bluuebunny/crossref_metadata_2025 huggingface dataset:
The transcription factor HIF1α controls expression of the cytokine IL-22 in CD4 T cells
Authors: Zenewicz Lauren A , Budda Scott A , Girton Alanson
Published: January 2023

IL-22 is expressed by activated lymphocytes and is important in modulation of tissue responses during inflammation. The cytokine induces proliferative and anti-apoptotic pathways in epithelial cells allowing enhanced cell survival. This can have positive effects, such as in the maintenance of epithelial barriers in the gastrointestinal tract, but also negative effects, such as contributing to colorectal tumorigenesis. As IL-22 can be dual-natured, we hypothesized that its biological activity should be tightly regulated in order to limit IL-22 expression to the sites of inflammation. One such environmental cue could be low oxygen, which often accompanies inflammation. We show that in CD4 T cells IL-22 expression is upregulated in hypoxia, and this upregulation is dependent on the transcription factor hypoxia-inducible factor 1α (HIF1α). This finding has implications on the regulation of Il22 gene expression and the cytokine’s presence in different inflammatory environments.
Wnt/β-catenin signaling regulates IL-15-mediated terminal maturation and interferon-gamma production of invariant NKT cells.
Authors: Pyaram Kalyani , Chang Cheong-Hee
Published: January 2023

The Wnt/β-catenin signaling pathway is important for various cellular functions including survival and proliferation. The indispensable role of this pathway in conventional T cell development is well established but little is known about its role in innate T cell development. In this study, we investigated the role of the Wnt/β-catenin signaling in the development of invariant natural killer T (NKT) cells using mice that express a constitutively active form of b-catenin (CAT-Tg) or deficient in β-catenin (CAT-KO). Thymic NKT cells were increased in CAT-Tg and decreased in CAT-KO mice compared to their wild type littermates. Thymic NKT cells progress from Stage 0 to Stage 3 as they mature. In the absence of Wnt signaling (CAT-KO), we observed accumulation of Stage 0 NKT cells. In contrast, in CAT-Tg mice, the final maturation was compromised - more Stage 2 but less Stage 3 NKT cells. Thus, stage-specific regulation of Wnt signaling may be critical for the thymic NKT development. Indeed, endogenous β-catenin expression was high in early stages but low in late stages, suggesting that its reduction mounts the stage-2 to -3 NKT transition. The CAT-Tg mice also had lower number of IFN-gamma expressing NKT cells, which, in these cells, correlated with reduced expression of transcription factor Tbet and CD122 (IL-2R & IL-15R subunit), requisites for final maturation and IFN-gamma expression of NKT cells. Further, although PLZF, a master transcription factor for NKT cell development is expressed at high levels in CAT-Tg NKT cells, Wnt signaling-mediated NKT cell maturation was found to be independent of PLZF. Together, our data shows that regulation of Wnt/β-catenin signaling is necessary for terminal maturation and IFN-gamma production of NKT cells.
Bone marrow fat induces inflammation that inhibits B lymphopoiesis
Authors: Kennedy Domenick Edward , Knight Katherine L
Published: January 2023

Adipocytes accumulate in the bone marrow (BM) during aging and obesity, correlating with decreased B lymphopoiesis. We previously found that adipocyte factors induce MDSCs, which in turn inhibit B lymphopoiesis via IL-1 production. In this study, we asked what adipocyte factor(s) promotes MDSC accumulation. By cytokine array, we found that adipocyte conditioned medium (ACM) contains inflammatory cytokines known to induce MDSCs (ex. IL-6, G-CSF, C3a). These and other adipocyte factors can activate the NLRP3 inflammasome, leading us to test if blocking NLRP3 activation would prevent MDSC accumulation. Cultures of mouse BM progenitors with OP9 cells in the presence of ACM yielded many MDSCs and few B lineage cells. However, glybenclamide (NLRP3 inhibitor) treatment reduced MDSC accumulation and increased B lymphopoiesis, suggesting that adipocyte factors induce MDSCs through inflammasome activation. Adipocytes fill the BM of rabbits by 2 months of age, a time when B lymphopoiesis is arrested. To determine if the arrest of B lymphopoiesis could be due to adipocyte-initiated BM microenvironmental changes, we co-cultured BM progenitors and OP9 cells with or without conditioned medium generated from BM fat explants (BM fat-CM) taken from >2 month old rabbits. These cultures resulted in decreased numbers of B lineage cells and increased numbers of myeloid cells, similar to the myeloid skew seen in BM of >2 month rabbits. We conclude that rabbit BM fat can initiate the loss of B cell development. Strategies to modulate BM fat volume and inflammatory state could be useful for boosting B lymphopoiesis in the aging and obese populations.
Effects of treatment guidelines for the management of anaphylaxis on patient and caregiver adherence
Authors: Brown Duncan , Locklear Julie , Karjalainen Martin , Lehnigk Ulrike , Saathoff Friederike
Published: January 2023

OBJECTIVE

Treatment guidelines recommend a prescription for two doses of an epinephrine auto-injector (EAI) be given at discharge following an anaphylactic event and patients carry these at all times to ensure timely access to epinephrine. However, previous research suggests approximately 30% of patients will not carry any EAI, and of those that do, approximately 10% have expired. This web-based US survey aims to understand patients’ and caregivers’ adherence to these proposed treatment guidelines.

METHODS

159 patients aged 18–65 years (M= 29.7; 32.1% male) and 215 caregivers of individuals under aged 18 years (M = 23.3; 39.5% male) participated in the survey. All participants had been prescribed an EAI for self-administration or for administration as a caregiver. The survey took place between November 15 and November 30, 2015.

RESULTS

18.2% of patients and 15.4% of caregivers did not store a single EAI at home; 61.0% of patients and 62.8% of caregivers indicated only one EAI was available at home. 83.0% of patients did not have access to an EAI at work and 35.8% of caregivers reported their child did not have access to an EAI at school. Only 8.0% of caregivers indicated two EAIs were available for their children at school. Caregivers were more likely to identify lack of training as the cause of an error compared to patients (p=0.02). No difference was identified for EAI compliance between those treating anaphylaxis with an EAI and those who have not used an EAI.

CONCLUSION

This survey revealed a suboptimal adherence to proposed treatment guidelines on the part of patients and caregivers. The findings bring into question the reliability of current education systems and safety information delivered by healthcare providers and retained by EAI users.
Transcriptional profiling of mast cell and basophil responses to differential stimuli identifies unique gene signatures.
Authors: Chhiba Krishan D , Hsu Chia-Lin , Berdnikovs Sergejs , Bryce Paul J
Published: January 2023

Mast cells (MC) and basophils (BΦ) are key effector cells in allergy that are developmentally related. Functionally, they both release mediators like histamine and granule proteases. However, studies also suggest that MC and BΦ have non-redundant roles in allergic diseases. Their ability to respond to IL-33—an epithelium-derived cytokine that helps initiate allergic inflammation—is not well understood. To fully characterize the transcriptional responses and heterogeneity of MC and BΦ, we performed large-scale comparative microarrays of bone marrow-derived mast cells (BMMCs) and basophils (BMBs) either at rest, upon IgE activation, or upon IL-33 activation. Hierarchical clustering demonstrated that BMMCs had activation-specific transcriptional signatures. IgE-crosslinking upregulated 1803 unique genes including Ccl7, Fxyd6, and Cd33; while IL-33 stimulation induced 1918 genes including Il1b, Cd72, and Tnfaip2. Focused bioinformatics pathway analysis demonstrated IgE-activation aligned with pathways relating to complement activation and CD137 signaling, while IL-33 initiated responses in the NF-κB and IL-10 signaling pathways. Furthermore, BMBs activated via IgE-crosslinking induced type 2 immune response genes like Il2, Il4, Ccl17 and Il13 which were surprisingly absent in IL-33 stimulated BMBs. Further analysis revealed cell-specific transcriptional signatures with 4360 genes that were significant in BMBs but not in BMMCs after IgE activation, and 1645 after IL-33 stimulation. Here we show that MC and BΦ have cell-specific and activation-specific transcriptional responses, and our data suggest novel gene networks and pathways that may shape how the immune system responds to allergens and innate cytokines.
Biological markers of infection may assist in the identification of early stage viral infection and serve as a surrogate biomarker to identify asymptomatic Ross River or Barmah Forest virus infection
Authors: Dean Melinda M , Dunford Melanie , Flower Robert L , Faddy Helen M
Published: January 2023

Background and Aims

Although Ross River virus (RRV) and Barmah Forest virus (BFV) pose a risk to transfusion safety, in Australia, blood donations are not screened for either. RRV and BFV pathogenesis is poorly understood; however, mannose binding lectin (MBL) levels have been associated with RRV disease severity. We investigated biological markers to identify early stages of infection in asymptomatic RRV or BFV infection.

Methods

Samples from 7001 blood donations from donors at risk for RRV and/or BFV were tested for anti-RRV (IgM) and/or anti-BFV (IgM). MBL level was assessed in 139 anti-RRV (IgM) positive samples, 143 anti-BFV (IgM) positive samples and clinical samples (10 RRV, 10 BFV). MCP-1, MIG, TNF-α, IFN-α, IFN-γ, IL-8, IL-10 and IP-10 were quantified in these samples and 50 seronegative samples.

Results

Anti-RRV (IgM) was detected in 2.3%, and anti-BFV (IgM) in 2.4%, of donations, consistent with asymptomatic infection. MBL deficiency was not associated with seropositivity, but higher MBL levels were evident in RRV patients. IP-10, MIG, MCP-1 and IL-8 were elevated in RRV patients and IFN-γ, MCP-1 and IL-8 higher in BFV patients. For both anti-RRV (IgM) and anti-BFV (IgM) positive donations, IL-8 was elevated compared to IgM negative donors. All statistical analysis unpaired T-test, 95% CI.

Conclusions

Asymptomatic RRV or BFV infection occurs in blood donors. The frequency of MBL deficiency was similar between samples from clinical and presumed asymptomatic RRV or BFV infection. Measurement of IL-8 may assist in the identification of early stage viral infection and function as a surrogate biomarker for early stage RRV or BFV infection.
Chronic STING-dependent activation of TBK1 suppresses mTORC1 activity and dysregulates cellular metabolism in mice
Authors: Gonugunta Vijaya Kumar , Hasan Maroof , Dobbs Nicole , Ali Aktar , Palchik Guillermo , Calvaruso Maria , DeBerardinis Ralph J , Yan Nan
Published: January 2023

Trex1−/− mice suffer from systemic inflammation caused largely by chronic activation of the cGAS-STING-TBK1-IRF3 signaling pathway. We showed previously that Trex1-deficient cells have reduced mTORC1 activity, although the underlying mechanism is unclear. Here, we performed detailed metabolic analysis in Trex1−/− mice and cells that revealed defects in mitochondrial respiration, reduced adiposity, increased energy expenditure and glycolysis. We also found that Trex1−/− mice and cells exhibit chronically suppressed mTORC1 activity. Furthermore, we provide genetic evidence corroborated with cellular and biochemical validation to show that metabolic dysregulation in Trex1−/− mice is caused by STING-dependent activation of TBK1, but not the downstream IRF3-mediated signaling and inflammation, and that TBK1 binds to the mTORC1 complex and inhibits its activity in Trex1−/− cells. Our data thus establish chronic STING-dependent activation of TBK1 as a novel regulatory axis of mTORC1 and cellular metabolism.
Macrophage-mediated lysine methyltransferase, Setdb2, regulates inflammation in wound healing
Authors: Kimball Andrew S , Joshi Amrita , Allen Ron , Bermick Jennifer , Schaller Matthew , Carson Beau , Kunkel Steven L , Gallagher Katherine
Published: January 2023

Epigenetic regulation of macrophages can alter their function and impact tissue inflammation and wound repair. Unregulated inflammation at the wound level in inflammatory diseases like type 2 diabetes (T2D) leads to significant morbidity and mortality. Recent work by our group and others has shown that chromatin-modifying enzymes influence inflammatory gene expression in macrophages and other immune cells. Here we show that Setdb2, a histone methyltransferase, is induced in macrophages during the inflammatory phase of wound healing. Specifically, Setdb2 repressed expression of NF-κB-induced inflammatory genes, including IL-1B. This coincided with occupancy by Setdb2 at the IL-1B promoter, which in the absence of Setdb2 displayed diminished trimethylation of histone 3 lysine 9 (H3K9me3) in bone marrow derived macrophages (BMDM). Mice with the inability to upregulate setdb2 in their macrophages (Setdb2f/fLyz2cre+) demonstrated impaired healing during the inflammatory phase of wound healing. This corresponded with decreased H3K9me3 at the promoter of IL-1B and other inflammatory genes in macrophages isolated from the wounds of these animals. Further, LPS was able to upregulate setdb2 expression in BMDM and hence TLR4 signaling may play a role in regulation of inflammation in wound tissue. This suggests that a Setdb2-mediated alteration of NF-κB pathways represents an important mechanism for successful wound healing.
Thiostrepton inhibits psoriasis-like inflammation induced by TLR7, TLR8, and TLR9
Authors: Lai Chao-Yang , Yeh Da-Wei , Lu Chih-Hao , Liu Yi-Ling , Huang Li-Rung , Kao Cheng-Yuan , Chen Huan-Yuan , Huang Chi-Ying F. , Chang Chung-Hsing , Luo Yunping , Xiang Rong , Chuang Tsung-Hsien
Published: January 2023

Toll-like receptors 7, 8 and 9 (TLR7-9) comprise a subfamily of TLR. Activation of these TLRs has been linked to the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and psoriasis. Thus antagonists of these TLRs are being investigated for their therapeutic applications on these diseases. Bortezomib is a proteasome inhibitor known to suppress activation of these TLRs. This drug is approved for the treatment of multiple myeloma, and its inhibitory effects on autoimmune disorders such as psoriasis, RA, and SLE have also been investigated in animal models. In an attempt to develop novel TLR7-9 inhibitors, we searched the Gene Expression Omnibus database for gene expression profiles in cells treated with bortezomib. These profiles were then used as an input to screen the Connectivity Map database for chemical compounds with similar functions as bortezomib. Here we report that the antibiotic thiostrepton is a novel TLR7–9 inhibitor. Like bortezomib, thiostrepton effectively inhibits TLR7–9 activation in cell-based assays and dendritic cells. In contrast to bortezomib, thiostrepton is less cytotoxic to dendritic cells, and its inhibitory activity is more specific to TLR7–9. Thiostrepton inhibits TLR9 localization in endosomes for activation via two mechanisms. One mechanism is similar to the proteasome inhibitory function of bortezomib, and the other is through inhibition of endosomal acidification. In different animal models, thiostrepton attenuated LL37- and imiquimod-induced psoriasis-like inflammation. These results indicated that thiostrepton is a novel and specific inhibitor to TLR7–9.
IL-10 Producing Regulatory B cells are Decreased and Functional Impaired in Myasthenia Gravis
Authors: Sheng JianRong , Soliven Betty
Published: January 2023

Myasthenia gravis (MG) is a prototype of antibody-mediated neuromuscular disease causing fluctuating weakness and disability. Experimental autoimmune MG (EAMG) is an animal model of human MG. In our previous study, adoptive transfer of CD1dhiCD5+B cells expanded in vivo by GM-CSF prevented and suppressed EAMG; and IL-10 production within CD1dhi CD5+ B cells played an important role in immune regulation of EAMG. In this study, we aimed to analyze the frequency and function of these regulatory B (Bregs) subsets (CD38hi CD24hi and CD5+ CD1dhi) in MG patients and healthy controls. We found that MG patients had lower frequencies of Breg subsets (CD38hi CD24hi and CD5+ CD1dhi), and lower percentages of B10 cells within each subset compared to controls. Depletion of CD38hi CD24hiB cell subset from the PBMCs of MG patients did not lead to any significant increase in the percentage of CD4+ IFNγ and CD4+ TNFα, in contrast to healthy controls. We concluded that IL-10 producing regulatory B cells were decreased and functional impaired in MG patients, which might suggest a crucial role of Bregs in the development of MG.
Mouse Fcgamma Receptor IV dependent inflammatory cytokine and chemokine response contributes to progression of atherosclerosis in apoE hyperlipidemic mice
Authors: Nagarajan Shanmugam , Zhu Xinmei , Feck Doug , Popovic Bane , Velayutham Murugesan
Published: January 2023

Fcgamma receptors (FcγRs) are classified as activating (FcγRI, III, and IV) and inhibitory (FcγRII) receptors. Others and we have demonstrated that combined deficiency of all the three activating FcγRs (I, III and IV) in apolipoprotein E (apoE)-Fcγ chain double knockout (dKO) mice decreased atherosclerosis. In this report, we investigated the independent role of FcγRI and FcγRIV in the progression of atherosclerosis. We tested the hypothesis that FcγRI and FcγRIV exacerbate atherosclerosis using apoE-FcγRI dKO and apoE-FcγRIV dKO mice. Our results show that arterial lesions were not different between apoE-FcγRI dKO and apoE knockout (apoE KO) mice. Interestingly, arterial lesions were significantly decreased in a regular chow or a high-fat diet fed apoE-FcγRIV dKO male and female mice, relative to apoE KO mice. Bone marrow chimeras were used to address the relative contribution of FcγRIV expressed on hematopoietic cells including macrophages and dendritic cell. ApoE KO mice transplanted with apoE-FcγRIV dKO marrow showed significantly reduced arterial lesions relative to recipient mice transplanted with apoE KO marrow. Next, we investigated whether pro-inflammatory response contributed to the pro-atherogenic effect of FcγRIV. Activated CD4+ T cells of apoE-FcγRIV dKO mice showed increased secretion of IL-10, whereas IFN-γ and IL-17 by T cells were decreased. Interestingly, dendritic cells at the lesion-prone vascular site from apoE-FcγRIV dKO mice induced increased IL-10 secretion by LDL-specific T cells. Collectively, our findings suggest that the pro-inflammatory responses initiated by FcγRIV, one of the activating FcγRs, contribute to the progression of atherosclerosis. Supported by NIH grant HL086674.
Single-walled carbon nanotubes disturbed the immune and metabolic regulation function on 13-weeks after a single intratracheal instillation
Authors: Park Eun-Jung , Hong Young-Shick , Lee Byoung-Seok , Yoon Cheolho , Jeong Uiseok , Kim Younghun
Published: January 2023

Due to their unique physicochemical properties, the potential health effects of single-walled carbon nanotubes (SWCNTs) have attracted continuous attention together with their extensive application. In this study, we aimed to identify local and systemic health effects following long pulmonary persistence of SWCNTs. As expected, SWCNTs remained in the lung on 13-weeks after a single intratracheal instillation (50, 100, and 200 μg/kg). In the lung, the number of total cells and the percentages of lymphocytes and neutrophils were significantly raised at 200 μg/kg dose compared to the control, and Th1-polarized immune response was induced accompanying an enhanced expression of tissue damage-related genes and an increased release of chemokines. Additionally, SWCNTs enhanced the expression of antigen presentation-related proteins on the surface of antigen-presenting cells, but maturation of dendritic cells was inhibited by their long persistence. As compared to the control, the significant increase in the percentage of neutrophils in WBC and the remarkable decrease of BUN and potassium level were observed in the blood of mice treated with the highest dose accompanied by the down-regulation in the expression of antigen presentation-related proteins on splenocytes. Moreover, protein and glucose metabolism was disturbed with an up-regulation of fatty acid β-oxidation. Taken together, we conclude that SWCNTs may induce adverse health effects by disturbing the immune and metabolic regulation function in the body. Therefore, careful application of SWCNTs is necessary for the enforcement of safety in nano-industries.
Early microbial changes affect mucosal immunity in autoimmune arthritis
Authors: Kuhn Kristi , Hendrickson Jason D. , Mehta Gaurav , Adhikari Sumitra , Banda Nirmal , Robertson Charles E. , Frank Daniel N. , Demoruelle M. Kristen , Deane Kevin D.
Published: January 2023

Multiple lines of evidence now suggest that autoimmune disease may result from microbial-mucosal interactions. Microbial dysbiosis occurs in new onset seropositive rheumatoid arthritis (RA); and during the preclinical phase of RA, there is an expansion of mucosal autoantibodies and plasmablasts. These findings strongly suggest that a mucosal site may drive the initial break in tolerance that leads to RA. Using the murine collagen-induced arthritis model of RA, we find that microbiome diversity changes quickly after immunization with bovine type II collagen, at the time when disease-related autoantibodies develop, but before clinical CIA is evident. Significant alterations in taxa clustered in Clostridia families occur in CIA. Interestingly, using IgA-seq in human at-risk for RA study groups we find that those subjects with autoantibodies have similar alterations in Clostridia families. Furthermore, during the development of murine CIA, increased intestinal permeability and altered cytokine profiles paralleled disease development and microbiota alterations. Taken together, these data suggest a model in which early microbial alterations stimulate mucosal immune responses that drive the development of pathogenic autoantibodies. Future studies are aimed at identifying specific bacterial species and elucidating the pathway by which they stimulate autoantibody production.
Langerhans cells and CD11b+ dermal dendritic cells are involved in skin Treg generation
Authors: Tordesillas Leticia , Mondoulet Lucie , Benhamou Pierre-Henri , Sampson Hugh A , Berin M. Cecilia
Published: January 2023

Antigen applied to the skin can induce immune tolerance in naïve mice and can be used as epicutaneous immunotherapy (EPIT) to treat experimental food allergy. In previous unpublished work, we have shown that EPIT induces Tregs, including a population of intestinal-homing LAP+ Tregs that can suppress mast cell activation and anaphylaxis. In this extension of that work, our goal was to determine how dendritic cells (DCs) in the skin contribute to the induction of LAP+ Tregs. Epicutaneous antigen exposure of mice that have received DO11.10 T cells with Viaskin®-OVA patches for 48 h induced OVA-specific LAP+ Tregs to a similar extent in naïve and sensitized mice. Therefore all additional studies were done in naïve mice. FACS sorting of DC subsets from skin-draining lymph nodes of Viaskin®-treated mice and then co-cultured with DO11.10 T cells demonstrated that CD11b+ CD103− dermal DCs and CD11b+EpCAM+ Langerhans cells (LCs) were both capable of antigen capture and presentation to CD4+ T cells, as well as Treg generation. PDL2 was found on both subsets and effectively discriminated between cells with and without antigen capture and presentation capacity. PDL2+ DCs presented a more mature phenotype, with higher expression of MHCII, CD86 and CD80 than PDL2− DCs. PDL2+ LCs and PDL2+ CD11b+ DCs were sorted separately and expression of regulatory molecules (GM-CSF, IL-27, IL-10, TGF-b, ALDH2 and IDO) were assessed by RT-PCR. PDL2+ LCs uniquely expressed IL-27, while PDL2+ CD11b+ DCs were enriched for ALDH2, which was then confirmed by flow cytometry. In vivo depletion of LCs using anti-CSF1 did not influence generation of Tregs in vivo. These results indicate that induction of Tregs through the skin is not restricted to one DC subset.
DMBA specific induces Treg proliferation in draining lymph node
Authors: Tang Hua , Kang Li , Cheng Yi , Wang Meixiang , Gao Jiming , Zhang Shanlong , Song Wengang
Published: January 2023

DMBA, a carcinogenic agent which can induce organism tumor production, was used as a tumor inducer to analyze the draining lymph node T cell development in the skin of mice. The results indicated that antigen-specific T cell proliferation was significantly increased in draining lymph node after DMBA treatment, and CD4+ Foxp3+ Treg measurement results showed that the percentage of Treg increased. Compared with the treatment of acetone, OVA or CpG the proliferation of CD4+ Foxp3+ Treg was carcinogenic agent dependence. More importantly, CD4+ Foxp3+ Helios+ T proliferation assay and CD4+ Foxp3-GFP+ T and CD4+ Foxp3-GFP− T adoptively transfer result indicated the proliferation of Treg was from nTreg but not iTreg. Gene chips assay showed that the expression of energy metabolism relative protein, such as Myo1f and Myl4, and cellular growth regulation protein, such as Anxa2, was significantly up-regulated, which provide a potential target to suppress Treg proliferation during tumor development.

This study was supported by the National Natural Science Foundation of China (81272315)
Autophagy-dependent TIGIT+ Treg are critical for the maintenance of tolerance
Authors: MacDonald Kelli PA , Le Texier Laetitia , Leveque-ElMouttie Lucie , Lineburg Katie , Nicholls Jemma , Guimaraes Fernando Souza-Fonseca , Alexander Kylie , Clouston Andrew , Blazar Bruce R , Hill Geoff R
Published: January 2023

Regulatory T cells (Treg) play a crucial role in the maintenance of peripheral tolerance. Quantitative and/or qualitative defects in Treg result in diseases such as autoimmunity and graft-versus-host disease (GVHD), a serious complication of bone marrow transplantation (BMT). We recently reported that G-CSF mobilisation of stem cells in donor mice increases autophagy related genes in Treg and it is associated with an improved GVHD outcome after BMT. Autophagy is a self-degradative process for cytosolic components which promotes cell homeostasis and survival. Here, we demonstrate that the lineage-specific disruption of autophagy within FoxP3+ Treg (Atg7fl/fl.FoxP3cre+ mice) resulted in a profound loss of Treg within the bone marrow (BM). This resulted in dysregulated effector T cell activation and expansion, and in aged mice, the development of enterocolitis and severe scleroderma. We show that the BM compartment is highly enriched in TIGIT+ Treg and that this subset is differentially lost in the absence of autophagy. Moreover, G-CSF administration to naive mice results in the mobilization of BM TIGIT+ Treg explaining the autophagy signature of peripheral Treg in G-CSF treated mice. Importantly, lethally irradiated B6D2F1 recipients of splenic grafts from G-CSF mobilized B6.Atg7fl/fl.FoxP3cre+ donors failed to reconstitute the Treg compartment within the BM. This resulted in GVHD exacerbation and reduced survival compared to recipients of B6.WT.FoxP3cre+ grafts. Collectively, these data indicate that autophagy-dependent TIGIT+ Treg in the BM are critical for the maintenance of tolerance and to control GVHD; thus the induction of autophagy may represent an attractive immune-restorative therapeutic strategy.
Antigen-specific induced T regulatory cells (iTregs) inhibit dendritic cell (DC) function by capturing peptide-MHCII complexes from the DC surface
Authors: Akkaya Billur , Akkaya Munir , Holstein Amanda H , Kamenyeva Olena , Kabat Juraj , Shevach Ethan M
Published: January 2023

Tregs are critical mediators of peripheral immune tolerance. A majority of the mechanisms utilized by antigen-specific Tregs to suppress immune responses, suggest an initial inhibition of dendritic cell (DC) function that results in diminished activation of T effector cells (Teff) specific for the homologous target as well as diminished activation of other antigen-specific Teff cells that recognize different antigens presented by the same DC (bystander suppression). In an adoptive transfer model in vivo, we demonstrated that OTII transgenic iTregs specific for Ovalbumin323–339 markedly inhibited the expansion of OTII Teff, but did not inhibit the expansion of co-transferred SMARTA Teff (specific for LCMV NP61–76) even though the transferred DCs displayed both peptides. Analysis of iTreg-DC co-cultures in vitro using flow cytometry and confocal microscopy demonstrated that specific peptide-MHCII complexes were captured and internalized by iTregs leaving DCs with decreased levels of antigen. Polyclonal iTregs, naïve, and activated antigen-specific Teff did not capture peptide-MHCII complexes. Intravital microscopy of the popliteal lymph node showed that adoptively transferred OTII iTregs made larger clusters around the DC for a longer period of time compared to activated Teff and increased the speed of co-transferred naïve OTII Teff cells. Taken together these studies suggest that antigen-specific iTregs inhibit immune responses locally in an antigen-dependent fashion by forming firm interactions with DCs leading to a stripping of peptide-MHCII complexes from the DC surface by a transendocytosis-based mechanism.
The role of PML in the activation and the differentiation of T cells and B cells
Authors: Lai Peng-Yeh , Lo Yu-Hsun , Lai Ming-Zong
Published: January 2023

Promyelocytic leukemia protein (PML) is a tumor suppressor with diverse activities in cell growth, cell death, stress responses or senescence. PML is a member of the tripartite motif (TRIM) family protein, which contains RING, B-box and coiled-coil domains. Recent studies showed that large number of TRIM family proteins positively regulate innate immune signaling pathways by acting as E3 ubiquitin ligases. To investigate the role of PML in adaptive immunity, we examined the population of different T cell and B cell subsets in PML-knockout mice. The T cells population including CD4+, CD8+, CD25+, effector T, naïve T, and memory T cells were comparable between WT and PML-deficient mice. The B cells population including follicular B, transitional type 1 and 2 B cells, and marginal zone B cells were also comparable between WT and PML-knockout mice. We also determined whether the activation of T cells and B cells were affected by PML-deficiency. It was found that the activation of PML-deficient T and B cells was lower than that of WT cells. We further determined the differentiation of induced regulatory T (iTreg) cells, and observed that iTreg differentiation was comparable in WT and PML-deficient cells. Therefore, PML is not apparently involved in the development of T cells and B cells, and is modesty required for the full activation of T lymphocyte and B lymphocytes. The exact contribution of PML to the activation of T cell and B cell awaits further investigation.
NLRP6 function in inflammatory monocytes reduces susceptibility to colitis in mice
Authors: Chen Grace Yi , Golovchenko Natasha , Schaf Bryan , Chen Jiachen , Eaton Kathryn , Seregin Sergey
Published: January 2023

NLRP6 is a member of the Nod-like receptor (NLR) family, whose members are involved in the recognition of microbes and/or tissue injury. NLRP6 was previously shown to regulate IL-18 secretion and is important for protecting mice against chemically-induced colitis and colitis-associated colon cancer. However, the cellular mechanisms by which NLRP6 reduces susceptibility to colonic inflammation remain unclear. Here, we determined that NLRP6 expression is specifically upregulated in Ly6Chi inflammatory monocytes that infiltrate into the colon during dextran sulfate sodium (DSS)-induced inflammation. Adoptive transfer of WT Ly6Chi inflammatory monocytes into Nlrp6−/− mice was sufficient to protect them from mortality, significantly reducing intestinal permeability and damage. NLRP6-deficient inflammatory monocytes were specifically defective in TNFα production, which was important for reducing DSS-induced mortality and dependent on autocrine IL-18 signaling by inflammatory monocytes. Our data reveal a previously unappreciated role for NLRP6 in inflammatory monocytes, which are recruited during colitis to promote barrier function and limit bacteria-driven inflammation. This study also highlights the importance of early cytokine responses, particularly NLRP6-dependent and IL-18-dependent TNFα production, in preventing chronic dysregulated inflammation that occurs in patients with inflammatory bowel disease.
Identification of two MCMV immunoevasins that modulate NK cell recognition via the NKR-P1B:Clr-b axis
Authors: Aguilar Oscar A , Rahim Mir Munir , Reichel Johanna , Lau Timothy , Samaniego Jackeline , Sampaio Isabella , Krmpotić Astrid , Jonjić Stipan , Makrigiannis Andrew , Allan David S.J. , Carlyle James R
Published: January 2023

Natural killer (NK) cells are a subset of innate lymphoid cells (ILC) capable of recognizing pathological target cells through germline-encoded receptor-ligand interactions. Murine cytomegalovirus (MCMV) is a betaherpesvirus that has co-evolved with its natural host, and as such, a significant portion of its genome encodes immunoevasins that directly target NK cell receptor-ligand interactions. Here, we identify two putative immunoevasins that modulate host-pathogen interactions via the inhibitory NKR-P1B:Clr-b recognition system. First, we identify an m145 family member, m153, that actively prevents MCMV infection-mediated Clr-b downregulation. Ectopic expression of m153 in mouse fibroblasts increases Clr-b expression at the cell surface. In contrast, infection with a virus deficient in m153 shows more substantial Clr-b loss at the cell surface compared to wild-type MCMV. Importantly, enhanced Clr-b loss upon infection with the m153-mutant could be reversed upon m153 complementation by overexpression. Secondly, we have identified an m02 family member, m12, that directly interacts with the NKR-P1B inhibitory receptor in reporter cell assays. Notably, the interaction between NKR-P1B and m12 is both Clr-b/β2m-independent, and infection of fibroblasts with m12-mutant MCMV abrogates NKR-P1B ligation on reporter cells. This interaction, suggestive of decoy ligand function, is also both MCMV strain-dependent and host NKR-P1B allele-specific, suggesting that polymorphisms have evolved at both the pathogen and host levels that affect NK cell recognition of infected target cells.
Murine hemophagocytic lymphohistiocytosis can occur in the absence of interferon-gamma.
Authors: Burn Thomas Niels , Rood Julia Elizabeth , Weaver Lehn , Kreiger Portia A , Behrens Edward M
Published: January 2023

Hemophagocytic lymphohistiocytosis (HLH) is an ultimately fatal disease manifesting as a result of a dysregulated systemic hyperinflammatory response. Infection of perforin knockout (PKO) mice with an acute strain of lymphocytic choriomeningitis virus (LCMV) mimics the human disease. Based on this murine model, the current dogma suggests that disease is driven by excessive production of interferon gamma (IFNγ) by hyper-proliferative, LCMV specific CD8+ T cells. Evidence in humans has however suggested that HLH can develop in the absence of IFNγ signaling. To test this possibility we developed a double knock-out (DKO) mouse lacking both perforin and IFNγ, and show that DKO mice develop multiple HLH disease characteristics to a similar extent to the PKO mice, including weight loss, elevated sCD25, cytopenia, and hepatitis. DKO mice had equivalent numbers of LCMV specific CD8+ T cells in the spleen, and increased numbers of CD8+ T cells displaying a terminal effector phenotype (p<0.05). Interestingly, DKO mice had extensive neutrophilia in the blood, liver, and spleen compared to PKO mice, correlating with elevated serum IL-6 (p<0.01) and IL-1β (p<0.05). We hypothesized that DKO CD8+ T cells drove HLH through an aberrant, type-17 skewing. Indeed, following ex vivo re-stimulation, there was a significant population of IL-17a producing CD8+ T cells, corroborated by an increase in IL-17a and IL-17f transcript levels in purified DKO CD8+ T cells. Overall, these data suggest that perforin deficient HLH can occur independently of IFNγ, which is important to consider as IFNγ blocking therapies begin clinical trials. Additionally we have potentially unveiled an important regulatory mechanism for IFNγ in the control of CD8+ T cell fate decisions.
miR-146a restricts germinal center B cell responses through targeting CD40 signaling pathway
Authors: Cho Sunglim , Lee Hyang-Mi , Choi Youn Soo , Lin Ling-Li , Kang Seung Goo , Xiao Changchun , Crotty Shane , Lu Li-Fan
Published: January 2023

The germinal center (GC) reaction is the hallmark of B cell-mediated immune responses to T cell-dependent (TD) antigens. Engagement of CD40 by its ligand, CD154, which is expressed on activated CD4+ T cells, follicular helper T (Tfh) cells in particular, is crucial for TD humoral immunity. Previously, it has been demonstrated that miR-146a serves as a molecular break in limiting Tfh cells and the resultant GC B cell responses. Here, by taking the mixed bone marrow chimeras as well as the cell-type-specific miR-146a deletion approaches, we show that like Tfh cells, elevated levels of miR-146a in B cells are equally important in controlling humoral immunity as miR-146a deficiency leads to increased GC B cell differentiation. Moreover, deletion of miR-146a specifically in isotype-switched B cells also resulted in augmented GC responses, suggesting that continued miR-146a expression in GC B cells is required for the maintenance of optimal GC reactions. Mechanistically, we found that miR-146a-ablated B cells exhibit elevated CD40 expression and that enhanced CD40 signaling is accounted for the dysregulated GC responses in mice with B cell-specific miR-146a ablation. Together, our study demonstrates an indispensable role of miR-146a in restricting GC B cell responses and suggests that miR-146a is a critical negative regulator in controlling both T and B cells, two key arms in orchestrating protective humoral immunity.
ROLE OF LIPID RAFT-BASED MEMBRANE ORDER IN SIGNALING THROUGH THE ANTIGEN RECEPTOR IN CD4+ T CELLS: INVESTIGATING THE MECHANISM
Authors: Bamezai Anil K , Bakun Walter
Published: January 2023

Plasma membrane of all animal cells are highly organized into liquid ordered (Io) and liquid disordered (Id) phases. While Io phase is enriched in saturated lipids, cholesterol and lapidated proteins, the unsaturated lipids and some proteins are the major constituents of the Id phase. Lipid rafts nano-domains on the membrane that are enriched in cholesterol and saturated lipids, constitute, in-part, the ordered phase of the membrane. We have previously reported the role of membrane order in spatiotemporal signaling in primary CD4+ T lymphocytes expressing αβ T cell receptor with specificity to chicken ovalbumin323–339 peptide (cOva323-339) (Schieffer et. al., 2014, BMC Immunology 15:58). Primary DO11 CD4+ T cells with disrupted lipid raft-based membrane order lose their proliferative capacity to clonally expand in response to cOva323-339. Moreover, integrity of membrane order within 120 minutes of antigen exposure is critical for antigen-specific clonal expansion of CD4+ T cells. Since early events of T cell activation are initiated at the plasma membrane we examined the effect of membrane disorder on key signaling proteins participating in early CD4+ T cell signaling, such as, p56lck, Zap70, LAT, PLC-γ1, and Akt. We show that disruption of lipid raft-based membrane order does not inhibit the early phosphorylation events associated with aforementioned signaling proteins after engaging the TCRαβ/CD3 complex on primary CD4+ T cells. We are currently assessing the contribution of accessory molecules/adhesion proteins expressed on CD4+ T cells whose biological function may depend on integrity of lipid raft-based membrane order.
PD-1 inhibits the TCR signaling cascade by sequestering SHP-2 phosphatase, preventing its translocation to lipid rafts and facilitating Csk-mediated inhibitory phosphorylation of Lck.
Authors: Bardhan Kankana , Patsoukis Nikolaos , Weaver Jessica , Freeman Gordon , Li Lequn , Boussiotis Vassiliki A
Published: January 2023

PD-1 inhibits T cell responses but the biochemical mechanisms are poorly understood. PD-1 cytoplasmic tail contains an ITIM and an ITSM motif. SHP-2 tyrosine phosphatase interacts with the ITSM and has a critical role in PD-1-mediated inhibition. Although phosphatases are considered negative regulators of activation, SHP-2 deficient T cells display impaired activation of MAPK in response to TCR/CD3 stimulation. Conversely, SHP-2 mutations leading to gain of function induce activation of Ras-MAPK pathway in AML. Thus, SHP-2 may activate some signaling pathways. We investigated how PD-1: SHP-2 interaction inhibits T cell activation. A key event required for activation of the TCR/CD3 signaling cascade is the downregulation of Csk-mediated inactivating phosphorylation of Lck on Tyr-505. Growth factor stimulation of epithelial cells recruits SHP-2 to the plasma membrane and abrogates Csk-mediated inactivating phosphorylation of Src kinase. We determined that TCR/CD3 stimulation downregulated Lck phosphorylation on Tyr-505 but this was prevented by PD-1 ligation. Because Lck is localized in membrane lipid rafts, we examined whether SHP-2 might be recruited to the rafts. TCR/CD3 stimulation resulted in translocation of SHP-2 to the lipid rafts. In contrast, PD-1 ligation recruited SHP-2 to the PD-1 ITSM abrogating translocation of SHP-2 to the rafts and resulted in enhanced Lck phosphorylation on Tyr-505. Our results unravel a previously unidentified mechanism via which PD-1 inhibits T cell activation by sequestering SHP-2 and preventing its translocation to the site on the plasma membrane where SHP-2 plays a key role in activating the TCR signaling cascade by reversing Csk-mediated inactivating phosphorylation of Lck.
A calcium-dependent PKC, independent of NF-κB, is required for proper initiation of chromatin decondensation during T-cell activation.
Authors: Rawlings Jason S , Swatzel Hayley , Ward Audrey , Eikenbusch Sarah , Shields Molly , Meredith Jenna , Mitchell Taylor , Bingham Kellie , Lee Megan
Published: January 2023

Following antigen engagement at the T-cell receptor (TCR), there are two known signaling pathways that are involved in the activation of T-cells, initiated by DAG and IP3. These signaling pathways ultimately lead to chromatin decondensation, which is required for subsequent T-cell proliferation mediated by IL-2. It has previously been demonstrated that intracellular calcium is necessary for proper chromatin decondensation but that calcium does not act through transcription factor NFAT. In this study, we demonstrate that PKC, activated through DAG, is also required for proper chromatin decondensation. Furthermore, we show that calcium is required for PKC mediated chromatin decondensation, suggesting that a calcium dependent isoform of PKC is involved in the process. Interestingly, PKC but not IP3 is required for subsequent competence of T-cells to respond to IL-2 signaling. Finally, we show that PKC does not act downstream through NF-κB to decondense chromatin and our data suggest that NF-κB is not required for IL-2 competence. Collectively, these data could be suggestive of multiple mechanisms to control the activation and proliferation of T-cells during an immune response.
T cell receptor signaling and Toll-like receptor signaling converge to amplify T cell responses
Authors: Young Mary H , Navarro Garnet , Askovich Peter , Aderem Alan
Published: January 2023

Toll-like receptors (TLRs) are an essential part of the innate immune system. Their function on antigen presenting cells helps drive efficient T cell responses to pathogens. However, T cells also express several TLRs. Additionally, many of the downstream signaling components of TLRs are shared with those of the T cell receptor. Our hypothesis is that these two pathways work in concert to exert unique and potent T cell responses. We have found that several TLRs are able to enhance T cell responses, including TLR3, TLR4, TLR7, and TLR9. We have found that TLR7 signaling, in particular, synergizes with TCR activation and improves T cell effector functions. Priming T cells with synthetic and natural TLR7 ligands before TCR ligation further boosts T cell responses. The combination of TLR7 and TCR stimulation in T cells enhances cytokine production and proliferation in T cells. We identified several overlapping components between TLR7 and TCR activation pathways by gene expression, which may provide combinatorial increases in T cell activation. In addition, we found increases in anti-viral response pathways that are atypical in T cells. This response was unique to the combination of TLR and TCR stimuli. Thus, we wanted to determine if TLR7 signaling in T cells is important during an acute infection. During influenza, T cell-specific loss of TLR7 alters infection dynamics in the T cell population, resulting in significant activation delays and abnormalities. These findings show that TLR7 is important in normal T cell responses to viral infections. Understanding the contribution of TLR signaling to T cell activation will be useful in guiding enhanced vaccine strategies.
IL-4 sensitivity shapes the peripheral CD8+ T cell pool and response to infection
Authors: Renkema Kristin , Lee June-Yong , Lee You-Jeong , Hamilton Sara , Hogquist Kristin Ann , Jameson Stephen
Published: January 2023

Previous studies have revealed that a population of innate memory CD8+ T cells is generated in response to IL-4, first appearing in the thymus and bearing high expression levels of Eomesodermin (Eomes) but not T-bet. However, the antigen specificity and functional properties of these cells is poorly defined. Here we show that IL-4 regulates not only the frequency and function of innate memory CD8+ T cells but also regulates Eomes expression levels and functional reactivity of naïve CD8+ T cells. Lack of IL-4 responsiveness attenuates the capacity of CD8+ T cells to mount a robust response to LCMV infection, with both quantitative and qualitative effects on effector and memory antigen specific CD8+ T cells. Importantly, our data revealed that these effects of IL-4 exposure occur prior to, not during infection. Together, these data show that IL-4 influences the entire peripheral CD8+ T cell pool, influencing expression of T-box transcription factors, functional reactivity and the capacity to respond to infection. These findings indicate that IL-4, a canonical Th2 cytokine, can sometimes promote rather than impair Th1-type immune responses.
Smad4 Signaling is Important for Generation of Teff Cells but not Protection
Authors: Hu Yinghong , Suarez-Ramirez Jenny , Cauley Linda S
Published: January 2023

Ideally, vaccines for pulmonary infections should generate protective populations of anti-viral memory CD8 T cells without accompanying responses by pathogenic effector T (Teff) cells. With this goal in mind, we examined the influence of transforming growth factor β (TGFβ), and the downstream molecule Smad4, during CD8 T cell differentiation. CTLs lacking the TGFβ receptor preferentially expressed KLRG1 after influenza virus infection indicating the presence of highly differentiated Teff cells, while differentiation of CD103+ tissue resident memory T (Trm) cells was completely abrogated. In contrast, Smad4-deficient CD8 T cells preferentially acquired a KLRG1-negative phenotype, while CD103 was aberrantly expressed on CTLs in other tissues. Smad4-deficency caused slightly delayed viral clearance after primary IAV infection, but was not required for protective immunity upon reinfection with another strain. Our data indicate that Smad4 dictates the fate decision of activated CTLs by integrating signals from multiple environmental stimuli to promote TEFF generation.