RAG-/-) induced rapid onset of colitis suggesting that neither the PPs, MLNs nor the spleen are required for induction of disease. The mechanisms by which naïve T-cells are converted to colitogenic effector cells in this model are currently under investigation.
Dietary vitamin B9 is a survival factor of intestinal regulatory T cells (166.2)
Authors: Kunisawa Jun , Hashimoto Eri , Ishikawa Izumi , Kiyono Hiroshi
Published: January 2023

It is well known that dietary factors regulate versatile immunological functions, but the detail molecular and cellular mechanisms remain to be investigated. Here we show that vitamin B9 is a survival factor of regulatory T (Treg) cells in the intestine when it is supplied from diet. In vitamin B9-null condition, naïve T cells could differentiate into Treg cells, but differentiated Treg cells failed to survive. This effect was dose-dependent and associated with the decreased expression of anti-apoptotic Bcl-2, but independent from the IL-2- and CD28-mediated survival signals. Treg cells thus showed normal responses against these survival stimulations even in vitamin B9-null condition, suggesting that vitamin B9 is the third survival factor of Treg cells. Consistent with these findings, in vivo depletion of dietary vitamin B9 resulted in the reduction of intestinal, but not serum vitamin B9, which led to the specific decrease in the numbers of intestinal but not splenic Treg cells. These findings provide new links between dietary vitamin B9 and the gut immune system, which could be used for the development of new mucosal immunotherapy by controlling intestinal vitamin B9-mediated Treg cell network.
HDAC-mediated repression of IFN-γ responses is conserved in mouse and human trophoblast cells (166.20)
Authors: Murphy Shawn , Sullivan Danielle , Weise Paige , Choi Jason , Bushway Meghan
Published: January 2023

Successful pregnancy requires that the semi-allogeneic conceptus evades maternal immune-mediated rejection, while the maternal immune system simultaneously protects mother and fetus from infection. IFN-γ is a pro-inflammatory cytokine that activates innate and adaptive immunity, induces apoptosis and inhibits cell proliferation. During normal pregnancy, IFN-γ is secreted in both the human and mouse uterus. Placental trophoblast cells (TBCs) are the only cells derived from the fertilized egg that contact maternal blood, and they play important roles in controlling maternal immunity to the conceptus. Human and mouse TBCs are resistant to both IFN-γ-mediated apoptosis and activation of MHC class II expression. Moreover, while epithelial cells can clear Toxoplasma infections when exposed to IFN-γ, TBCs cannot. These collective results suggest that TBCs are resistant to IFN-γ. We recently demonstrated that IFN-γ-inducible gene expression is inhibited in mouse TBCs by histone deactylases (HDACs). Our current studies utilizing selective HDAC inhibitors and siRNA suggest that HDAC-2 and/or -3 are responsible for repressing IFN-γ-inducible gene expression in mouse TBCs. Importantly, HDAC-mediated repression of IFN-γ responses is conserved in Jar human TBC-derived choriocarcinoma cells. In contrast, HDACs activate these same genes in human cells of non-trophoblastic origin. These collective results suggest that HDACs function by a novel mechanism to repress IFN-γ-responses in TBCs.
Exercise prior to pregnancy enhances Treg-mediated suppression and reduces asthma severity in offspring in a mouse model of asthma (166.25)
Authors: Kunz Hawley , Lowder Thomas
Published: January 2023

We have previously shown that exercise significantly reduces airway hyperresponsiveness (AHR) and Th2 inflammatory cytokines in the lung while significantly enhancing regulatory T cell (Treg)-mediated in vitro suppression and in vivo expression. Others have shown that OVA-treated nursing dams confer protection against AHR and inflammation to pups exposed to an OVA-driven model of asthma. In the present study, we wanted to determine if exercise prior to pregnancy conferred the same effect. Male pups from exercise-trained (E) or sedentary (S) female mice were OVA-sensitized and challenged. While not significant, we saw a decrease in AHR in E pups compared to S pups. When co-cultured with CD4+CD25- effector cells, Tregs from E pups reduced proliferation from 8.4 to 10% at increasing titers, demonstrating an enhanced suppression in vitro. We saw a significant increase in the percentage of CD4+ thymocytes in the pups from E mice compared to S mice (95.1 +/- 2.7, 83.95 +/- 3.7, respectively), with a significant decrease in percent of CD8+ splenocytes and mediastinal lymph node (MLN) cells. Although not significant, there was a more than doubling of CD4+FoxP3+ thymocytes in E versus S pups. There was a trend (p=0.08) for higher CD4+FoxP3+ MLN in S compared to E pups. We show for the first time that exercise prior to pregnancy can confer protection against asthma in an OVA-driven mouse model.
Obesity compromises the ability of skin γδ T cells to regulate skin homeostasis and barrier function (166.6)
Authors: Jameson Julie , Costanzo Anne , Taylor Kristen
Published: January 2023

Within the epidermis of the skin resides a unique population of γδ T lymphocytes that play key roles in skin homeostasis and wound repair. During obesity and related metabolic disease the skin-resident γδ T lymphocytes become dysfunctional due the chronic inflammatory environment. This dysfunction contributes to the non-healing wounds associated with disease. We are now investigating how skin γδ T cell dysfunction impacts normal keratinocyte homeostasis. Interestingly, in lean mice we have determined that the number of keratinocytes in the epidermis is directly proportional to the number of closely neighboring γδ T cells. Once obesity and related metabolic disease ensues, the keratinocyte numbers resemble areas that are devoid of γδ T cells in lean mice and keratinocytes are unable to maintain normal homeostatic numbers. Furthermore, keratinocytes in obese mice exhibit diminished proliferation and accelerated differentiation. This results in a dysregulated epidermis in which fewer basal keratinocytes express the proliferation marker keratin 5 and more exhibit early expression of the differentiation marker keratin 1. Additionally, skin γδ T cells downregulate CD103 in obese mice, while keratinocytes alter expression of its ligand E-cadherin. Together these studies demonstrate that dysfunctional γδ T cells in the epidermis of obese mice contribute to impaired keratinocyte homeostasis ultimately resulting in compromised skin barrier function.
Rip2 plays a critical role in regulating intestinal inflammation (166.8)
Authors: Zaki Md. , Kanneganti Thirumala-Devi
Published: January 2023

Human inflammatory bowel disease (IBD) constitutes a major health problem in developed countries. Genome wide association studies suggested that mutation in a gene encoding the intracellular pathogen sensor NOD2 is a predisposing factor for development of IBD. However, not all IBD patients exhibit the NOD2 mutation. This leads to postulate that genes downstream of NOD2 could also be mutated in some IBD patients. Upon activation by bacterial cell wall component muramyl dipeptide, NOD2 activates NF-kB and MAP kinase pathways through adaptor molecule RIP2. However, the role of RIP2 in IBD pathogenesis has not been examined yet. Therefore, we studied the role of RIP2 in intestinal inflammation using a mouse model of colitis induced by dextran sulphate sodium (DSS). We observed that both Nod2-/- and Rip2-/- are susceptible to DSS-induced colitis, however, more pronounced disease phenotypes developed in Rip2-/- mice manifested by increased weight loss, diarrhea, rectal bleeding and exacerbated histological changes compared to wild-type and Nod2-/- mice. Investigation on the nature of the immune response suggests that certain cytokines critical in IBD pathogenesis are differentially regulated in Rip2-/- mice as compared to Nod2-/-. These novel findings underscore the divergent role Rip2 play in maintaining intestinal homeostasis and protecting intestinal inflammation, and may open new avenues in the understanding of IBD pathogenesis.
Egr-2 regulates inflammatory plasticity of effector T cells (167.1)
Authors: Raymond Meera , Wang Ping
Published: January 2023

Effector CD4 T cells can orchestrates different immune cells to induce a protective immune response, but a disregulated response can result in immune pathology or autoimmune diseases. Therefore, the levels of activation and the production of effect cytokines have to be controlled. In addition to the extrinsic regulation of effector T cells by Treg, Early Growth Response 2 gene (Egr-2) has been found to regulate T cell tolerance intrinsically. Egr-2 deficiency in T cells results in the development of autoimmune diseases. We have found that Egr-2 is a negative regulator for the expression of pro-inflammatory cytokines. In the absence or over expression of Egr-2, T cells have normal effector function such as proliferation and production of IL-2 and IFNg. However, the production of IL-17 and differentiation of Th17 cells were reciprocally regulated in the presence or absence of Egr-2 in CD4 T cells. The disregulation of Th17 was associated with severity of the EAE in these mice suggesting that Egr-2 is important for the control of inflammatory plasticity of the effector T cells. We have discovered that Egr-2 interacted with Batf a transcription factor from AP1 family and suppressed its function. Batf partially represses AP1 activation, but activates IL-17 expression and Th17 differentiation. These findings conclude that Egr-2 controls Th17 mediated inflammation at least partly through inhibition of Batf function.
MicroRNAs (miR16 and miR146a) regulation of lupus development in NZB/W F1 mouse model (167.11)
Authors: Yuan Yao , Kotenko Serguei , Laurindo Fernanda , Salerno Erica , Raveche Elizabeth
Published: January 2023

SLE is a heterogeneous autoimmune disease and the specific cause remains unknown. IFNα has well defined positive effects in lupus disease development and by delivering exogenous IFNα to NZB/W F1 mouse accelerated lupus manifestations. Yet the effects of IFNλ, sharing partially common signaling pathways with IFNα, remains unknown in lupus. In the study, we treated B/W mice with IFNα or IFNλ for 12 week period and IFNα treated group developed much earlier and more severe lupus-disease than others. Moreover, combination with both cytokines enhanced the effects of either single treatment. At the time when only IFNα treated group developed proteinuria, this group of mice showed a pronounced decrease in regulatory B cells (B10)than others, in contrast, IFNλ treated group showed an expansion of B10. Furthmore, we wanted to investigate whether or not, as consequences of IFNs treatment, any specific microRNAs could be responsible for the cell type changes. In these B/W mice, splenic RNA were taken for microRNA microarray analysis. Results showed a few miRNAs up or downregulated in IFNs treated groups. To validate the result, miR16 and miR-146a were selected for realtime PCR.We found that almost in consistence with microarray, IFNλ treated had elevated level of both miRNAs than control, however, IFNα treated group showed underexpression of both miRNAs. Thus, it is likely that abnormal miRNAs changes due to IFNs treatment may drive the disease development in the lupus-prone B/W mice.
Induction of tolerance in the NOD mouse by WE14, a peptide of Chromogranin A (167.14)
Authors: He Jing , Delong Thomas , Bradley Brenda , Haskins Kathryn
Published: January 2023

Type 1 diabetes (T1D) is a chronic autoimmune disease mediated by autoreactive T cells. We recently reported that a new autoantigen, chromogranin A (ChgA), is targeted by diabetogenic T cell clones in the NOD mouse (Stadinski, Delong et al, Nat Immunol 2010). To investigate the induction of antigen-specific tolerance by WE14, a natural proteolytic cleavage product of ChgA, we tested whether this peptide could protect against adoptive transfer of diabetes in the NOD mouse model. Our data indicate that the production of inflammatory cytokines is less in both CD4 and CD8 T cells from NOD mice after WE14 immunization. Furthermore, co-transfer of spleen cells from NOD mice immunized with WE14 delayed disease transfer by spleen cells from diabetic NOD mice. We also demonstrated that when the WE14 peptide is coupled to antigen-presenting cells (APCs) by a chemical cross-linker, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI), transfer of disease into NOD recipients by the pathogenic Th1 T cell clone BDC-2.5 was totally prevented. These results suggest that WE14 may be a tolerogenic peptide in T1D. We are currently investigating the mechanism of this antigen-specific tolerance through analysis of T cells from the pancreas after adoptive transfer.
Fas and p21 suppress autoimmunity by regulating cell cycle progression of effector/memory T cells (167.19)
Authors: Balomenos Dimitrios , Daszkiewicz Lidia , Vazquez-Mateo Cristina
Published: January 2023

Fas drives activated T cell apoptosis in vitro, but lymphadenopathy and lupus development in Fas-deficient (lpr) mice cannot be explained only on the basis of the lpr T cell apoptotic defect. Unexplained also remains the in vivo hyperproliferation of T cells in lpr mice, including CD4+, CD8+ and CD4-CD8-TCR+ cells. To date, the effect of lpr T cell-hyperproliferation in both lymphadenopathy and lupus autoimmunity is not known. We investigate this point by generating T cell-specific p21-trangenic -lpr mice. p21, a cell cycle inhibitor and a lupus-like autoimmunity suppressor, regulates the expansion of memory but not of naïve T cells. p21 overexpression in lpr T cells did not affect primary T cell responses, but profoundly diminished lpr T cell proliferation after secondary activation, without affecting the compromised apoptotic cell death of lpr T cells. Importantly, in vivo, p21 overexpression restrains lpr T cell memory expansion, and results in reduced autoantibody production, glomerulonephritis and lymphadenopathy in lpr mice. p21 overexpression did not affect T cell memory in wt mice, pointing to a specialized role for p21 in hyperproliferating T cells. Overall we conclude that lpr T cell hyperproliferation is essential for the increased T cell memory, the autoimmunity and the lymphadenopathy development in lpr mice. Thus, decreasing the hyperproliferation of pathogenic T cells may be of therapeutic value for both Lupus and the Autoimmune Lymphoproliferative Syndrome.
α9β1 integrin-mediated signaling serves as an intrinsic regulator of pathogenic Th17 cell generation (167.2)
Authors: Kanayama Masashi , Morimoto Junko , Matsui Yutaka , Yoshida Toshimichi , Uede Toshimitsu
Published: January 2023

Th17 cells are recognized as primary pathogenic cells in autoimmune disorders such as rheumatoid arthritis. The differentiation of Th17 cells are tightly regulated by cytokines derived from APCs, receiving various signals including toll-like receptor (TLR) stimuli. Previously, we have reported that the interaction between matricellular proteins such as tenascin-C (TN-C) and osteopontin (OPN) and α9 integrin was capable of inducing the several cytokines from synovial cells, result in the development of arthritis. In this study, we have used collagen induced arthritis (CIA) model and found that increased numbers of α9 integrin positive conventional dendritic cells (cDCs) and macrophage were detectable in draining lymph node (dLN) shortly following first immunization, and these cells produced both TN-C and OPN. Alpha9 integrin-mediated signaling, induced by TN-C and OPN, enhanced Th17-related cytokine productions including IL-6 by cDCs and macrophages in synergy with TLR2 and 4 signaling, results in the promotion of Th17 generation at dLN. Moreover, Th17 cells, generated under blocking of α9 integrin-mediated signaling showed low level of CCR6 expression and impaired migration ability toward CCL20. In fact, the functional blockade of α9 integrin ameliorated the development of CIA. Thus, we have identified α9 integrin-mediated signaling by TN-C and OPN as a novel intrinsic regulator of pathogenic Th17 cell generation that contributes to the development of autoimmune arthritis.
Cross-regulation between TLR7, TLR8, and TLR9 in autoreactive B cells. (167.22)
Authors: Green Nathaniel , Debatis Michelle , Marshak-Rothstein Ann
Published: January 2023

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies against intracellular targets, frequently nucleic acid associated proteins. Toll-like receptors (TLR) 7 and TLR9 (which recognize single stranded RNA and DNA respectively) have been shown by our lab to be critical in the activation of autoreactive B cells. Genetic deletion of TLR7 significantly reduces disease, whereas genetic removal of TLR9 leads to exacerbated disease. To understand the basis for this difference, we examine the in vitro responses elicited by DNA- and RNA-containing immune complexes (ICs). TLR9-/- autoreactive B cells respond more strongly than TLR9+/+ B cells to RNA ICs, however TLR7-/- and TLR7+/+ autoreactive B cells respond comparably to DNA ICs. Since it has been shown that TLR7 mediated responses can significantly increase with a two-fold change in expression, we examined whether the absence of TLR9 changed the expression levels of TLR7, or vice versa. While TLR7 and TLR9 expression were unaffected by the absence of the other TLR, TLR7 knockout B cells expressed greatly enhanced levels of TLR8, which is thought to be suppressive. In fact, TLR7-/- B cells show a repressed response to DNA ICs in the presence of TLR8 ligands. This inhibitory effect of TLR8 on TLR9 mediated responses in TLR7 knockouts could partially explain the dramatically decreased disease seen in TLR7 deficient autoimmune prone mouse models.
Aicda mediates central tolerance in B cells (167.24)
Authors: Kuraoka Masayuki , Holl T. , Liao Dongmei , Womble Mandy , Kelsoe Garnett
Published: January 2023

Activation-induced cytidine deaminase (AID), the product of Aicda gene, is required for somatic hypermutation (SHM) and class-switch recombination (CSR) of immunoglobulin genes. Although crucial for humoral immunity, these processes exact genomic damage that often results in cell death, and occasionally oncogenesis. In mice, AID expression, SHM and CSR are thought to be restricted to mature B cells in germinal centers. We reported that developmentally immature B cells in mice and humans express low levels of AID, which support limited SHM and CSR. However, the physiological significance of AID expression and activity in these B-cell compartments is controversial. Here we show that low levels of AID in immature mouse bone marrow B cells suppress the development of autoreactivity. Aicda deficient mice exhibit significantly increased serum autoantibody and reduced capacity to purge autoreactive immature/transitional B cells; the clonal deletion and heavy chain receptor editing characteristics of autoimmune 3H9 VDJ knock-in mice is significantly impaired in the absence of AID expression. In vitro, AID deficient immature/transitional B cells are more resistant to anti-IgM induced apoptosis than their normal counterparts. Thus, early AID expression in immature B cells plays a fundamental and unanticipated role in purging self-reactive B cells during their development in the bone marrow. We are currently investigating signals that induce AID expression in immature B cells in vitro.
Trp53 negatively regulates autoimmunity via the STAT3-Th17 axis (167.27)
Authors: Cui Yan , Zhang Shuzhong , Zheng Mingquan , Kibe Ryoko , Huang Yunping , Marrero Luis , Zieske Arthur , Iwakuma Tomoo , Kolls Jay
Published: January 2023

Emerging evidence suggests that the tumor suppressor p53 is also a crucial regulatory factor for many physiological processes. Previous observations indicate that p53 negatively regulates inflammation via suppressing inflammatory cytokine production by antigen presenting cells. However, the role of p53 in regulating autoimmune effector T cells, such as IL-17 producing Th17 cells, is largely unknown. Here, we demonstrate that p53nullCD45.1 mice spontaneously develop autoimmunity associated with a significant increase in Th17 cells in their lymphoid and pathological tissues as well as elevated levels of inflammatory cytokines IL-6 and IL-17 in serum. This increase in Th17 response results largely from an increased sensitivity of p53nullCD45.1 T cells to IL-6 induced STAT3 phosphorylation. Administration of a STAT3 inhibitor to p53nullCD45.1 mice reduced the frequency of Th17 cells and alleviated autoimmune pathology. This is the first report, to our knowledge, revealing the contribution of T-cell p53 in the suppression of autoimmunity by controlling Th17 differentiation. This study suggests that p53 serves as a guardian of proper immunological functions and that the p53-STAT3-Th17 axis might be a therapeutic target for autoimmunity.
Negative feedback regulation of encephalitogenic Th1 cells by IFNγ in experimental autoimmune encephalomyelitis (167.28)
Authors: Sestero Christine , Naves Rodrigo , Singh SimerPreet , De Sarno Patrizia , Raman Chander
Published: January 2023

Ifngr1-/- mice develop more severe experimental autoimmune encephalomyelitis (EAE) with delayed onset compared to wild-type (WT) mice. Enhanced disease severity in Ifngr1-/- mice was associated with elevated numbers of both Th1 and Th17 cells in the central nervous system. Elevated Th17 population was expected, but the expanded numbers of Th1 cells in Ifngr1-/- mice contradicts the paradigm that IFNγ signaling is a positive feedback loop for Th1 expansion. Furthermore, cultures of encephalitogenic T cells isolated from these mice restimulated under Th1 skewing conditions contained several fold greater numbers of IFNγ-expressing Th cells than WT T cell cultures. These observations led us to investigate the possibility that IFNγ participates in a negative feedback loop associated with Th1 differentiation. Addition of suboptimal levels of neutralizing anti-IFNγ mAb during in vitro polarization of encephalitogenic WT T cells to Th1 cells led to at least a two-fold greater number of CD4+IFNγ+ T cells. Suboptimal neutralization of IFNγ didn’t alter the generation and/or expansion of Th1 cells when encephalitogenic T cells were obtained from Ifngr1-/- or Stat1-/- mice, confirming the requirement for intact IFNγ signaling. Contrary to the traditional dogma, these data suggest that IFNγ can negatively regulate Th1 differentiation, which may partly explain the opposing biology of IFNγ in EAE and multiple sclerosis.
CD5 modulates regulatory T cell function, proliferation, and survival. (168.12)
Authors: McGuire Donald , Sestero Christine , De Sarno Patrizia , Raman Chander
Published: January 2023

We previously showed that CD5-/- mice develop less severe experimental autoimmune encephalomyelitis (EAE). This is unexpected since CD5-/- T cells are hyperresponsive to myelin peptide immunization. The decreased severity is partially due to loss of CD5-dependent survival signals that are mediated by the activation of the serine/threonine kinase CK2. EAE in CD5 mutant knock-in mice (CD5ΔCK2BD), in which the ability to bind/activate CK2 is abrogated, was also less severe and recapitulates the findings in CD5-/- mice. CD5-/- Treg cells are hypersuppressive in in vitro suppression assays and therefore may also contribute to lower EAE severity in the CD5-/- mouse. This led us to address if CD5 regulates Treg cell function through activation of CK2. In contrast to the hypersuppressive activity of Treg cells from CD5-/- mice, Treg cells from CD5ΔCK2BD mice were less efficient in their ability to suppress responder CD4+ T cells than WT Treg cells. The proliferation of CD5-/- Tregs in response to stimulation with anti-CD3 in the presence of APCs and IL-2 was greater than WT and CD5ΔCK2BD. From these results we infer that the CD5-dependent CK2 activation pathway is a positive regulator of Treg suppressive activity. In contrast, the inhibitory (ITIM) domain is a negative regulator of Treg biology and proliferation. The combined signaling pathways from these two domains of CD5 balance Treg function and maintain homeostasis.
Controlling viral immunoinflammatory lesions by modulating aryl hydrocarbon receptor signaling (168.19)
Authors: Veiga Parga Tamara , Suryawanshi Amol , Rouse Barry
Published: January 2023

Ocular HSV-1 infection can cause a blinding CD4+ T cell orchestrated immunoinflammatory lesion called Stromal Keratitis (SK). Lesion severity is affected by the balance of proinflammatory effector T and regulatory T cells (Treg). In this report we show that expanding Treg activity using TCDD (2, 3, 7, 8- Tetrachlorodibenzo-p-dioxin), a ligand for the AhR receptor, results in the differential expansion of Treg in the eye and a resultant reduction in the extent of inflammatory lesions. Treatment of HSV infected mice with TCDD either on day 1 or day 5 pi, diminished the severity of SK lesions as well as the extent of corneal angiogenesis. The observed effect was mainly due to the changed balance of CD4+ T cells in the cornea that were IFN-γ or IL17 cytokine producers compared to those that were Foxp3+ Tregs. The change was explained by the significantly increased apoptosis of activated effector T cells but not of Tregs. Additionally, effector T cells from HSV infected mice proliferated significantly less in TCDD treated mice compared to control mice. Furthermore the addition of TCDD to the culture of antiCD3/CD28 stimulated naïve CD4+ T cells caused significant induction of Tregs. In conclusion, our results demonstrate that modulation of AhR signaling by TCDD represents a valuable approach to expand Treg and inhibit Th1 and Th17 cells to control HSV induced blinding lesions.
Regulatory T cells enhance mast cell production of IL6 via a TGFβ dependent innate mechanism (168.20)
Authors: Ganeshan Kirthana , Bryce Paul
Published: January 2023

Recently, Tregs have been proposed to limit mast cell degranulation upon antigen stimulation. However, mast cells also produce many cytokines that regulate both innate and adaptive immunity. Here we show that Tregs inhibit mast cell (BMMC) degranulation but, surprisingly, promotes IL6 in response to innate (LPS or PGN) and adaptive (IgE) activation. This required cell-cell contact and did not require activation via the TCR. Blockade of OX40/OX40L pathway inhibited the Treg effects on BMMC degranulation but had no effect on IL6 production. Conversely, blockade of surface bound-TGFβ ablated IL6 production, suggesting that Tregs influence mast cell responses via distinct mechanisms. Furthermore, TGFβ was sufficient to promote enhanced IL6 but had no effect on degranulation. In innate immunity, IL6 and Tregs have been suggested to influence neutrophil clearance and resolution of inflammation. In an LPS-driven model of lung injury, Treg depletion elevated lung neutrophilia in response to LPS. Similarly, mast cell deficient mice displayed significantly elevated neutrophilia in the lung that was fully corrected upon reconstitution with wildtype mast cells but not by IL6 deficient mast cells, suggesting a critical role for mast cell derived IL-6 in neutrophil clearance during innate inflammation. Our data suggests that Tregs are important in limiting the severity of the inflammatory response during innate activation and that they may do so by promoting mast cell production of IL-6.
Functional plasticity of regulatory T cells (168.7)
Authors: R. Pillai Meenu , Collison Lauren , Finkelstein David , Rehg Jerold , Boyd Kelli , Workman Andrea , Griffith Thomas , Ferguson Thomas , Vignali Dario
Published: January 2023

Regulatory T cells (Tregs) play a pivotal role in maintaining immune homeostasis, preventing autoimmunity, controlling exaggerated immune responses against infections and suppressing anti-tumor immunity. Tregs utilize multiple mechanisms to mediate their function. However, the number of mechanisms critical to exert maximal suppressive activity is unclear. It is conceivable that suppressive mechanisms could be tightly regulated by cross-regulatory networks. It has been shown that interleukin 10 (IL10) and IL35 have critical role in Treg function. Therefore, we assessed the functional capacity of Tregs lacking the ability to secrete IL10 and IL35. Surprisingly, our study revealed that Tregs lacking both IL10 and IL35 (Ebi3-/-Il10-/- or Il12a-/-Il10-/-) were fully functional and were able to suppress Tconv cells both in vitro and in vivo . Absence of IL10 and IL35 was compensated by an increase in cathepsin E (CTSE), influencing the release of soluble TRAIL and thereby resulting in suppression of target cells in a TRAIL-dependent manner. Inhibiting TRAIL activity disrupted the function of Ebi3-/-Il10-/- Tregs. The loss of suppressive function was maximal in Tregs lacking IL10, IL35 and TRAIL (Ebi3-/-Il10-/-Tnfsf10-/-Tregs). These data suggests the existence of cross-regulatory networks which controls the utilization of suppressive factors, thereby maintaining Treg functional plasticity.
Surfactant Protein-A modulates induction of regulatory T cells in the lung via TGFβ (168.8)
Authors: Mukherjee Sambuddho , Giamberardino C. , Thomas J. , Ledford J. , Evans K. , Pastva A. , Wright Jo Rae
Published: January 2023

TCR signaling plays a critical role in regulatory T cell (Treg) development. However, the mechanism for tissue specific induction of Tregs in the periphery remains unclear. Surfactant protein A (SP-A) is a major immune defense protein in the lung. To determine if SP-A contributes to the normal non-inflammatory status mediated by Tregs in the lung, T cells were activated with various stimuli. Compared to WT mice, on ex vivo culture, T cells from lungs of SP-A deficient mice have impaired expression of Foxp3 and fewer CD25+Foxp3+ Treg phenotype cells. These phenotypic effects could be rescued by the addition of exogenous SP-A. Even as cell cycle progression is blocked, both IL-2 levels as well as active TGFβ levels increase on extended culture. In addition, kinetic suppression assays demonstrate that SP-A enhances the frequency of functional Foxp3+Tregs in responder T cell populations in a TGFβ dependent manner. To induce Tregs in the lung in vivo, we modified the extended LPS model described by d’Alessio et al (1). While the proportion of Tregs increased ≈160% in WT mice ~8d post-exposure, SP-A-/- mice showed a much more modest 50% increase over their near identical baselines. We also observed a strong correlation between FR4 and Foxp3 expression in functional Tregs induced by SP-A. Together, these findings suggest that SP-A exerts long-term effects on T cell immune function by the induction of Tregs during activation. (1) D’Alessio et al J Clin Invest. (2009) 119: 2898
NIK overexpression intrinsically inhibits regulatory T cell function (168.9)
Authors: Polesso Fanny , Murray Susan , Rowe Alexander , Parker David
Published: January 2023

Activation of the non-canonical, or alternative, NF-κB pathway depends on NF-κB-inducing kinase (NIK). We have shown that NIK is intrinsically essential for T cells to cause lethal graft-versus-host disease and to respond to OX40 costimulation. We went on to investigate the effects of NIK overexpression in T cells by crossing inducible NIK transgenic mice to CD4-Cre inducer mice and found that modest NIK overexpression limited to T cells (CD4-NIKtg) caused fatal autoimmunity by 4-weeks of age, resembling the phenotype described in FoxP3-/- and scurfy mice. We hypothesized that NIK overexpression in regulatory T cells (Treg) affects their suppressive phenotype. Here we show that NIKtg Treg cells obtained from healthy bone marrow chimeras fail to suppress WT conventional T cell (Tconv) proliferation in vitro. In addition, NIKtg Tconv obtained from these mice are resistant to suppression by WT Treg cells. Transient NIK overexpression in vitro also represses Treg cell function, and preliminary experiments suggest that NIK overexpression destabilizes the Treg phenotype. Lastly, we investigated the effect of more extreme NIK overexpression using a mutant NIK transgene that is not targeted for degradation (NIKΔTRAF3). Contrary to modest NIK upregulation, excessive NIK accumulation is lethal to T cells. Together, these results show that NIK plays a critical T-cell intrinsic role in both Tconv and Treg, and that its tight regulation is crucial for T cell regulation and survival.
Intracranial administration of human activated NK cells in a xenogeneic model of orthotopic glioblastoma (169.18)
Authors: Ames Erik , Hagino Takeshi , Gorin Frederic , Fragoso Ruben , Murphy William
Published: January 2023

Glioblastoma multiforme (GBM) is a highly invasive end-stage glioma which conveys an extremely poor prognosis. Based on our in vitro data showing very powerful anti-tumor activity of cytokine activated human natural killer cells against the GBM cell line U87MG, we sought to develop a xenograft model of GBM engraftment into NSG mice which would be treated with an intracranial administration of human NK cells. Such NK cell-based therapies allow for autologous NK cells to be utilized, thus minimizing toxicities. While rhIL-2 administered every other day significantly increased NK cell engraftment compared to saline treated controls, a single hydrodynamic injection of human IL15-encoding plasmid produced a far superior engraftment in the brain, spleen, liver, bone marrow and peritoneal fluid 4 days after injection with no toxicities observed. Activated human NK cells were then administered to mice bearing U87MG-luciferase gliomas five days after the orthotopic injection of tumor cells along with IL-2. While NK cell therapy provided a significant tumor volume reduction in the first several weeks after administration, this therapy was unable to protect mice in the long-term from tumor growth and death. Future studies will utilize IL-15 as well as IL-2 in a similar model. These combined data demonstrate the feasibility of NK cell-based therapies for gliomas as well as their potential role as a secondary treatment to target chemotherapy- and irradiation-resistant tumor cells.
DRAK2 blockade leads to transplant tolerance by simultaneous deletion of effector T cells and enrichment of regulatory T cells (169.3)
Authors: Weist Brian , Walsh Craig
Published: January 2023

DRAK2 is a serine/threonine kinase that has previously been shown to negatively regulate activation of T cells through the T cell receptor. Additionally, T cells lacking Drak2 are hypersensitive to low levels of stimulation, but are resistant to T cell mediated autoimmunity. Furthermore, Drak2-/- mice have no defects in viral immunity, making DRAK2 a novel target to replace current non-specific immunosuppressants used following transplantation. We hypothesized that Drak2 deficient T cells die after activation, and tested the ability of these T cells to respond to allogeneic stimulation. Using in vitro mixed lymphocyte reactions, Drak2-/- CD8+ T cells exhibited defects in accumulation after 5 days of allostimulation compared to wildtype T cells in vitro. Additionally, in vivo, allogeneic tumors grew uncontrolled by Drak2-/- T cells, and allogeneic skin graft rejection was significantly delayed. Overexpression of anti-apoptotic Bcl-xL in Drak2-/- T cells restored rejection of allogeneic tumors. Additionally, Drak2-/- mice had increased proportions of Tregs, and depletion of Tregs in a skin transplant model significantly enhanced Drak2-/- alloresponses. As a consequence of deletional tolerance, allogeneic memory was drastically reduced post transplant in Drak2-/- mice. Overall, these results demonstrate that DRAK2 plays a significant role in allograft responsiveness and highlight this kinase as a potential pharmacologic target in efforts to block allograft rejection.
Altered T cell profiles in uterine draining lymph nodes of mice with preterm labor (169.31)
Authors: Nijagal Amar , Wegorzewska Marta , Le Tom , Tucker Andrew , Tang Qizhi , MacKenzie Tippi
Published: January 2023

Background: Preterm labor (PTL) is a devastating consequence of fetal interventions such as in utero hematopoietic cell transplantation (IUHCTx). We previously found that PTL after allogeneic IUHCTx is decreased in mice lacking T cells. We hypothesized that the T cell profile in the uterine draining lymph nodes (uLN) would be altered in mothers with PTL. Methods: Mothers carrying semi-allogeneic fetuses underwent IUHCTx at E14.5. At E18.5, we quantified maternal CD4+, CD8+, and CD4+Foxp3+ T cells and measured the production of IL2, IL4, IFN gamma, and IL17 in the uLN, spleen, or non-draining lymph nodes (ndLN). Results: We found equivalent numbers of CD4+ and CD8+ T cells when comparing mothers with and without PTL. We observed a significant increase in the proportion of CD4+Foxp3+ T cells only in the uLN of mothers who experienced complete fetal loss. We also observed equivalent IL2, IFNγ, and IL17 production between all groups. IL4 production by CD4+Foxp3+ T cells was, however, significantly elevated in the uLN of mothers who aborted their entire litter. Conclusions: PTL after fetal intervention leads to expansion of the CD4+Foxp3+ T cell pool in the uLN. The production of IL4 by CD4+Foxp3+ T cells in the uLN suggests that the conversion of these cells to Th2 T effectors may be one potential mechanism by which maternal T cells participate in PTL. Our results support the role of maternal T cells in the pathogenesis of PTL.
IL6 is necessary for the progression of experimental autoimmune myocarditis to dilated cardiomyopathy (171.11)
Authors: Legault Jillian , Baldeviano G. , Barin Jobert , Wu Lei , Ong SuFey , Talor Monica , Cihakova Daniela , Rose Noel
Published: January 2023

Previous findings in our lab have showed that IL17A deficient animals are protected from the progression of experimental autoimmune myocarditis (EAM) to dilated cardiomyopathy (DCM). These findings showed an increase of systemic IL6 in the WT animals that progressed to heart failure, whereas the protected IL17A deficient animals had lower IL6 levels. This was the only cytokine shown to differ consistently between the two groups at both day 14 and 21. We sought to address the question of whether systemic IL6 levels can mediate disease and is therefore critical for DCM development. To investigate the IL17A requirement for DCM development through its induction of IL6, we used two experimental approaches. First, we over-expressed IL6 in WT animals with EAM through hydrodynamic gene delivery on day -1 prior EAM induction. WT mice, which over-expressed an IL6 insert, showed worsening of heart function by echo on day 54 compared to controls with empty plasmid. Second, we blocked IL6R signaling using an anti-IL6R monoclonal antibody (mAb) in WT BALB/c mice with EAM. In mice administered the anti-IL6R mAb on days 6 and 13 of EAM, the mice were protected from heart failure and DCM development by day 60 of disease compared to animals receiving isotype control antibodies. Our data suggest that IL6 early during EAM is necessary for the development of DCM.
Effects of apoptotic cell accumulation caused by Mer deficiency on germinal center B cells and follicular helper T cells (171.12)
Authors: Khan Tahsin , Wong Eric , Rahman Ziaur
Published: January 2023

Mer (MerTK) is a member of the Tyro-3/Axl/Mer (TAM) subfamily receptor tyrosine kinases which facilitates phagocyte clearance of apoptotic cells (ACs). We previously showed that Mer expression in germinal centers (GCs) occurs predominantly on tingible body macrophages (TBMϕs). Furthermore, deficiency in Mer (Mer-/-) results in accumulation of ACs in GCs leading to augmented antibody-forming cell (AFC), GC and Th1-skewed IgG2 Ab responses. Here we show that elevated AFC, GC and IgG2 Ab responses in Mer-/- mice were due to increased proliferation of B cells and higher number of TFH cells in Mer-/- GCs. AC accumulation in Mer-/- GCs was also associated with elevated long-lived primary and secondary AFC, GC B cell, TFH cell and IgG2 Ab responses compared to controls. Interestingly, AC accumulation in Mer-/- GCs was not due to increased B cell apoptosis, rather due to a defect in AC clearance. Finally, by evaluating GC TBMϕ expression of several other bridging molecules and receptors (i.e., Axl, Tyro-3, Mfge-8 and Tim-4) involved in AC clearance, we ruled out the contribution of the potential alteration in the expression of these molecules/receptors to AC accumulation in Mer-/- GCs. These data indicate that accumulation of ACs in Mer-/- GCs is primarily due to Mer deficiency. Together these results highlight the important role of AC clearance by Mer in regulating GC B cell and TFH cell responses and in maintaining peripheral B cell tolerance.
Pain and inflammation coincide in a collagen-induced arthritis model in rats (171.17)
Authors: McCarthy Richard , Goess Christian , Rundell Lian , Cuff Carolyn , Prisca Honore , Andrew Long
Published: January 2023

Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation, swelling and destruction. In patients diagnosed with RA, these symptoms manifest as pain and lead to fatigue, physical impairment and loss of function. This can be modeled pre-clinically using collagen induced arthritis (rat CIA). Historically, increased swelling of the hind paws measured by plethysmography, and inflammation measured by histology in the ankle joints of arthritic rats have been used to evaluate the effects of potential anti-inflammatory therapies. In the present study, we set out to understand the relationship between joint inflammation and pain. To evaluate knee inflammation, we employed a technique that involves lavaging the knee joint to determine the characteristics of the inflammation (cells and cytokines). Knees of arthritic animals had increased cellularity and pro-inflammatory cytokines including IL-1b and IL-6, as well as increases in the neutrophil chemokine KC. To evaluate pain, we used Von Frey monofilaments to elicit mechanical allodynia as measured by paw withdrawal. We initially observed increased paw withdrawal activity on day 11, with maximal pain responses by day 15 that were maintained through day 18. These findings indicate that mechanisms of pain and inflammation are coordinate in the rat CIA model. Measurement of pain in preclinical models of RA will aid in the development of future effective therapies.
Potential roles for soluble MD-1 in disease progression of autoimmune prone MRLlpr/lpr mice (171.2)
Authors: Nagai Yoshinori , Yanagibashi Tsutomu , Watanabe Yasuharu , Ikutani Masashi , Kariyone Ai , Tsuneyama Koichi , Hirai Yoshikatsu , Takatsu Kiyoshi
Published: January 2023

MD-1 is a secreted protein that forms a complex with radioprotective 105 (RP105) on B cells, macrophages and dendritic cells. This complex plays a crucial role in lipopolysaccharide (LPS) recognition by B cells. Disease progression is known to improve in RP105-deficient lupus-prone MRLlpr/lpr mice. Furthermore, a soluble form of the homologous MD-2 protein is present in the plasma of septic patients and can opsonize gram-negative bacteria in cooperation with Toll-like receptor 4. We have now established a flow cytometry-based assay to detect endogenous soluble form of murine MD-1 (sMD-1) and explored potential roles in autoimmunity. The assay was quantitative and validated with sera from MD-1-deficient mice. Interestingly, heat-inactivated murine serum diminished the ability of sMD-1 to bind RP105. The sMD-1 was secreted by bone marrow-derived macrophages from C57BL/6 mice. Autoimmune prone MRLlpr/lpr mice had higher levels of serum sMD-1 than control MRL+/+ mice, and levels markedly increased with disease progression. Expression of MD-1 but not MD-2 mRNA increased with age in the liver and kidney of MRLlpr/lpr mice. Finally, immunohistochemical analyses revealed that MD-1 was present in infiltrated macrophages within perivascular lesions of the MRLlpr/lpr kidney. This correlation suggests that sMD-1 may contribute to pathogenesis in this autoimmune disease model mouse. Also, sMD-1 may be feasible to monitor autoimmune disease severity.