bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of myeloid-derived suppressor cells in type 1 diabetes (167.16)
Authors: Yen Chun-Yu , Chen Shu-Hsia , Pan Ping-Ying , Pan Chien-Yuan , Yang Wen-Chin
Published: January 2023

Type 1 diabetes is an autoimmune disease which are mainly caused by the autoimmune destruction of pancreatic islets. One of the most effective immunotherapy for type 1 diabetes (T1D) relies on the induction of peripheral tolerance. Accumulating data show that myeloid-derived suppressor cells (MDSCs) can suppress immune response through multiple strategies. In the study, we first showed MHC-dependent Ag presentation is required in MDSC-mediated Treg induction or T-cells suppression. The mechanism of action of MDSCs included the secretion of anti-inflammation cytokines, induction of CD4+CD25+Foxp3+ Treg and suppression of T-cell proliferation to create immune tolerance. Next, we examined the role of MDSCs in murine models. Adoptive transfer of MDSCs reduced diabetes by 75% compared with control group in RIP-HA/Rag2-/- mice. We further confirmed the protective role of MDSCs in NOD/scid mice. NOD/scid mice were injected with inflammatory T cells from diabetic NOD mice in the presence of MDSCs. Consistently, single-dose treatment of MDSCs showed significant long-term protection (60% remained diabetes free) and the protection effect is dose-dependent. Overall, the data suggest that MDSCs can suppress T1D via regulation of T cell-mediated tolerance. MDSCs have great potential to treat T1D and other autoimmune diseases.
B-cells are a therapeutic target for IFNβ in relapsing-remitting multiple sclerosis (167.17)
Authors: Marcus Karen , Ramgolam Vinod , Sha Yonggang , Choudhary Neelima , Troiani Luigi , Chopra Manisha , Markovic-Plese Silva
Published: January 2023

Background: IFNβ-1b is a first-line immunomodulatory therapy for relapsing remitting multiple sclerosis (RRMS), however its effects on B-cells have not been characterized. Methods: We have characterized the effect of the IFNβ-1b administered to RRMS patients on the B-cells’ antigen presenting capacity using allogenic MLR and cytokine secretion using ELISA. B-cells were derived from 7 treated and 7 untreated RRMS patients Results: B-cells derived from RRMS patients treated with recombinant subcutaneously injected IFNβ-1b revealed that they induced a significantly lower proliferative response in allogenic mixed lymphocyte reaction (MLR) than the B-cells from untreated patients (p=0.018). Further confirming the IFNβ-1b in-vitro induced changes in B-cell cytokine secretion that collectively inhibit Th17 cell differentiation, B-cells derived from the IFNβ-1b-treated patients secreted significantly lower levels of IL-1β(p=0.046) and IL-23(p=0.028), and higher levels of IL-12(p=0.046) and IL-27(p=0.028) in comparison to the B-cells derived from untreated RRMS patients.Conclusion: IFNβ-1b exerts its therapeutic effects in part by inhibiting B-cells’ antigen presenting capacity and by modifying their secretion of Th17-regulatory cytokines, which contribute to the autoimmune pathogenesis of RRMS.
CRP-mediated reduction of EAE severity is FcγRIIb dependent (167.18)
Authors: Wright Tyler , Jones Nicholas , Ramos Theresa , McCrory Mark , Szalai Alexander
Published: January 2023

Blood levels of human C-reactive protein (CRP), the prototypical acute phase protein, elevate in response to inflammatory stimuli. Previously, our lab has shown that elevation of transgene-expressed human CRP is protective in mice with experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis (MS). Specifically, EAE onset is delayed and its clinical severity reduced in human CRP transgenic mice (CRPTg) compared to wildtype mice. Since human CRP is known to bind human and mouse Fc receptors (FcR), we sought to determine which FcRs, if any, might be required for human CRP-mediated protection from EAE. To do so, we induced EAE in CRPTg mice with normal FcR expression versus CRPTg mice deficient in expression of FcγRI, FcγRIIb, or FcγRIII. Time of EAE onset and clinical severity of disease were monitored for 30 days. We observed that in CRPTg mice deficient in the excitatory Fc receptors, FcγRI and FcγRIII, CRP’s full protective effect was still manifest. In stark contrast, for CRPTg mice deficient in the inhibitory Fc receptor, FcγRIIb, no human CRP dependent protection was evident. Furthermore, in wildtype mice with established EAE subcutaneous administration of purified human CRP stalled disease progression, whereas the same treatment did not impede EAE progression in FcγRIIB deficient mice. These results suggest that a human CRP→mouse FcγRIIB axis is responsible for the observed protection of human in the CRPTg model.
Indispensable role of B cells in mediating protective effects of estrogen against EAE (167.20)
Authors: Bodhankar Sheetal , Wang Chunhe , Vandenbark Arthur , Offner Halina
Published: January 2023

Increased remissions in multiple sclerosis (MS) during pregnancy suggest elevated levels of sex steroids exert immunoregulatory activity. Estrogen (E2) protects against experimental autoimmune encephalomyelitis (EAE), but its mechanism is unclear. Depletion of B cells prior to induction of EAE exacerbates disease severity. The purpose of this study was to evaluate role of B cells in E2-induced protection against EAE. B cell deficient (μMT-/-) mice were used to study whether absence of B cells abrogated E2-mediated protection against EAE. Mice were sham-treated or implanted with E2 pellets, 8 days prior to immunization with mMOG35-55 peptide with CFA/Ptx and the disease course was followed. EAE-protective effects of E2 were totally lost in μMT-/- mice, with no reduction in disease severity, cellular infiltration or pro-inflammatory factors in CNS compared to controls. E2 treatment of WT mice selectively increased percentage expression of PD-L1 on B cells and IL-10-producing CD1dhighCD5+ regulatory B cells. Direct treatment of B cells with E2 significantly reduced proliferation of MOG35-55-specific T cells that required ERα. Thus, B cells play an indispensable role in E2-mediated protection involving direct E2 effects on regulatory B cells. E2-primed B cells may play a crucial regulatory role in MS and have strong implications in current MS therapies that cause B cell-depletion.
Adipose tissue-derived mesenchymal stem cells ameliorate experimental autoimmune hearing loss (167.23)
Authors: Zhou Bin , Yuan Jingdong , Zhou Yixuan , Ghawji Maher , Yoo Tai
Published: January 2023

Objective Autoimmune inner ear disease (AIED) is characterized by progressive, bilateral although asymmetric, and sensorineural hearing loss; there are no specific therapeutic strategies to treat AIED. However, human adipose-derived mesenchymal stem cells (hASCs) are a promising approach for the treatment of autoimmune diseases due to their immune modulatory effects and regenerative potential. Methods BALB/c mice underwent β-tubulin immunization to develop EAHL; and then administered hASCs or PBS intraperitoneally once a week for 6 consecutive weeks. Results Systemic infusion of hASCs significantly improved hearing function in established EAHL. hASCs decreased the proliferation of antigen-specific Th1/Th17 cells and induced the production of anti-inflammatory cytokine IL-10. hASCs also induced the generation of antigen-specific CD4+CD25+Foxp3+ Treg cells, with the capacity to suppress autoantigen-specific T cell responses. Moreover, we tested the presence of HLA-ABC by confocal analysis found that hASCs migrated to inner ear including the stris vascularis and spiral ligament. By histology, we confirmed that hASCs treatment correlated with the repair process of morphological recovery in the inner ear, while the cochlea of control mice remained seriously damaged. Conclusion We show that hASCs have therapeutic potential by a bimodal mechanism, by suppressing the autoimmune response of EAHL as well as by inducing repair of cochlea in animals with established disease.
Programmed death-1 promotes tolerance by blocking the T cell receptor-induced stop signal. (167.26)
Authors: Fife Brian , Pauken Kristen , Obu Takashi
Published: January 2023

Programmed death-1 (PD-1) and CTLA-4 are inhibitory receptors expressed on activated T cells that are critical for preventing autoimmunity. However, the biological context in which PD-1 and CTLA-4 control T cell tolerance is not fully understood. In this study we used an antigen specific diabetes model and T cell dynamic motility during the induction and maintenance of tolerance in vivo by two-photon imaging. Using this system we demonstrate that unlike naive or activated islet antigen-specific T cells, tolerized islet antigen-specific T cells moved freely and did not swarm around antigen-bearing dendritic cells (DCs) in pancreatic lymph nodes. However, blocking interactions between PD-1 and PD-L1 altered the dynamic T cell motility and caused the breakdown in peripheral tolerance resulting in autoimmune diabetes. Neutralizing CTLA-4, on the other hand, did not impact tolerized T cell motility in vivo or clinical disease. The ability of PD-1 to influence the T-cell stop signal was antigen dependent, since T cell motility and T cell-DC contact times were unaffected by PD-L1 neutralization in non-draining (inguinal) LNs. These data provide in vivo evidence that PD-1 -PD-L1 interactions are critical for the maintenance of peripheral tolerance and prevention of autoimmunity. Further, the effects of PD-1 are fundamentally distinct from those of CTLA-4 and suggest unique mechanisms of action for these two inhibitory receptors.
miRNA modulation of T cell phenotype in Multiple Sclerosis (167.3)
Authors: Guerau-de-Arellano Mireia , Smith Kristen Marie , Godlewski Jakub , Lawler Sean , Whitacre Caroline , Racke Michael K. , Lovett-Racke Amy E.
Published: January 2023

Pro-inflammatory T cells mediate autoimmune demyelination in Multiple Sclerosis (MS). However, the factors driving their development and MS susceptibility are obscure. We investigated how miRNAs, newly described as post-transcriptional regulators of gene expression, contribute to pathogenic T cell differentiation in MS. We profiled miRNA expression in naïve CD4+ T cells of healthy (n=8) and untreated MS (n=22) donors. miR-128, miR-27a/b and miR-340 were increased in naïve CD4+ T cells from MS patients. miR-128, -27a/b and -340 directly suppressed the pro-Th2 factor Bmi1, resulting in decreased GATA-3 expression. miR-340 additionally suppressed the Th2 cytokine IL-4. Overall, these miRNAs suppressed Th2 and enhanced pro-inflammatory Th1 responses. In addition, transfection of myelin-specific T cells with these miRNAs worsened EAE while treatment of MS patient cells with miRNA inhibitors led to the restoration of Th2 responses. These findings link miR-128, -27ab and -340 overexpression in MS patients’ naïve CD4+ T cells to the pro-inflammatory T cell differentiation observed in MS and illustrate the biological significance and therapeutic potential of these miRNAs.
Altered miRNA expression in splenic lymphocytes in autoimmune-prone murine models with active lupus disease (167.9)
Authors: Dai Rujuan , Heid Bettina , Khan Deena , Ahmed S. Ansar
Published: January 2023

There is markedly increased recognition of physiological and pathological roles of miRNAs in immune system that impacts health and disease. While numerous dysregulated miRNAs were identified in autoimmune diseases, the significance of most disease-related miRNAs remains elusive. We have recently reported that selective miRNAs including the miR-182-96-183 cluster were commonly dysregulated in splenocytes from MRL-lpr, C57BL/6-lpr and NZB/WF1 mice when compared to control mice. Also, we have shown that the dysregulation of these miRNAs was associated with lupus disease development. In current study, we investigated the potential pathogenic contribution of lupus-associated miRNAs. The previous reports have shown that miR-182 and miR-96 target Foxo1 and Foxo3a, which play critical roles in controlling T lymphocyte homeostasis and tolerance. Consistent with the upregulation of miR-182-96-183, we found that Foxo3a and Foxo1 protein levels were inhibited in splenocytes from MRL-lpr mice when compared to MRL mice. However, the Real-time RT-PCR revealed that only Foxo1 mRNA, but not Foxo3a mRNA level was decreased in splenocytes from MRL-lpr mice, suggesting a post-transcriptional regulation of Foxo3a in splenocytes from lpr mice. Together, our data suggested a potential pathogenic contribution of miR-182 to lupus by targeting Foxo1 and Foxo3a, which could conceivably lead to the abnormal lymphocytes activation and autoimmunity development.
Immune regulatory mechanism in mouse IBD model (168.26)
Authors: Cho Kyung-Ah , Suh Jee Won , Woo So-Youn
Published: January 2023

Inflammatory bowel disease (IBD) in humans, such as Crohn’s disease and ulcerative colitis, is characterized by chronic inflammation with dysregulated mucosal immune tolerance. Some reported that immune responses to intestinal microbiota are critical to develop IBD and NOD-like receptor-mediated inflammasome is related to epithelial integrity. In order to investigate dysregulated mechanism focusing on the relationship between inflammasome and regulatory cells in IBD, we used a 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model in WT-C57BL/6 mice and caspase-1-/- mice. In addition, to observe Th2-dependent immune responses in IBD mouse, we also used WT-Balb/c mice and eosinophil-deficient ΔdblGATA mice. As a result, mice treated TNBS had significant changes in clinical parameters, weight loss, and colonic weights compared with those of sham control mice. Histologic analysis also showed severe inflammation in intestines of TNBS treated mice. We found that deficiency of eosinophil or IL-1β had resistance in initial stage of disease progression, represented by survival score and body weight loss and we also noted that proportion of Foxp3+ cells and myeloid-derived suppressor cells (MDSCs) increased in above TNBS-treated ΔdblGATA and caspase-1-/- mice. These results provide us another mechanism of immune modulation in the pathogenesis of intestinal inflammation.
In vitro exposure to the common water contaminant Atrazine inhibits CD4+ T cell proliferation and increases the frequency of FoxP3+ cells (168.5)
Authors: Wetzel Scott , Thueson Lindsay
Published: January 2023

Atrazine (ATR) is the second-most widely applied herbicide used in the United States. The US EPA has reported that ATR and its primary metabolite are the most common ground water contaminant in the US. While the majority of the US population routinely ingests ATR, much remains to be learned about its biological activity. Atrazine is a potent phosphodiesterase inhibitor and is classified as an estrogen-disrupting compound that has been linked to reproductive abnormalities and cancer development. Previous studies have also implicated the immune system as a target of ATR toxicity. In this study we examined the impact of ATR on antigen-driven, in vitro activation, phenotype and expansion of primary murine TCR transgenic CD4+ T cells. In the presence of ATR there were minimal changes T cell activation marker expression, although kinetics of CD69 and CD25 up-regulation and down-modulation of the TCR were slightly delayed. However, ATR treatment resulted in a significant inhibition of T cell proliferation and effector cytokine production. Rather than rescuing the T cell proliferation defect, addition of exogenous IL-2 resulted in a more pronounced proliferation defect in the ATR-treated cultures. We subsequently observed that the frequency of Foxp3+ T cells significantly was increased in the ATR-treated cultures by day 4. Studies are underway to determine mechanism of the ATR-mediated inhibition of T cell proliferation along with the increase in FoxP3+ T cell frequency.
Role of donor liver steatosis on immune responses to Hepatitis C, self antigens and allograft fibrosis following human liver transplantation (169.11)
Authors: Subramanian Vijay , Seetharam Anil , Tiriveedhi Venkataswarup , Angaswamy Nataraju , Anderson Christopher , Shenoy Surendra , Crippin Jeffrey , Chapman William , Mohanakumar Thalachallour
Published: January 2023

Hepatitis C virus (HCV) recurrence following liver transplantation (OLT) is associated with increased allograft fibrosis. Severe donor graft steatosis results in poor early post-operative outcomes but steatosis resolves within 3 weeks. Goal of this study is to characterize the role of donor liver steatosis on HCV immunity, fibrosis and outcome post OLT. Methods: HCV OLT recipients were enrolled at 1 year. Based on donor steatosis, grafts were divided into: Group 1-No steatosis (0-5% steatosis, n=18), Group 2 - Mild (5-33% steatosis, n=15), Group 3 - moderate (>33% steatosis, n=10). Mononuclear cells were stimulated with HCV antigens, and IL-17 and IL-10 cells were enumerated by ELISpot. Serum antibodies (abs) to Collagen (Col) 3 were measured by ELISA. Results: Patient demographics were similar in all groups. Group 3 OLT had a higher incidence of fibrosis (Batts Ludwig grades 3 or 4) when compared to groups 2 and 1 (93%, 34%, 15%, p<0.05). Group 3 demonstrated higher HCV specific IL17 (10±2.1, 12.2±5.2, 23±3.3 spots per million cells (spm) p<0.05) and IL10 secreting cells (107±23.1, 259.6±30.5, 402.3±37.3 spm, p<0.05). Groups 2 and 3 also had higher abs to Col 3 (11±1.1, 13±1.7, 17±1.5 µg/ml, p=0.05). Patient survival at 2 years was 100%, 95% and 84% among the three groups. Conclusion: Donor graft steatosis significantly influenced immune responses to HCV following OLT leading to increased IL17 and IL10 as well as increased incidence of autoimmunity and poorer outcome.
Recipient CD73-generated adenosine plays a preventive role in graft-versus-host disease by regulating allogeneic T cell activation (169.16)
Authors: Thompson Linda , Tsukamoto Hiroki
Published: January 2023

Graft-versus-host disease (GvHD) is a systemic disease in which donor T cells attack the allogeneic recipient. Here we investigated the role of CD73 in GvHD induced by transplantation of BALB/c bone marrow and spleen cells into lethally irradiated C57BL/6 recipients. CD73 is an enzyme that generates extracellular adenosine, a nucleoside with anti-inflammatory and immunosuppressive activity mediated by adenosine receptors. Survival curves showed more severe GvHD in CD73-/- → CD73-/- transplants than in CD73+/+ → CD73+/+ transplants and serum IFNγ and IL-6 were higher in CD73-/- recipient mice. CD4+ and CD8+ T cells from CD73-/- donors proliferated more in vivo than those from CD73+/+ donors as assessed by CFSE dye dilution and cell numbers in spleens. However, irradiation-induced activation of dendritic cells as assessed by CD86 up regulation was similar in CD73+/+ and CD73-/- mice. In vivo anti-C57BL/6 CTL activity was higher in CD73-/- transplant recipients than in CD73+/+ recipients. Additional CD73-/- → CD73+/+ and CD73+/+ → CD73-/- transplants revealed that more severe GvHD is caused primarily by a lack of CD73 in recipients. The adenosine receptor antagonist caffeine exacerbated GvHD in CD73+/+ recipients, consistent with the idea that the protective effect of CD73 depends upon its adenosine-generating activity. In conclusion, recipient CD73 plays a primary preventive role in GvHD by regulating allogeneic donor T cell activation in an adenosine-dependent manner.
Chemokine independent transendothelial migration of effector and memory T cells to vascularized allografts (169.2)
Authors: Walch Jeffrey , Oberbarnscheidt Martin , Li Qi , Hoffman Rosie , Camirand Geoffrey , Williams Amanda , Lakkis Fadi
Published: January 2023

Memory T cells play a central role in the rejection of transplanted organs. The migration of memory T cells to allografts is thought to depend on chemokine stimulation of G-protein coupled receptor (GPCR) signaling. This paradigm was revisited by testing whether B6 (H-2b) effector or memory T cells generated against BALB/c (H-2d) alloantigens can migrate to and reject BALB/c cardiac allografts despite treatment with pertussis toxin (PTx), which blocks GPCR signaling. Flow cytometry analysis revealed no significant difference between the number and proliferation of PTx treated and non-treated effector or memory T cells recovered from cardiac allografts at 1, 3 and 6 days after i.v. transfer. There was no significant difference in the survival of cardiac allografts in recipients of either PTx treated or non-treated memory T cells. Neither naive nor natural memory T cells harvested from unimmunized mice migrated in significant numbers to cardiac allografts. To our knowledge these are the first results indicating that effector and memory T cells migrate to allografts independent of chemokine stimulated GPCR signaling, which is central to the traditional model of leukocyte migration. These findings suggest that targeting chemokine receptors may prove an unsuccessful therapy to prevent T cell mediated transplant rejection and that alternate mechanisms exist for the migration of T cells into non-lymphoid tissues.
Inflammasome-derived IL-1β regulates the production of GM-CSF by CD4+ T cells and γδ T cells (171.24)
Authors: Lukens John , Kanneganti Thirumala-Devi
Published: January 2023

Recent findings have demonstrated an indispensable role for granulocyte-macrophage colony-stimulating factor (GM-CSF) in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). However the signaling pathways and cell populations that regulate GM-CSF production in vivo remain to be elucidated. Our work demonstrates that IL-1 signaling centrally regulates GM-CSF production during EAE. IL-1R1 is required to induce GM-CSF production following polyclonal T cell stimulation under both TH17 (i.e. IL-23) and TH1 (i.e. IL-12) stimulatory conditions. Furthermore, we demonstrate that γδ T cells are also potent producers of GM-CSF during the inflammatory response and that their secretion of GM-CSF is similarly dependent on IL-1 signaling. Interestingly, we find that the expression of GM-CSF by myelin-reactive T cells during EAE is not restricted to discrete TH1 and TH17 cell lineages. Rather GM-CSF can be co-expressed with both IFNγ and IL-17. Inflammasome-derived IL-1β was identified to be an upstream amplifier of GM-CSF producing T cells during the priming phase of EAE. Finally, we show a central role for MYD88 in the control of GM-CSF production by both CD4+ T cells and γδ T cells. These findings highlight the importance of inflammasome-derived IL-1β and the IL-1R1/MYD88 signaling axis in the regulation of GM-CSF production.
The adaptor protein CIKS/Act1 is essential for imiquimod-induced psoriasis. (171.25)
Authors: Ha Hye-Lin , Wang Hongshan , Siebenlis Ulrich
Published: January 2023

Psoriasis is a relapsing skin disease characterized by abnormal keratinocyte proliferation and differentiation and by an influx of inflammatory immune cells. The IL-23/IL-17 cytokines axis is thought to play an important role in the pathogenesis of psoriasis. The TLR7/8 ligand Imiquimod (IMQ) can induce/exacerbate psoriasis in patients and it induces a psoriasis-like condition in mice. In addition to IL-17A and IL-17F, which are produced primarily by Th17 and innate T cells, IL-17C has very recently been implicated in IMQ-induced psoriasis as well. To determine by what mechanisms IL-17 cytokines critically contribute to IMQ-induced psoriasis, we have begun to investigate a mouse model in which signaling by all IL-17 cytokines is abrogated due to the loss of the obligate adaptor protein CIKS/Act1. Importantly, the gene for this adaptor protein has also recently been identified as a strong susceptibility locus in psoriasis. Here we show that IMQ applications induced many markers of psoriasis in wild-type mice, but failed to do so in CIKS-deficient mice. These data demonstrate that signaling by IL-17 cytokines via the adaptor protein CIKS is essential for IMQ-induced psoriasis and thus the CIKS pathway may provide targets for therapeutic intervention in psoriasis. We will report on the progress in understanding the functions of IL-17 cytokines in development of psoriasis.
IL-23 inhibits autoimmunity by facilitating clearance of apoptotic bodies in the marginal zone (171.26)
Authors: Li Hao , Hsu Hui-Chen , Li Jun , Yang PingAr , Wu Qi , Cua Daniel , Oukka Mohamed , Mountz John
Published: January 2023

IL-23 helps expansion of Th17 cells which are important in the pathogenesis of spontaneous autoimmune disease in BXD2 mice. In this study, we surprisingly found that IL-23p19-deficient BXD2 mice (BXD2-Il23-/-) exhibit accelerated development of spontaneous germinal centers (GCs) and production of pathogenic autoantibodies, compared with wild-type BXD2 mice. Expression of IL-23 by adenovirus in BXD2-Il23-/- mice reversed these phenotypes. IL-23R was mainly expressed by marginal zone macrophages (MZMs) in the spleen of mice, as indicated by the GFP-IL-23R reporter. In BXD2-Il23-/-, however, there was an age-related loss of both MZMs and CD21hiIgMhi marginal zone (MZ) B cells. MZMs are a small subset of specialized splenic macrophages essential for clearance of apoptotic cells entering to the spleen from circulation to prevent generation of immunogenic autoantigens. MZM specific depletion by clondronate liposome severely accelerated BXD2 autoimmune disease progression. Absence of IL-23 correlates with downregulated expression of BAFF by MZMs which is essential for the differentiation of MZ B cells. Our results suggest a unique mechanism for IL-23 to limit autoimmunity in that IL-23 supports the cross-talk between MZM and MZ B cells. In this model, IL-23 acts on MZM to upregulate BAFF to promote the survival of MZ B cells, and MZ B cells help to maintain the survival of MZM. Loss of MZM is associated with defective clearance of apoptotic cells and acceleration of autoimmunity.
Defective Lysosomal Degradation of Immune Complexes in Lupus-Prone Mice (171.30)
Authors: Monteith Andrew , Kang Sun ah , Jacobson Ken , Costello M. , Moorman Nat , Vilen Barbara
Published: January 2023

The production of autoantibody and subsequent tissue deposition of immune complexes (ICs) contributes to the end-organ disease associated with systemic lupus erythematosus. What role ICs play in the autoimmune disease process remains unclear. In this study we found that low levels of ICs were displayed on dendritic cells (DCs), macrophages (MFs) and B cells from C57BL/6 (B6) mice via Fc receptors (FcRs). In contrast, cells from lupus-prone (MRL/lpr) mice had a 30-50-fold increase in surface-bound ICs. MRL/lpr MFs internalized ICs at a rate comparable to B6 however, they failed to degrade the ICs and consequently recycled them back to the cell surface within 48 hours. The recycling defect was not due to the failure of endosomes to properly mature, as cryoelectron microscopy showed equal numbers of ICs in secondary lysosomes. Rather, it appeared that a defect occurred in the ability of MFs to properly acidify lysosomes. Ratiometric imaging revealed that vesicles found in B6 MFs consistently acidified to a pH of 4, while those within MRL/lpr MFs failed to attain a pH lower than 5. These findings suggest that the recycling of ICs to the surface of MRL/lpr MFs results from defects within the endocytic pathway that prevent degradation of internalized cargo. Studies are underway to define the molecular events preventing acidification, and to assess whether the accumulated ICs activate autoreactive B cells, or promote excessive cytokine secretion by DCs and MFs from lupus-prone mice.
An IL15-dependent novel immune cell subset with multiple functionalities affecting the onset and severity of autoimmune disease (171.36)
Authors: Voynova Elisaveta , Bolland Silvia
Published: January 2023

Interferon-producing killer dendritic cells (IKDC) are a recently discovered innate cell subset that shares morphological and functional characteristics of NK and DC cells. The aim of this study is to investigate the role of IKDCs in terms of initiating autoimmune disease. We found that these cells are expanded in mice with multiple copies of the TLR7 gene and that the number of these cells correlates with disease activity. Expansion of this population is due to their cell intrinsic sensitivity to TLR7 stimulation. IKDCs are dependent of IL15, produce high levels of IFN type I and type II after stimulation with a TLR7 agonist, are capable of MHC class II antigen presentation, and can cross-present antigen to CD8 cells. Adoptive transfer of IKDC to wild type mice causes activation of naïve T cells, expansion of inflammatory monocyte populations and IFN-dependent CD8 activation. IKDC adoptive transfer also induces elevated inflammatory cytokine and IgG levels in the serum. We found that IFN type I and CD40/CD40L interactions are essential for the inflammatory effect of these cells. Taken together these results uncover a potential role for this cell populaiton in the development of autoimmune pathologies.
Sec61 is essential for antigen cross-presentation and the development of lupus nephritis (171.4)
Authors: Tsumiyama Ken , Shiozawa Shunichi
Published: January 2023

Objective: We repeatedly immunized mice normally not prone to autoimmune diseases with the same antigen to show that repeated immunization reproducibly led to the development of SLE, where full-matured CTL generated via antigen cross-presentation caused lupus nephritis. Here we clarify the molecular mechanism of antigen cross-presentation in relation to SLE. Methods: Bone marrow-derived dendritic cell (BMDC) from BALB/c mice was cultured with fluorescent-labeled OVA. Early endosome antigen 1 (EEA1), calnexin and Sec61 were detected using immunofluorescent staining. For in vivo study, BALB/c mice were repeatedly immunized with OVA to evoke SLE. Exotoxin A was co-immunized with OVA to inhibit Sec61. Proteinuria and IFNγ-producing CD8+ T cell were measured. Results: Upon culture of BMDC, OVA was co-localized with the endosomal marker EEA1, and subsequently separated from EEA1. OVA was never co-localized with the endoplasmic reticulum (ER) marker calnexin. Instead, OVA was co-localized with a translocon, Sec61, indicating that OVA was exported directly from endosome to cytoplasm via Sec61. Generation of both CTL and lupus nephritis was abrogated by in vivo treatment with exotoxin A, an inhibitor of Sec61. Thus, export of antigen from endosome to cytoplasm via Sec61 is essential for antigen cross-presentation. Conclusion: Direct export of antigen from endosome to cytoplasm via Sec61 is essential both for antigen cross-presentation and induction of lupus nephritis.
B1a cell expansion and autoimmunity promoted by cyclin-dependent kinase inhibitor Cdkn2c deficiency in a mouse model of lupus (171.41)
Authors: Potula Hari hara , Xu Zhiwei , Zeumer Leilani , Morel Laurence
Published: January 2023

By using a combination of genetic mapping and candidate gene analysis, we previously identified Cdkn2c, a gene encoding for cyclin-dependent kinase inhibitor p18INK4c (p18), as the top candidate gene for the lupus susceptibility locus Slec2c1 in the NZM2410 mouse. P18 is expressed at about 4 time lower levels in B6.Sle2c1 than in B6 B cells, leading to an increased cell cycle activity. Here, we showed that p18 deficiency results in a large expansion of the B1a cell compartment in the peritoneal cavity (Pc), which is the Slec2c1 primary phenotype. This was further confirmed by an increased production of polyreactive IgM (another feature of Sle2c1 phenotype) in the serum of B6.p18-/- similar to B6.Sle2c1 mice. Furthermore, B6.p18-/- mice produced anti-nuclear autoAbs and dsDNA IgG, which are serological markers strongly associated with lupus. We further showed p18 deficiency results in a significantly increased homeostatic expansion of Pc B1a cells but not B1b or B2 cells in B6.Rag mice. Finally, Cyclin D2 deficiency abrogates Sle2c1-induced B1a cell expansion, with the number of Pc B1a cells similar between B6.Sle2c1.D2-/- and B6 mice. This demonstrates a causal relationship between defective p18 gene expression, B1a cell expansion and development of murine SLE. These results provide concrete evidence that p18 plays a critical role in B1a cell self-renewal, and its impaired expression leads to accumulation of B1a cells, leading to autoimmunity either directly or indirectly.
Metabolomic analysis of macrophage activation and relevance of the cross-talk between energetic metabolism and polarized gene expression (172.12)
Authors: Bosca Lisardo , Través Paqui , Pimentel-Santillana María , de Atauri Pedro , Marin Silvia , Cascante Marta
Published: January 2023

Immune activation rapidly and substantially enhances metabolic outputs. Macrophage activation is followed by rapid changes in nutrient flux, which also seems to be necessary for immune activation, indicating that signals produced by immune cells might directly regulate their metabolism. Macrophages respond to a variety of extracellular stimuli developing patterns of gene expression that also encompass metabolic changes linked to the activation programs and that are not fully understood. Macrophages can be activated to two opposite end-points: M1-polarization accomplished by pro-inflammatory stimuli, and M2-polarization. The study of how energetic and redox metabolism of macrophages varies in accordance with recruitment and activation (M1, M2 or mixed combinations) and how these conditions are required for the specific functions of the macrophage (oxidative burst, phagocytosis, migration and infiltration, etc) are relevant to protect organ activity and to restrict the involvement of macrophages in chronic inflammation. We have studied the metabolic profile associated with relevant activation pathways using a [1,2-<13>C]glucose tracer-based metabolomic approach and bioinformatic tools to analyze changes in isotopomer distribution and changes in main energetic metabolites under M1/M2 conditions. Our data show the existence of specific metabolic signatures depending on the activation pattern elicited by the macrophages.
KIR mediated natural killer cell function modified by Tim3 expression level in early untreated HIV infection (172.18)
Authors: Brayton Bonnie , Crawford Timothy , Chew Glen , Ndhlovu Lishomwa , Barbour Jason
Published: January 2023

Tim-3 is a regulator of Natural Killer (NK) cell function. The polymorphic killer cell Ig-like receptor (KIR) expressed on NK cells is associated with HIV-1 progression rate. We examined Tim-3 expression level, NK cell function, and KIR genetics in 596 recently HIV-1 infected adults. We measured KIR genotype by SSO (One Lambda, Inc.) (n=596), and NK cell function (n=147) in an 11 color flow cytometry intra-cellular cytokine stain (ICS) assay, after stimulation with K562 cells, IL12/IL18, and HLA I transfectants. Tim-3 levels were lower on NK cells from KIR3DS1 gene carriers (all heterozygotes, hence KIR3DL1+ heterozygotes), [KIR3DS1+ Tim-3 median 84% (range 64-91%, N = 12) vs. KIR3DS1- median 91% (range 79-98%), p < 0.05]. Upon stimulation Tim-3+ NK cell responses did not differ in responses (IFN-g or CD107a) by KIR3DS1 gene carriage (p = NS), suggesting that while KIR3DS1 gene carriage relates to lower total Tim-3 expression levels on NK cells, KIR3DS1 gene carriage is not a potent regulator of effector activities of the Tim-3 bearing NK subset. Variation in NK function of KIR3DS1+ persons may be attributable to mixed KIR3DL1/3DS1 carriers whose variable level of KIR3DL/S1 expression, and lower level of Tim-3 expression mitigates differences in NK function. Tim-3, a molecule that restrains but does not abrogate NK effector activity, may play a key role in regulating KIR mediated effector functions of NK cells.
p38 MAPK but not ERK is required for eosinophil migration and degranulation (172.29)
Authors: Shamri Revital , Young Kristen , Weller Peter
Published: January 2023

Eosinophil-associated RNases (EARs); the human eosinophil-derived neurotoxin and eosinophilic cationic protein and their murine orthologs, are stored in eosinophil cytoplasmic granules and secreted upon inflammation as part of innate immune responses. In eosinophil-associated diseases, such as hyper eosinophilic syndrome, allergy and asthma, EARs play crucial roles in disease pathology and can be used as biomarkers for disease severity. However, secretion mechanisms of EARs are not fully understood, especially in mouse eosinophils, the major animal model for eosinophil-associated diseases. Our study aimed to understand the mechanisms of eosinophil EAR secretion following chemokine stimulation. Both ERK and p38 MAPK activation were found to increase in mouse eosinophils in response to CCL11. CCR3-mediated EAR secretion was blocked by p38 inhibitor (SB202190), confirming previous data in human eosinophils. However, a specific inhibitor for ERK, a MEK1-derived peptide, did not inhibit CCR3-mediated secretion or migration. These results are in contrast to previous studies in human eosinophils and our studies in mouse eosinophils, showing ERK is crucial for CCR3-mediated EARs secretion and migration using PD98059, a less specific inhibitor of the ERK-activating enzyme, MEK. Collectively, these data suggest a signaling pathway for CCR3-mediated EAR secretion that includes: PI3K and p38 but not ERK. These findings are pertinent to host defense and eosinophil-associated diseases.
mTORC2 negatively regulates DC PD-L1 and IL-10 through SOCS3 and STAT3 (172.34)
Authors: Rosborough Brian , Matta Ben , Turnquist Heth , Thomson Angus
Published: January 2023

The mammalian Target of Rapamycin (mTOR) is a central regulator of dendritic cells (DC) that functions in two independent complexes, mTOR complex (mTORC) 1 and 2. Rapamycin (RAPA) is a selective inhibitor of mTORC1, whereas novel ATP-competitive mTORC1/2 inhibitors (Torin1) inhibit both complexes. The immunoregulatory function of RAPA-resistant mTORC2 in DC is unknown. C57BL/6, PD-L1-/-, IL-6-/-, IL-10-/-, IL-12/23p40-/-, Ebi3-/-, interferon regulatory factor (IRF)1-/-, or FoxO1/3/4-/- bone marrow cells were cultured in GM-CSF and IL-4 to generate CD11c+ DC. RAPA or Torin1 was added on d2, and LPS added on d7. DC phenotype and signaling was assessed (d8) by flow cytometry and immunoblot, respectively. While RAPA reduced PD-L1, mTORC1/2 inhibition selectively upregulated PD-L1 expression on DC in a STAT3-dependent manner in unstimulated and LPS-stimulated DC. Whereas RAPA reduced IL-10 secretion, mTORC1/2 inhibition augmented IL-10 secretion following LPS stimulation. The PD-L1 regulator, IRF1, and FoxO, a STAT3 regulator, were not required for PD-L1 upregulation. PD-L1 upregulation on mTORC1/2-inhibited DC did not require autocrine IL-6, IL-10, IL-12/23, or IL-27. SOCS3, a negative regulator of STAT3, was reduced dramatically in mTORC1/2-inhibited, but not RAPA-exposed DC. mTORC2 negatively regulates STAT3 in DC through SOCS3. Blockade of mTORC2 signaling enhances PD-L1 expression and IL-10 secretion. These novel findings establish mTORC2 as a key regulator of DC function.
S4D-SRCRB, a soluble member of the group B scavenger receptor cysteine-rich superfamily, binds to both endogenous and exogenous structures (172.8)
Authors: Miró Cristina , Roselló Sandra , Padilla Olga , Yelamos José , Vila Jordi , Lozano Francisco
Published: January 2023

Human S4D-SRCRB is a soluble member of the Scavenger Receptor Cysteine-Rich Superfamily (SRCR-SF), and is composed of four group B SRCR domains separated by Pro-, Ser- and Thr-rich polypeptides, which are o-glycosilated. Northern blot analysis indicated that S4D-SRCRB is expressed as two major mRNA species: one of 2,8 kb, with a restricted tissue expression pattern (mainly kidney, placenta and HepG2 cell line), and another of 1,5 kb, with a broader distribution. To gain insight on the function of this molecule, we produced and affinity purified a recombinant form of this protein fused to a HA tag (rhS4D-HA) using an episomal expression system (pCep/Pu) in HEK 293-EBNA cells. Similarly to other members of the SRCR-SF (S5D-SRCRB, CD6 and DMBT1), rhS4D-HA binds to the surface of bacteria (both gram-positive and -negative) and fungi (both saprophytic and pathogenic), and this is mediated through recognition of lipotheicoic acid (LTA) or lipopolysaccharide (LPS), and lineal glucans, respectively. Interestingly, rhS4D-HA increased the secretion of IL-8 of HepG2 when exposed to bacterial cell wall components. Moreover, rhS4D-HA also binds to some (fibronectin and laminin) but not all (collagen) extracellular matrix proteins. Taken together the data suggest that S4D-SRCRB behaves as a dual-specific receptor for non-self and self-structures, likely playing a role in the innate defence and homeostasis of epithelial surfaces.
Mechanisms underlying T cell guided innate immunity after inhalation of Staphylococcus enterotoxin A (SEA) (173.1)
Authors: Kumar Sanjeev , Menoret Antoine , Vella Anthony
Published: January 2023

Inhalation of Staphylococcus aureus enterotoxin A (SEA) in mice induces strong pulmonary inflammation marked by vascular permeability and alveolitis, thereby modeling aspects of lung injury in humans. After SEA inhalation CD8+Vβ3+ T cells robustly expand but the early events of this response in the lung are unclear. Our new data show that Vβ3+ T cells are activated quickly after SEA inhalation since CD69 and the high affinity IL-2 receptor α-chain (CD25) were upregulated by 3 hours. This coincided with several changes in the innate immune system including recruitment of neutrophils, monocytes and NK cells into the lung, and imprinting an activation phenotype on macrophages. Importantly, in the absence of T cells all of these effects were abrogated. As a way to understand how T cells condition innate immunity, we analyzed cytokine production and found an unexpected role for TCRαβ T cells promoting IL-17 secretion by TCRγδ T cells. We demonstrated that TCRγδ cells did not produce IL-17 when tested in TCRβ-/- mice. Currently, we are defining how TCRγδ cells are induced to synthesize IL-17 by the TCRαβ cells. In sum, TCRαβ T cells mediate pro-inflammatory responses, not only as a direct source of cytokines, but also by guiding the recruitment and activation of innate cells. Thus, our data show role reversal between the adaptive immune and innate immune system.
Pathway Signature PCR Array: a novel method for studying inflammatory responses (173.11)
Authors: Wu Zhong , Schlumpberger Martin , Raza Sameen , Yu Jiaye , DiCarlo John , Wang Yexun , Devgan Vikram
Published: January 2023

Signaling pathways play central roles in cellular physiology, and assessing the state of pathways helps clarify molecular mechanisms of disease and inflammatory responses. Gene expression signatures of pathway activation status enable reliable measurement of pathway activity. Our group developed Pathway Signature PCR Arrays to analyze gene expression and determine pathway activity within a single real-time PCR experiment. This study identified a set of genes that provide a gene signature for evaluating IL6/STAT3 pathway activity. A set of 88 signature genes were derived from microarray profiling on HepG2 and MCF10A cells treated with IL-6 protein and STAT3-specific siRNA. Genes were selected on the basis of statistically significant expression changes in response to IL-6 and reversion of altered expression upon treatment with STAT3 siRNA. These 88 IL-6/STAT3 response genes were further verified by real-time PCR, using a panel of 14 different cell lines stimulated with IL-6 or inhibited with STAT3 siRNA. Using a mathematical classifier method, a subset of genes was identified as a gene expression signature for assessing IL-6/STAT3 pathway activity. These signature genes, along with IL-6 pathway-related genes identified by literature mining, constitute the IL-6 Pathway Signature PCR Array. This array provides a useful tool for studying regulation of pathway activity via gene expression changes during inflammatory responses. Array is for molecular biology applications only.
CD34 family proteins are key regulators of inflammatory cell migration and vascular integrity (173.17)
Authors: McNagny Kelly , Debruin Erin , Gold Matthew , Bennett Jami , Blanchet Marie-Renee , Maltby Steve , Hughes Michael
Published: January 2023

CD34 is a widely used marker of hematopoietic progenitors and vascular endothelia yet its function remains obscure. CD34 is the founding member of a family of three proteins (CD34, Podocalyxin and Endoglycan) with overlapping expression patterns and we have used gene targeting to investigate the function and redundancy of these proteins. We find that CD34 is uniquely expressed by mast cells, eosinophils and dendritic cells and that mice lacking CD34 are remarkably resistant to a variety of mucosal inflammatory diseases including allergic asthma, hypersensitivity pneumonitis and colitis. Resistance reflects defective inflammatory cell trafficking due to enhanced adhesion and impaired chemotaxis and these defects can be reversed through ectopic expression of a human CD34 transgene. Podocalyxin, in contrast, is uniquely expressed by a variety of embryonic tissues and inactivation of the Podxl gene leads to perinatal lethality due to a failure of tissues to undergo appropriate morphogenesis. To investigate the function of Podocalyxin (and CD34) on adult vasculature we have generated mice that selectively inactivate the encoding gene in endothelia. These exhibit excessive vascular leakage and develop an emphysema-like phenotype due to poor vessel patency in the lung. In summary, CD34 type proteins play critical roles in a variety of normal developmental processes and immunity. Mechanistically, they act as key regulators of cell adhesion, cell chemotaxis and morphogenesis.
Targeted PGE2 inhibition enhances anti-viral immunity through induction of Type I interferon in macrophages (P6180)
Authors: Divangahi Maziar , Coulombe François , Jaworska Joanna , Verway Mark , Tzalepis Fanny , Massoud Amir , Gillard Joshua , Wong Gary , Kobinger Gary , Joubert Philippe , Fritz Jörg , Xing Zhou , Powell William
Published: January 2023

The 1918 Spanish Influenza virus pandemic had a major impact on the rise in popularity of non-steroidal anti-inflammatory drugs (NSAIDs). Although these drugs are used extensively for the management of Influenza symptoms, their role in anti-viral immunity is poorly understood. NSAIDs act mainly by inhibiting cyclooxygenase enzymes involved in the synthesis of all prostaglandins, including PGE2. We now show that during Influenza infection, upregulated PGE2 inhibited macrophage type I interferon production and apoptosis via EP2 and EP4 receptors, thereby increasing virus replication. This inhibitory role of PGE2 was not limited to innate immunity since both antigen presentation and T cell mediated immunity were also suppressed. Inhibition of microsomal prostaglandin E-synthase 1 (mPGES-1), and thereby PGE2, in human Mϕ was protective against Flu infection. These data show that the mPGES-1/PGE2 pathway is specifically targeted by Influenza virus to evade host anti-viral immunity and propose that specific inhibition of PGE2 may serve as a new treatment for viral infection.
The length of the peptide in the MHC groove compartmentalizes the CD8+ T cell repertoire (P1186)
Authors: Miles John , van den Berg Hugo , Ekeruche-Makinde Julia , Skowera Ania , Cole David , Dolton Garry , Schauenburg Andrea , Tan Mai Ping , Pentier Johanne , Peakman Mark , Price David , Burrows Scott , Sewell Andrew , Wooldridge Linda
Published: January 2023

TCRs expressed at the CD8+ T-cell surface interact with short peptide fragments bound to MHC class I molecules (pMHCI). The TCR/pMHCI interaction is pivotal in all aspects of CD8+ T-cell immunity. However, the rules that govern the outcome of TCR/pMHCI engagement are not entirely understood, and this is a major barrier to understanding the requirements for both effective immunity and vaccination. Using large combinatorial peptide arrays, each containing between twenty billion to forty quadtrillion distinct peptides, we have discovered an unexpected feature of the TCR/pMHCI interaction by showing that all TCRs exhibit an explicit preference for an MHCI-bound peptide of defined length. Agonists of nonpreferred length were extremely rare, suboptimal, and often entirely distinct in sequence. Structural analysis indicated that alterations in peptide length have a major impact on antigenic complexity, to which individual TCRs are unable to adapt. This surprising finding, that the CD8+ T cell repertoire is rigorously "compartmentalized" by peptide length, has important consequences for cellular immune system research and vaccine development.