bluuebunny/crossref_metadata_2025 huggingface dataset:
TLR7 responsiveness of human transitional B cells
Authors: Giltiay Natalia V , Shu Geraldine , Wang Ting , Marken John , Durcan Laura , Clark Edward A
Published: January 2023

Systemic lupus erythematosus (SLE) is a complex autoimmune disease that is known to be associated with B cell hyperactivity and production of autoantibodies (Auto-Abs) against nucleoproteins. Auto-Abs reactive to RNA-containing antigens are found in a fraction of SLE patients, particularly those with severe disease activity. Toll-like receptor 7 (TLR7), an intracellular TLR that recognizes ssRNA, has been highly implicated in the generation of pathogenic anti-RNA autoantibodies in SLE; however, the cellular origin of anti-RNA Ab–secreting cells is not well defined. We have found that increased expression of TLR7 in TLR7Tg mice results in activation of their newly-formed (NF) transitional B cells which upon stimulation produced class-switched IgGs, including anti-RNA auto-Abs. Subsequent analysis of the B cell compartment in SLE patients revealed a correlation between the levels of TLR7 expression and increases in the frequencies of circulating CD20+CD27−CD10+CD38highCD24high NF B cells. SLE patients with dysregulation of NF B cells were also more likely to have Sm/RNP auto-Abs. IFNa treatment induced more than a 10-fold increase in the mRNA expression of TLR7, UNC93B and IRF7 in NF B cells isolated from umbilical cord blood. Furthermore, IFNa-primed NF B cells became highly responsive to in vitro stimulation with TLR7 ligand-R848 and differentiated into CD27+CD38hiCD24lo plasmablasts in the absence of any other stimuli. Human NF B cells are enriched in polyreactive specificities and increases of NF B cells have been described in SLE patients. Our studies suggest that human NF B cells might be an important source of auto-Ab-secreting cells and provide a new link between innate IFNa and TLR7 signaling and B cell activation in SLE.
MDSCs enhance Th17 differentiation and disease progression through arginase-1 production in patients with systemic lupus erythematosus
Authors: YI HUANFA , Wu Hao , Zhen Yu , Ma Zhanchuan , Li Huiumin , Yang Yong Guang
Published: January 2023

Expansion of myeloid-derived suppressor cells (MDSCs) has been documented in some murine models and patients with autoimmune diseases, but the exact role of MDSCs in this process remains largely unknown. Although MDSCs are supposed to be immunosuppressive, previous studies have shown that these cells may also enhance Th17 responses via an IL-1-dependent mechanism in murine autoimmune models, such as experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA). However, the role of MDSCs in the development of autoimmune responses in human remains largely unknown. Current study investigates this question in patients with systemic lupus erythematosus (SLE). We found that patients with active SLE (n=32) had a significant increase in HLA-DR−CD11b+CD33+ MDSCs, in the peripheral blood compared to healthy controls (HCs; n=25). The frequency of MDSCs was positively correlated with the level of serum arginase-1 (Arg-1) activity, Th17 responses and disease severity in SLE patients. Consistently, in comparison with MDSCs from HCs, MDSCs from SLE patients exhibited significantly elevated Arg-1 production, and increased potential to promote Th17 differentiation in vitro in an Arg-1-dependent manner that is likely mediated by multiple mechanisms involving RORgt, RORa and mTOR. Moreover, in a humanized SLE model, MDSCs were essential for the induction of Th17 responses and the associated renal injuries and the effect of MDSCs was Arg-1-dependent. Our data provide direct evidence demonstrating a pathogenic role for MDSCs in human SLE, and suggests that targeting MDSCs or Arg-1 may offer novel therapeutic strategies for the treatment of SLE and other Th17 cell-mediated autoimmune diseases.
Complement-tagged basophils and eosinophils in Systemic Lupus Erythematosus (SLE)
Authors: Liu Chau-Ching , McCarty Berryhill E. , McGeary Meaghan , Manzi Susan , Ahearn Joseph M.
Published: January 2023

Cell-bound complement activation products (CB-CAPs) on erythrocytes, reticulocytes, platelets, and lymphocytes have been recognized as biomarkers for SLE diagnosis, monitoring, and prognosis and to render such cells dysfunctional. Recently, basophils have been shown to contribute to the development of lupus nephritis and presence of eosinophils in the urine has been reported as a potential biomarker for lupus nephritis. This study was based on the hypothesis that basophils and eosinophils bearing C4d may serve as valuable biomarkers and participate in lupus pathogenesis.

To verify this hypothesis, a pilot study investigating basophil-bound and eosinophil-bound C4d levels was conducted using blood samples taken from 103 patients with SLE, 23 patients with other immune-inflammatory diseases, 17 patients with asthma or allergic rhinitis, and 17 healthy individuals. Peripheral blood leukocytes were isolated, and basophils and eosinophils therein were detected and analyzed for surface-bound C4d by flow cytometry.

Preliminary results showed that basophil-bound C4d (Baso-C4d) and eosinophil-bound C4d (Eos-C4d) levels were significantly elevated in patients with SLE, compared to patients with other immune-inflammatory diseases, patients with asthma/allergy, and healthy controls (all p < 0.05). As diagnostic tools, Eos-C4d and Baso-C4d, respectively, were 29% sensitive/96% specific and 22% sensitive/96% specific in differentiating SLE from other immune-inflammatory diseases. The results obtained indicate that Baso-C4d and Eos-C4d may serve as specific markers for SLE diagnosis and possibly for SLE global/organ-specific disease activity.
Autoantibody-mediated generation of platelet-bound C4d in Systemic Lupus Erythematosus (SLE)
Authors: Liu Chau-Ching , Schofield Travis , Manzi Susan , Ahearn Joseph M.
Published: January 2023

Premature and accelerated onset of atherothrombotic disease is prevalent in patients with SLE. Most, if not all, atherothrombotic diseases are likely to involve platelets and the complement system in some capacity. Previously, we discovered that platelets bearing complement activation product C4d (P-C4d) are present in approximately 20% of SLE patients and are significantly associated with anti-phospholipid (aPL) antibody positivity and stroke in SLE patients. The goal of the present study was to elucidate the role of aPL and other platelet-reactive autoantibodies in the generation of P-C4d.

To determine the association between P-C4d and aPL, the level of P-C4d in 180 SLE patients was measured by flow cytometry. Serum level of aPL, including anti-cardiolipin (aCL) and anti-b2 glycoprotein I (anti-b2GPI) antibodies, in these patients was measured by enzyme-linked immunosorbent assays. To investigate the role of aPL, and possibly other autoantibodies as well, in mediating the generation of P-C4d, in vitro 2-step transfer experiments using normal platelets in combination with plasma or immunoglobulin derived from SLE patients were performed. The results showed that the presence and levels of serum aCL and anti- anti-b2GPI were highly specific for SLE patients with positive P-C4d. The plasma and immunoglobulins purified from SLE patients who were positive for P-C4d and aCL/anti-b2GPI were able to induce C4d deposition on normal platelets in vitro. The capacity of SLE plasma in inducing P-C4d appeared to correlate proportionately to the serum aCL levels. Collectively, the results demonstrate that both aPL, particularly aCL, and other platelet-reactive autoantibodies participate in mediating the generation of P-C4d in SLE patients.
Increased cytokine expression of mesangial cells through altered glycosphingolipid catabolism in lupus nephritis
Authors: Nowling Tamara K , Sundararaj Kamala , Angel Peggi , Drake Richard
Published: January 2023

Previously we demonstrated that glycosphingolipid (GSL) levels and neuraminidase (NEU) activity are altered in the kidneys and/or urine of lupus mice and human patients with nephritis compared to their non-nephritic counterparts and healthy controls. We also showed that sterol regulatory element-binding protein (SREBP)-1 expression is increased in the kidneys of nephritic lupus mice and altered GSL levels were observed in the glomerular region. Therefore, we hypothesized that NEU-mediated GSL catabolism is regulated by SREBP-1 and contributes to the production of cytokines by mesangial cells (MC) leading to renal inflammation in lupus nephritis. We now show co-localization of GSL lactosylceramide (LacCer) with MCs and increased expression of IL-6, IFNγ and other cytokines following addition of LacCer to cultured MCs. Primary MCs from pre-nephritic lupus mice stimulated with heat aggregated IgG (a mimic of immune complexes), LPS, or lupus serum showed elevated GSL levels, IL-6 production, MCP-1 expression and Neu1 expression. We now demonstrate that SREBP-1c expression is significantly upregulated in the kidney prior to overt nephritis compared to healthy controls. Over-expressing SREBP-1c in a MC line upregulated Neu1 promoter activity by two-fold, increased endogenous NEU1 expression and increased LacCer levels. Moreover, over-expressing either NEU1 or SREBP-1c significantly increased IL-6 production. Together these results suggest that MC activation leads to increased cytokine expression in part through NEU-mediated GSL catabolism regulated by SREBP-1c in lupus nephritis.
Stromal cells from inflamed lymph nodes modulate B cell surface CD23 expression
Authors: Bottaro Andrea , Kuzin Igor , Sims Etty , Koshkareva Yekaterina , Bernstein Steven , Burack Richard , Schwarz Edward
Published: January 2023

CD23 is a trans-membrane protein belonging to the C-type lectin family, widely expressed in the immune system. Originally identified as the low-affinity receptor for IgE, CD23 was later shown to also bind CD21, MHCII and various integrins, and to have pleiotropic roles, including modulation of cell proliferation, differentiation, Ig and cytokine secretion. Expression of membrane CD23 (mCD23) is regulated at least in part by its shedding from the cell surface via activity of metalloproteinases, the most important of which is ADAM10. Interestingly, secreted CD23 (sCD23) isoforms can exert immunomodulatory functions as soluble factors. B cells upregulate mCD23 and serum sCD23 levels have been found to be elevated in inflammatory and autoimmune conditions, including rheumatoid arthritis, and in B cell chronic lymphocytic leukemia. However, the mechanisms responsible for CD23 modulation on B cells are still unclear. Here we show that mCD23 is upregulated on mouse and human B cells found in inflamed lymphoid tissues. Furthermore, in vitro experiments demonstrate that B cell mCD23 upregulation is strictly dependent on soluble signals provided by stromal cells from inflamed, but not non-inflamed lymph nodes, and can be reversed by ADAM10-specific inhibitors. We propose therefore that the balance between B cell mCD23 and sCD23 expression in inflammatory conditions is modulated by microenvironmental signals provided by stromal cells within lymphoid tissues.
Effects of different treatment regimens on T cell function in acute HIV infection.
Authors: Clutton Genevieve Tyndale , Xu Yinyan , Fernandez Maria Abad , Crooks Amanda , Turner Blair , Maponga Nicole , Cope Anna , Fiscus Susan , McGee Kara , McKellar Mehri , Kuruc JoAnn , Ferrari Guido , Margolis David , Hicks Charles , Eron Joseph , Gay Cindy , Goonetilleke Nilu
Published: January 2023

In HIV-infected individuals, anti-retroviral therapy (ART) is effective in maintaining low viral loads, restoring CD4+ T cell counts, and prolonging life. However, chronic inflammation and immune dysfunction persist even after years of successful ART. Previous studies have suggested that early initiation of ART may result in improved immune reconstitution, but whether more rapid suppression of viral load during early treatment also improves immunological outcomes is unknown. In this study we compare the effects of two regimens initiated during acute infection on T cell phenotype and function. Regimen 1 (n=30) is a non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimen and Regimen 2 (n=30) combines an integrase strand transfer inhibitor (INSTI) with nucleoside reverse transcriptase inhibitors (NRTIs). NRTIs and NNRTIs inhibit reverse transcription of viral RNA to DNA in the infected cell, while INSTIs block integration of viral DNA into the host genome. Since Regimen 2 targets multiple stages of the viral life cycle, we hypothesize that more rapid viral suppression may be achieved with this treatment schedule. In a cross-sectional study performed 96 weeks after treatment initiation, we are comparing the effects of the two regimens on T cell activation (CD25, CD69, CD38, HLA-DR), exhaustion (PD-1, Tim-3, CD160, T-bet, Eomes), and proliferation using standardized flow cytometry panels. This study will provide valuable insights into the relationship between viral decay kinetics and immune preservation during acute infection, informing the design of future ART regimens.
Ficolins promote fungal clearance during the early stage of pulmonary infection with Aspergillus fumigatus.
Authors: Genster Ninette , Cowland Jack , Cramer Elisabeth , Rosbjerg Anne , Pilely Katrine , Garred Peter
Published: January 2023

Aspergillus fumigatus is a fungal pathogen causing severe and usually fatal invasive infections in immunocompromised patients. The Ficolins are pattern recognition molecules of the lectin pathway of complement and in vitro studies suggest a role for ficolins in the defence against A.fumigatus. However, little is known about their significance in fungal infections in vivo. Thus, our aim of the current project was to establish a murine model of pulmonary A. fumigatus infection to study the relevance of ficolins in host protection against A.fumigatus. Wildtype and ficolin knockout mice were infected intranasally with a sublethal dose of A.fumigatus conidia and the lungs were removed at various time points post-infection to assess the fungal load and expression of pro-inflammatory cytokines. In addition, the concentration of pro-inflammatory cytokines was measured in the bronchoalveolar lavage. After 24 hours of A.fumigatus infection, the pulmonary fungal load was significantly higher in ficolin knockout mice compared to wildtype mice. In contrast, no significant difference in fungal load between ficolin knockout mice and wildtype mice was observed after 12, 48 or 72 hours of infection. Interestingly, the increased fungal load in knockout mice 24 post-infection was accompanied by a significantly impaired induction of pro-inflammatory cytokines compared to wildtype mice. These results show that ficolins promote fungal clearance and modulate the pro-inflammatory cytokine response, demonstrating the importance of ficolins during the initial phase of A. fumigatus infections in vivo.
Critical role of LIGHT/TNFSF14 in poly(I:C)-induced acute hepatitis
Authors: Wang Pu , Lu Xiaohua , Zheng Wei , Zou Junhui
Published: January 2023

LIGHT is a member of the TNF superfamily, which is known to be involved in liver pathologies. Toll-like receptor 3 (TLR3) is a critical component of the innate immune response to dsRNA viruses. The polyinosine-polycytidylic acid (Poly(I:C), a TLR3 viral mimetic, induces an acute hepatic injury and mimics a model of viral hepatitis. In this study, we investigated the role of LIGHT in acute hepatitis induced by poly(I:C). The results show that administration of poly(I:C)/D-galactosamine (D-GalN) in mice led to acute liver injury associated with the increased expression of LIGHT in liver. Blockade of LIGHT/HVEM signaling ameliorated liver injury. Pre-treatment with a NF-κB inhibitor dramatically reduced LIGHT expression and liver injury, suggesting the involvement of NF-κB in LIGHT expression in poly(I:C)/D-GalN-induced acute hepatitis.
Netrin-1 mediates macrophage retention in adipose tissue through AGAP-2 interaction with the dependence receptor UNC5B via miR-762 and miR-346
Authors: Mehrpouya-Bahrami Pegah , Zumbrun Elizabeth E. , Zameer Fahran , Nagarkatti Prakash S , Nagarkatti Mitzi
Published: January 2023

Retention of Macrophages in adipose tissue is associated with high expression of neuroimmune guidance cue Netrin-1 and its receptor UNC5B which are involved in chemotaxis. Cannabinoid receptor1 (CB1) receptors are known to regulate the immune response. We have shown previously that treatment of mice with SR141716A, a CB1 antagonist, attenuates obesity. To further investigate the mechanisms, Diet-Induced Obesity (DIO) was generated by feeding C57BL/6J mice with high-fat diet (HFD) whereas their lean, age-matched controls were fed low-fat diet (LFD). HFD-fed mice were treated with either SR141716A (10mg/kg/day) or vehicle by daily oral gavage for 4 weeks starting at week 12. Our data demonstrated that SR141716A suppressed diet intake transiently, however weight loss and reduction in fat mass was persistent. Suppression of pro-inflammatory macrophages was observed in SR141716A-treated HFD-fed group followed by down regulation of Netrin-1 and UNC5B in macrophages. In silico analysis demonstrated that miRs induced by SR141716A regulated several pathways including macrophage migration. miR-762 and miR-346 which were down regulated following SR141716A treatment were found to be complementary to the 3′-UTR of AGAP-2 gene. SR141716A regulated AGAP-2 expression and its association with UNC5B and inhibited UNC5B-provoked macrophage retention in adipose tissue, and subsequently ameliorated insulin resistance. Thus, for the first time we demonstrate that blockade of CB1 receptors leads to altered chemotaxis balance of adipose tissue macrophages leading to amelioration of obesity.
The IL-21 signaling pathway is enhanced in RA B cells and has the potential to alter development and cytokine production in RA B cells
Authors: Dam Elizabeth M , Buckner Jane H
Published: January 2023

B cells have been implicated in the development of rheumatoid arthritis (RA) and IL-21 plays a central role in B cell maturation. Thus, in this study, we examined the response of B cells to IL-21 from RA subjects to determine if alterations in this response contribute to perturbations in B cell development and function in RA.

Flow cytometric studies of PBMC from a cohort of RA and healthy subjects demonstrated an increase in the expression of the IL-21 receptor (IL-21R) on RA B cells as compare to those of healthy controls, and demonstrated a positive correlation between the IL-21R expression and the phosphorylation of STAT3 in response to IL-21. To further understand the molecular mechanisms involved in the altered signaling in RA B cells, we examined mRNA expression in B cell subsets with RNA flow cytometry. Using this approach we determined that IL-21R mRNA levels were enhanced in RA B cells and correlated with observed IL-21R protein expression. However, no alterations in the expression of SOC1 or SOCS3 were observed. Analysis of IL-21 induced activation of the Akt pathway demonstrated enhanced total FOXO1 protein expression in RA B cells with high IL-21R expression. This suggests that both STAT3 and Akt signaling pathways are enhanced in RA B cells that express elevated IL-21R levels. Further, we found that in vitro differentiation of naïve B cells to plasma cells was enhanced in RA B cells and that these plasma cells are IgM+ and produce increased quantities of IL-6.

Together these findings suggest a mechanism by which the IL-21/IL-21R pathway may be contributing to alterations in B cell development and function in RA and point to a role for therapeutics targeting this pathway in these individuals.
Cannabidiol (CBD) induces functional CD4+ CD25+ FOXP3+ Tregs
Authors: Dhital Saphala , Park Nogi , Seo Keun Seok , Kaplan Barbara L. F.
Published: January 2023

We previously reported that cannabidiol (CBD), one of the Cannabis sativa-derived cannabinoid compounds and a potent immunosuppressive agent, increased CD25 and FOXP3 expression on CD4+ cells under “ultrasuboptimal” T cell stimulation (1 nM PMA/0.0125 μM Ionomycin, designated “Us/o”). We also observed that CBD increased IL-10 and il10 mRNA expression under Us/o stimulation. Thus, we tested the hypothesis that CBD-induced CD4+ CD25+ FOXP3+ cells are functional Tregs. First, we determined that the expression of CD25 and FOXP3 was induced on CD4-enriched cells in response to CBD (10 μM) plus Us/o activation as compared to CBD plus low level anti-CD3/28 stimulation (soluble anti-CD3 and anti-CD28 antibodies at 400 or 800 ng each) for 5 days. Second, we determined the functionality of the CBD-induced Tregs using a mixed lymphocyte response assay. For this study, splenocytes were treated with CBD (10 μM) plus Us/o activation for 5 days. CBD-induced CD4+ CD25+ and CD4+ CD25− cells were then purified and treated with mitomycin C (MMC). Fresh, naïve responder splenocytes were activated with anti-CD3/28-coated beads to induce proliferation of the responder cells, then mixed with the MMC-treated CD4+ CD25+ or CD4+ CD25− cells. CBD-induced CD4+ CD25+ cells, but not CBD-induced CD4+ CD25− cells, suppressed responder cell proliferation. Thus, CBD induced functional Tregs under conditions of low level activation, which is consistent with other studies demonstrating that CBD can induce Tregs under optimal stimulation conditions.
Ndfip proteins control Treg cell fate and function to prevent autoimmune disease.
Authors: Layman Awo Akosua Kesewa , Deng Guoping , Oliver Paula M
Published: January 2023

Regulatory T cells (Tregs) are key immune cells for enforcing peripheral tolerance against self. Supporting this, in humans and in mice, a defect in Treg number or function results in severe autoimmune disease. Tregs limit autoimmunity by restraining the activation and cytokine production of conventional T cells (among other mechanisms). Our data reveal how two E3 ubiquitin ligase-activating proteins, Ndfip1 and Ndfip2, regulate Treg fate and function. Ndfip (Nedd4 family interacting proteins) 1 and 2 are known to activate several members of the Nedd4-family of E3 ligases, such as Itch, that have known roles in T cells. We generated mice that constitutively lack Ndfip2 and conditionally lack Ndfip1 in Foxp3+ cells (Treg cDKO mice). These Treg cDKO mice, display severe spleen and lung pathology marked by pulmonary arterial fibrosis, and ectopic lymphoid follicles. Additionally, Treg cDKO mice have higher circulating antibody levels. Since Foxp3 is an X-linked gene, we expect female DKO Treg mice to have both normal and Ndfip-deficient Tregs. Surprisingly, we find that even these female mice are characterized by splenomegaly, pulmonary arterial fibrosis, and these mice show circulating autoantibodies by 16 weeks of age. We will present data explaining why both male and female Treg cDKO mice get sick and discuss the roles of Ndfip-proteins in Treg activation, Treg cytokine production, PD-1 expression, and FoxP3 stability.
Regulatory T cell expression of β1-integrin is critical to the maintenance of CD8+ T cell homeostasis and memory development in mice after infection.
Authors: Da Costa Andreia , Garza Esteban , Lund Jennifer M
Published: January 2023

Regulatory T cells (Tregs) are recognized as suppressors of autoimmunity however; Tregs also play an important although variable role in the development of an immune response to infection. The mechanism of Treg modulation of and migration to immune activation sites during viral infections is not yet clearly defined. As such, we investigated the effect of Treg migratory protein expression on the development of an effective viral immune response.

To test this hypothesis, we utilized an ITGb1Flox x FoxP3Cre conditional knockout mouse model. These mice generate Tregs that lack the ability to express β1-integrin, a transmembrane protein necessary for migration to sites of inflammation. Through flow cytometric analyses, we found that wild type (WT) Treg expression of β1-integrin correlated with an activated, migratory phenotype as indicated by increased surface expression of CD44, CXCR3, and CCR5. In addition, we found that when the balance of β1-integrin +/− Tregs is altered, the expression of suppressive proteins such as CD73 and CTLA4 is significantly decreased. Despite this potential loss in Treg functionality, there is no corresponding increase in mortality following primary infection. However, mice that received CD8+ T cells derived from previously infected β1-integrin Treg conditional knockouts succumbed to secondary infection at much greater rates than WT Treg recipients. This increase in mortality after secondary (but not primary) infection indicates that virus-specific memory CD8+ T cell development is compromised in β1-integrin conditional knockout mice.

In sum, these data suggest that Treg migratory capability is critical for the maintenance and development of a functional CD8+ T cell response after infection.
Pathogenic regulatory T cells promote immunopathology during infectious myositis
Authors: Jin Richard M , Blair Sarah J , Heffner Reid R , Blader Ira J , Wohlfert Elizabeth A
Published: January 2023

Immune dysfunction is a central event in the pathogenesis of idiopathic inflammatory myopathies, yet its etiology remains elusive. Skeletal muscle repair is reliant on the temporally distinct transition from pro-inflammatory (M1) to pro-regenerative (M2) macrophage-mediated phases of repair. Regulatory T cells (Treg) facilitate this transition and its dysregulation results in debilitating disease. Infection of C57Bl/6 mice with Toxoplasma gondii is an ideal model to study perturbations of Treg as infection causes their systemic collapse and high levels of Th1 inflammation. During infection, we observe that M1 persist in skeletal muscle, which at steady state is dominated by M2. Skeletal muscle Treg frequencies dramatically decrease and become Th1-skewed. Therefore, we hypothesized that infection alters Treg function and their ability to promote M2 in skeletal muscle. To test the effect of chronic interferon-gamma (IFNγ) exposure on Treg function, we adoptively transferred Treg cultured with and without IFNγ. Surprisingly, adoptive transfer of IFNγ-Treg significantly increased M1 in infected skeletal muscle, while transfer of non-polarized Treg did not affect M1 or M2 numbers. Furthermore, systemic depletion of Treg in infected mice increased muscle fiber regeneration through increased M2:M1 ratios without an increase in inflammation or immunopathology. Collectively, we show T. gondii infection promotes pathogenic Treg in the skeletal muscle. As Treg-directed therapies gain traction, a deeper understanding of the consequences of chronic inflammation on Treg physiology and tissue-specific reparative programing will be a powerful asset in producing safe and efficacious therapies.
A tissue-resident macrophage specific coinhibitory molecule promotes regulatory T cell differentiation and stability
Authors: Fu Wenxian
Published: January 2023

CD4+ Foxp3+ regulatory T (Treg) cells are key players in immune tolerance and tissue homeostasis. Their impact on tissue homeostasis has been further reinforced by recent studies showing that Tregs in tissue control non-immunological processes. However, how Treg cell differentiation and function are influenced by tissue-derived signals remain poorly understood. We here show that a specific subset of tissue-resident macrophages can promote the differentiation and stability of Tregs. This subset of tissue-resident macrophages distinctively express complement receptor of the immunoglobulin superfamily (CRIg). The phenotype of these macrophages are F4/80hi CRIg+. Our previous studies have found that CRIg acts a coinhibitory molecule to suppress T cell proliferation. We find that in addition to dampening effector T cell proliferation, CRIg promotes the conversion of induced Treg cells (iTreg), by synergizing with TGFβ. CRIg also stabilizes Foxp3 expression in induced Tregs. In vivo CRIg-Ig fusion protein treatment increases Treg proportion, particularly in tissue (islets) of NOD mice, a commonly used animal model for autoimmune diabetes. Together, these data reveal a novel mechanism of shaping Treg pool and function by tissue-resident macrophages. CRIg may represent a novel means to generate ‘stable’ iTregs, which remains unmet need in Treg-based therapy.
Regulatory B10 cells control immune responses via antigen-specific pathways
Authors: Candando Kathleen , Kamata Masahiro , Kountikov Evgueni , Lykken Jacquelyn M , Yoshizaki Ayumi , Miyagaki Tomomitsu , Tedder Thomas
Published: January 2023

B cells secrete antigen-specific antibodies during immune responses to neutralize pathogens and other threats. Despite this well-characterized pro-inflammatory B cell function, a rare subset of B cells (B10 cells) in both humans and mice are known to produce the inhibitory cytokine interleukin 10 (IL-10) and provide negative regulation of inflammation and T cell-dependent autoimmunity. B cell receptor (BCR)-derived signals are essential to regulatory B cell function as B10 cell suppression of disease is antigen-specific and requires cognate interactions with T cells. In addition, B10 cell development is blunted in mice with fixed BCR specificities, underscoring the key role that BCR signaling plays in the acquisition of B cell IL-10 competence. Given the importance of the BCR in both B10 cell development and function, we hypothesized that antigen-specific B10 cells are required for optimal disease suppression. Here we show by single-cell PCR that B10 cells derived from naive mice as well as from mice exposed to antigen express a diverse set of both heavy and light chain immunoglobulin genes that are predominantly without mutations. In addition, we demonstrate using IL-10 reporter mice that antigen-specific B10 cells are reliably expanded in vivo and that these antigen-specific B10 cells are required for optimal disease suppression upon adoptive transfer. Thus, B10 cells provide robust antigen-specific regulation of immune responses in vivo through diverse, unmutated BCRs.
SHIPi alters innate immunity in VAT to control obesity
Authors: Srivastava Neetu , Iyer Sonia , Sudan Raki , Youngs Christie , Engelman Robert W , Howard Kyle T , Russo Christopher M , Chisholm John D , Kerr William G
Published: January 2023

Low-grade chronic inflammation has been established as the key etiological phenomenon responsible for the initiation and perpetuation of obesity and its associated metabolic disorders. Novel therapeutic approaches that can specifically target inflammatory pathways are very much needed to avert this looming epidemic of obesity & diabetes. Genetic & chemical inhibition of SH2 domain containing inositol phosphatase (SHIP)1 has been associated with systemic expansion of immunoregulatory cells that promote a lean-body state however its function in immunometabolism has never been assessed. This led us to investigate the role of SHIP1 in metabolic disorders during excess caloric intake in mice. Using a small molecule inhibitor of SHIP (SHIPi), here we show that SHIPi treatment in mice significantly reduces body-weight and fat content and improves control of blood glucose and insulin sensitivity as well as increases energy expenditure, despite continued consumption of a HFD. Additionally, SHIPi reduces age-associated fat accumulation in mice. We find that SHIPi treatment reverses diet-associated obesity by attenuating inflammation in the visceral adipose tissue (VAT). SHIPi treatment increases IL4-producing eosinophils in VAT and consequently increases both AAM and MDSC. In addition, SHIPi decreases the number of γ-IFN producing T-cells and NK-cells in VAT. Thus, SHIPi represents a novel approach that permits control of obesity and diet-induced metabolic syndrome without apparent toxicity that could benefit the health of millions worldwide.
Fluvastatin induces mast cell apoptosis and autophagy: effects on primary and transformed mast cells
Authors: Paez Patrick A , Kolawole Elizabeth Motunrayo , Kiwanuka Kasalina N , Ndaw Victor S. , Taruselli Marcela T , Paranjape Anuya R , Abebayehu Daniel , Haque Tamara , Ryan John J
Published: January 2023

Mast cells are best known for their role in inflammatory and allergic disease. Mast cell leukemia is a rare subtype of acute myelogenous leukemia, most of which express the c-Kit mutation, D816V, that yields factor-independent growth. Understanding and treating mast cell hyperplasia and leukemia could be beneficial to both inflammatory and malignant diseases. Recently the family of statin drugs, widely employed as HMG CoA Reductase inhibitors to lower serum cholesterol, have also been found to be immunosuppressive. However, statin effects on mast cell survival are not known. We report that Fluvastatin induces apoptosis in mouse bone marrow-derived mast cells (BMMC) and P815 mastocytoma cells in a dose- and time-dependent manner. Apoptosis was evidenced by DNA fragmentation and caspase activation. Importantly, the mastocytoma line P815, which expresses mutant c-Kit, was readily killed by Fluvastatin, with an IC50 of 5–10μM and apoptosis peaking at 72 hours. An up-regulation of the mutated c-Kit receptor was seen at 12 hours, followed by mitochondrial membrane instability and Caspase-9 activation within 24–28 hours. Finally, preliminary evidence of autophagy was detected during fluvastatin treatment. Interestingly, autophagy appeared to be cytoprotective in primary mast cells but cytotoxic in P815 mastocytoma cells. These preliminary data support the theory that statin family drugs induce mast cell autophagy and apoptosis, lending new therapeutic approaches for suppressing growth of both normal and transformed mast cells.
The role of histone deacetylases in the innate immune response to Mycobacterium tuberculosis infection
Authors: Campo Monica , Seshadri Chetan , Stein Catherine M. , Peterson Glenna , Wells Richard D. , Mayanja-Kizza Harriet , Boom W Henry , Hawn Thomas
Published: January 2023

Most of household contacts of pulmonary TB cases, develop latent Mtb infection (LTBI). In a large TB household contact study in Uganda, we found that 9.1% of contacts remained persistently TST negative in 2 years of follow up. We hypothesized that resistance to Mtb infection is mediated by monocytes. We compared genome-wide mRNA profiles of Mtb-infected monocytes from those who are resistant or susceptible to Mtb infection. Using Gene Set Enrichment Analysis, we recently identified a butyrate stimulated gene set that distinguished these two groups. Butyrate inhibits histone deacetylases (HDAC), a gene family that regulates transcription as well as innate immune responses to microbes. We found that treatment of peripheral blood monocytes with class I HDAC inhibitors, depsipeptide and sodium butyrate, led to reduced IL6 and TNF secretion after Mtb infection compared to untreated cells. In contrast, IL1beta levels were increased. Together, these data indicate that class I HDAC inhibitors modulate proinflammatory cytokines after Mtb infection. Future studies will examine how HDAC inhibitors regulate inflammasome-dependent IL-1beta and IL6 secretion differently. HDACs likely regulate multiple pathways that modulate Mtb pathogenesis and mediate clinical to Mtb infection. HDACs are plausible candidate human Mtb resistance genes and promising targets for host-directed therapeutics.
NKT Cell-Mediated Inhibition of Inflammation
Authors: Setzu Nicole , Molina-Limon Noel , Chaidez-Sandoval Antonio , Devoll Renada , Ramos Mireya G. , Spencer Charles T.
Published: January 2023

Francisella tularensis is an emerging pathogen stimulating an overactive pro-inflammatory response from the immune system leading to death of the host. Serum cytokine analysis of infected mice shows high pro-inflammatory cytokine production consistent with the overactive immune response in response to F. tularensis infection that correlates with death. Therefore, we sought to control this overwhelming inflammation using F. tularensis as the model trigger. Natural Killer T (NKT) cells are a subset of white blood cells with both pro-inflammatory and regulatory functions that could potentially control this lethal response. Therefore, mice sufficient and deficient in NKT cells were infected with F. tularensis and their survival and cytokine response was compared. Mice lacking NKT cells were more sensitive to the infection and produced higher cytokine levels than mice having NKT cells. These data suggest a regulatory role for NKT cells in controlling the F. tularensis--elicited inflammatory responses. In vitro studies of the effector mechanism controlling this tempering of inflammation suggest the engagement of a surface expressed effector molecule. Ultimately, these investigations will lead to a better understanding of the role of NKT cells in this and other pro-inflammatory infections.
Activation-induced cell death in GM-CSF differentiated dendritic cells is dependent on Sphingosine kinase 1
Authors: Schwiebs Anja , Friesen Olga , Katzy Elisabeth , Bouzas Nerea Ferreirós , Pfeilschifter Josef M , Radeke Heinfried H
Published: January 2023

Sphingosine 1-phosphate (S1P) is an immune modulatory lipid mediator and has been implicated in numerous pathophysiological processes. S1P is produced by sphingosine kinase 1 (Sphk1) and Sphk2. Dendritic cells are central for the direction of immune responses and crucially involved in autoimmunity and cancerogenesis. In this study we examined the function and survival of dendritic cells under long-term inflammatory stimulation. We observed that GM-CSF differentiated dendritic cells undergo activation-induced cell death upon LPS stimulation with an increased metabolic activity after short-term stimulation followed by a rapid activation of caspase 3 and subsequent augmented apoptosis. Importantly, we highlighted a profound role of Sphk1 in secretion of inflammatory cytokines and survival of dendritic cells that might be mediated by a change in sphingolipid level as well as by a change in STAT3 expression. Cell expansion in Sphk1-deficient cells treated with the functional S1P receptor antagonist FTYP was reduced. Importantly, in dendritic cells we failed to find a compensatory upregulation of Sphk2 mRNA in SphK1-deficient cells. Instead, we observed a massive increase in Sphk1 mRNA level upon long-term stimulation with LPS in wild type cells that might function as an attempt to rescue from inflammation caused cell death. Taken together, in this investigation we describe details of a crucial involvement of sphingosine, S1P and Sphk1 in activation-induced cell death during long-term immunogenic activity of dendritic cells.
Galectin-9 Controls the Activity of DR3 (TNFRSF25) in Suppressing Immunity
Authors: Madireddi Shravan , Eun So-Young , Mehta Amit K , Birta Aruna , Zajonc Dirk M , Nishi Nozomu , Niki Toshiro , Hirashima Mitsuomi , Schreiber Taylor H , Podack Eckhard R , Croft Michael
Published: January 2023

TNFR superfamily members are cell surface glycoprotein receptors that are either induced and/or constitutively expressed on various immune cell types. Previously we have shown that the membrane proximal extracellular region of the TNFR superfamily member 4-1BB, interacts with Galectin-9, and in turn facilitates oligomerization and/or clustering of 4-1BB molecules on the cell surface in a manner that allows efficient signaling. Galectin-9 is a member of the beta-galactoside binding lectin family of proteins that contain homologous carbohydrate recognition domains and typically bind to specific glycans of a glycoprotein. Consequently, a search for other TNFR superfamily glycoproteins that might be regulated by Galectin-9 revealed an interaction with the extracellular region of DR3 (TNFRSF25). DR3 is expressed on conventional CD4+ T cells and CD4+ Foxp3+ Treg cells and acts as a stimulatory receptor when ligated. Functional studies in vitro showed that the stimulatory activity of DR3 was in part dependent on Galectin-9 in that Galectin-9−/− CD4 T cells were less responsive when DR3 was ligated. Furthermore, in vivo, where an agonist of DR3 can transiently induce Treg cell expansion in wild-type mice, we found that this effect of anti-DR3 was significantly lost in Galectin-9−/− mice. Additionally, in EAE as well as Asthma models, where an agonist of DR3 can suppress disease via control of CD4+ Foxp3+ Treg cells, we found that this protective effect of anti-DR3 was lost in Galectin-9−/− mice. Thus, our data demonstrate a novel function of Galectin-9 in facilitating signaling through DR3, and suggest that Galectin-9 may allow DR3 to oligomerize and/or cluster in a manner that allows efficient engagement of intracellular signaling pathways.
Immune phenotype and IL-6 deficiency modulate the inflammatory response in a mouse model of Irritant Contact Dermatitis
Authors: Calhoun Kaitlin , Luckett-Chastain Lerin , Gallucci Randy
Published: January 2023

Skin diseases are the second most common occupational illness with associated per annum costs exceeding $1 billion. Irritant contact dermatitis (ICD), the most common occupational skin disease, is characterized as an acute inflammatory response that manifests as a result of topical irritant exposure. Immune phenotype, such as Th1- and Th2- dominance, is postulated to contribute to the variability seen between patients. In addition, interleukin 6, which is known to have variably both Th1 and Th2 properties, has been shown to play a key role in ICD. C57BL/6J (“Th1 dominant”), Balb/c (“Th2 dominant”), and IL-6KO mice were utilized to investigate ICD induced by exposure to the occupational irritants, benzoalkonium chloride (BKC) and JP-8 jet fuel. Histopathology revealed epidermal thickening and dermal cellular infiltration in dermatitis lesions, with IL-6KO skin exhibiting the most severe damage. Analysis of skin cytokine and chemokine protein expression showed that IL-1β, TNF-α, TGF-β, CCL2, CCL3 and CXCL1, were elevated in mice exposed to BKC regardless of immune phenotype. In contrast, IL-1β, TNF-α, TGF-β, and CXCL1 varied between mouse strains when JP-8 was compared to control exposure. Flow cytometric analysis and immunohistochemistry indicated neutrophil and monocyte presence within dermatitis lesions; however, IL-6KO showed a greater amount of neutrophils following BKC exposure and decreased neutrophil presence after JP-8 exposure when compared to C57 mice. Overall, it appears that immune phenotype and IL-6 deficiency alters the skin inflammatory response in ICD via differential cytokine and chemokine expression that influence the type of inflammatory cells that infiltrate into the skin.
The microglial cell response to sterile nerve injury in the brain is regulated by IRF8
Authors: Campbell Iain L , Xie Rui Dan , Villacampa Nàdia , Almolda Beatriz , Gonzalez Berta , Castellano Bernardo
Published: January 2023

Microglia, the resident immune cells of the central nervous system (CNS), respond vigorously to insults such as nerve injury. We have shown previously in the normal CNS the properties of microglia are dramatically altered in the absence of IRF8. Here we examined whether IRF8 is involved in the microglial cell response to sterile nerve injury. Facial nerve axotomy (FNA) was performed in wild type (WT) and IRF8−/− (KO) mice and the brain removed at different days post-injury (dpi). In brains from IRF8 KO mice, nucleoside diphosphatase (NDPase) histochemistry revealed gross alterations in the morphology of microglia, which were stunted and hypertrophied but were present in comparable numbers with WT. In WT mice, a progressive increase in microglial cell activation was observed peaking at 7 dpi and was accompanied by dense staining for lectin, NDPase and CD11b. By contrast, in IRF8 KO mice, the microglial cell activation was grossly attenuated with the density of staining for lectin, NDPase and CD11b reduced significantly. The attenuated microglial cell activation in IRF8 KO mice was paralleled by a significant decrease in microglial cell proliferation at 7 and 14 dpi. The wrapping of individual motor neuron cell bodies in the axotomised facial nucleus by microglia involved in synaptic stripping and phagocytosis was incomplete in IRF8 KO mice and was accompanied by an increase in degenerated neurons. These findings show that in addition to maintenance of homeostatic properties, IRF8 also has an important role in regulating a number of key processes in the microglial cell response to neuronal injury.
Role of M1-polarized Macrophages in Parkinson’s Disease Models
Authors: Qin Hongwei , Yan Zhaoqi , Lee Kevin S , Gibson Sara A , Li XInru , Harms Ashley S , Buckley Buckley A , Standaert David G , Benveniste Etty N
Published: January 2023

Parkinson’s Disease (PD) is an age-related, chronic neurodegenerative disorder. Activated microglia/macrophages and subsequent neuroinflammation is associated with the pathogenesis of PD. We have recently demonstrated that inhibiting the JAK/STAT pathway prevents neuroinflammation and neurodegeneration in an α-synuclein (α-syn) overexpression PD model by suppressing activation of immune responses. Our previous studies demonstrated that deletion of SOCS3, a negative regulator of the JAK/STAT pathway, in the myeloid cells (LysM-CreSOCS3fl/fl mice) resulted in polarization of the pro-inflammatory M1 macrophage phenotype, which have been implicated in contributing to PD pathogenesis. To better understand the function of M1 polarized macrophages in PD, we utilized the α-syn overexpression model in LysM-CreSOCS3fl/fl mice. Our results indicate that stimulation of SOCS3-deficient macrophages with a combination of α-syn and IFN-γ promotes a more pronounced M1 phenotype and higher levels of expression of MHC Class II, LRRK2, CD40, iNOS, TNF-α and IL-6, compared to SOCS3fl/fl cells. In the in vivo setting, the numbers of activated and infiltrating macrophages are increased in the midbrain of LysM-CreSOCS3fl/fl mice compared to SOCS3fl/fl mice at 4 weeks of a-syn transduction. Furthermore, there is enhanced MHC Class II expression and STAT3 activation in these activated macrophages from LysM-CreSOCS3fl/fl mice. Our novel findings demonstrate that dysregulation of the JAK/STAT pathway in myeloid cells contributes to neuroinflammation in PD by promoting the M1 macrophage phenotype. As such, targeting the JAK/STAT pathway to abrogate neuroinflammation and neurodegeneration may be a therapeutic strategy for PD patients.
Quantitating dendritic cell clustering in the lymph node.
Authors: Byrum Janie Rae , Fricke Matthew , Tafoya Justyna , Moses Melanie , Cannon Judy L
Published: January 2023

The efficiency of the T cell search for antigen presented by dendritic cells (DCs) in lymph nodes (LNs) is a determinant of the overall timing of the T cell immune response to infection. While there is suggestion that DCs are clustered in LNs, there has been little quantitative analysis done to precisely analyze DC positioning in LNs. We present the quantitation of murine DC motility, surface area, and volume in the lymph node. We also use computational analysis of 2 photon microscopy images of explanted murine lymph nodes from CD11c-YFP mice to determine the degree of clustered-ness of DCs. Our analysis indicates a degree of DC clustering within the lymph node. Previously our lab identified sites in the lymph node visited with greater frequency by T cells than would be expected by random motility. We hypothesize such sites demonstrate that DC clusters may actively attract T cells. Elucidating whether T cell motility around DC clusters is distinctive from T cell motility in areas where DCs are non-clustered will help decipher T cell search strategy and the timing of the adaptive immune response.