bluuebunny/crossref_metadata_2025 huggingface dataset:
Elevated adipose tissue expression of CCL19 in obese individuals with or without type-2 diabetes: Its association with metabolic inflammation
Authors: Alrashed Fatema , Kochumon Shihab , Tuomilehto Jaakko , Sindhu Sardar , Ahmad Rasheed
Published: January 2023

Adipose tissue produces inflammatory molecules considered to be involved in the development of chronic low-grade inflammation in obesity and type 2 diabetes (T2D). The adipose tissue expression of CCL19 or macrophage inflammatory protein (MIP)-3β in obesity is poorly understood. We, therefore, determined CCL19 gene expression in the adipose tissue samples from lean, overweight, and obese individuals with or without T2D. Subcutaneous adipose samples were collected from 58 non-diabetic (ND) (27-obese, 21-overweight, and 10-lean) and 49 T2D (34-obese, 11-overweight, and 4-lean) individuals. Gene expression of CCL19 and other inflammatory markers was determined using real-time RT-PCR. Plasma CRP and adiponectin were measured by ELISA. CCL19 expression was significantly higher in obese compared with lean individuals (ND: P<0.04; T2D: P<0.02). Notably, CCL19 expression was relatively higher in T2D than ND individuals regarding each body mass index (BMI) group. CCL19 expression correlated with BMI (ND: r=026, p=0.04; T2D: r=0.45, P=0.001). In relation to immune markers, CCL19 expression had association with CXCL10, IL-12A, and TLR2 in ND individuals, and with IL-6, IL-1β, TGF-β, TNF-α, TLR9, CCL11 and IRAK-1 in T2D individuals, while it associated with MyD88, CCL5, CCR2, and CRP in both populations. However, CCL19 gene expression associated negatively with adiponectin in ND individuals. With regard to clinical markers, in both populations, CCL19 expression correlated positively with triglycerides, fasting blood glucose, and HbA1C, whereas it had negative association with HDL cholesterol. These data support that increased adipose tissue expression of CCL19 may represent an immune signature of metabolic inflammation.
The damage signal IL-33 facilitates focal immune responses to Toxoplasma gondii in the brain
Authors: Still Katherine M. , Thompson Jeremy , Batista Samantha , Hayes Nikolas , Harris Tajie H.
Published: January 2023

An intact immune system is critical for survival of hosts chronically infected with T. gondii. We observe clusters of immune cells surrounding regions of free parasite within brain tissue. Many of these cells are monocyte-derived and have parasite-limiting capacity, but the initial cues which instruct their proximity to parasite are not well understood. One potential mechanism of broad relevance is the incitement of damage. We explored the importance of IL-33, a protein released upon cell damage, in the recruitment of monocytes in our model. We found IL-33 to be expressed by oligodendrocytes during T. gondii infection, and observed regions of oligodendrocyte loss in infected mouse and human brain tissue, implying ample opportunity for IL-33 release. Upon infection, induction of the monocyte chemoattractant, CCL2, was dependent on IL-33 signaling, as visualized using CCL2-mcherry reporter mice. CCL2 signal was focal and expressed by astrocytes surrounding immune cell foci. The concept that IL-33 could be acting on astrocytes directly was supported by bone marrow chimeras which implicated a radio-resistant responder to IL-33. In alignment with CCL2 induction, IL-33 was required for infiltration of monocyte-derived myeloid cells, and expansion of focal myeloid cell-derived iNOS, an anti-parasitic mechanism crucial for survival during chronic infection. Consequently, intact IL-33 signaling was necessary for control of brain parasite burden. These results expand our knowledge of alarmin signaling in the brain, an environment which is likely unique to the periphery in immune response initiation due to the structural challenge of the BBB, and demonstrate the importance of one damage signal in focal control of T. gondii infection.
T cell restricted TGF-βR3 (betaglycan) null mice develop exacerbated experimental autoimmune encephalomyelitis (EAE) associated with expanded generation of Th1 cells
Authors: Duesman Samuel J. , Ren Clark , Chellappan Rajeshwari , Sestero Christine , Kesterson Robert A. , De Sarno Patrizia , Soldevila Gloria , Raman Chander
Published: January 2023

TGF-β receptor 3 (TGFβ-R3), also known as betaglycan, promotes high-affinity binding of TGF-β (1, 2 and 3) to TGFβ-R1 and TGFβ-R2 and is also a receptor for bone morphogenetic proteins (BMP) and inhibins. The embryonic lethality of TGFβ-R3 knock-outs hindered the understanding of its mechanistic function in immune response. To interrogate the function of TGFβ-R3 in mature T cells (T-Tgfbr3KO), we generated a conditional Tgfbr3f/f mouse and bred it with the dLckCre mouse. We found EAE in mice with T-Tgfbr3KO developed with greater severity and mortality than WT littermate mice in both males and females. The onset of disease was similar. The greater severity in T cell T-Tgfbr3KO mice was associated with increased proportion of Th1 cells in the CNS with no difference in numbers of Th17 cells. OT2 T-Tgfbr3KO CD4 T cells also polarized more efficiently to Th1 cells than T-Tgfbr3WT CD4 T cells. The lack of TGF-βR3 had no significant effect on differentiation of Th17 cells. Since our results indicates that TGF-βR3 regulates differentiation to IFN-γ expressing T cells, we interrogated if T-dependent B cells responses to IFN-γ switching will be altered in mice immunized with TNP-KLH. We found that IFN-γ switch dependent anti-TNP IgG2c and IgG3 was greatly enhanced in T-Tgfbr3KO mice compared to WT littermates. All other isotypes were similar in both groups. Our data reveal a novel role for TGFβ-R3 in regulating differentiation of naïve CD4 T cells to IFN-γ expressing cells.
IL-7 promotes naïve T cell motility and regulates T cell-dendritic cell contacts in the lymph node via JAK/STAT signaling.
Authors: Byrum Janie Rae , Torres David J. , Mrass Paulus , Oruganti Sreenivasa Rao , Cannon Judy L.
Published: January 2023

Naïve T cells contact dendritic cells (DCs) presenting antigen in lymph nodes to become activated. T cell motility is a key step to enabling T-DC interactions by bringing T cells into close proximity with DCs. We find a novel role for interleukin 7 (IL-7) in regulating T cell motility in lymph nodes. We show using 2 photon microscopy that IL-7 promotes T cell speed and increases the search area for individual T cells. Downstream of IL-7R, IL-7-mediated T cell motion required JAK/STAT activation, but IL-7R-mediated T cell motility occurred independently of mTOR signaling. Inhibition of IL-7-IL-7R signaling resulted in reduced contact with DCs in the T cell zone of the lymph node. Using computational modeling, we show IL-7-IL-7R is specifically important for T cell contacts with unique DCs. Our results show a new role for IL-7-IL-7R in regulating T cell function by controlling T cell motility in the lymph node. Thus, IL-7-IL-7R axis promotes not only T cell development, survival, and proliferation, but also T cell motion, leading to enhanced T cell interaction with DCs.
The lung hematopoietic niche supports bone marrow-independent hematopoiesis and contributes to local eosinophil expansion during allergic inflammation.
Authors: Jeong Brian M. , Coden Mackenzie , Doan Ton C. , Chiarella Sergio , Rodriguez Raul , Berdnikovs Sergejs
Published: January 2023

Bone marrow is classically viewed as the major source of differentiated leukocytes infiltrating tissues from circulation. Recent evidence shows that the lung can potentially function as a hematopoietic organ on its own. This is a novel concept and tissue factors driving in situ leukocyte production are not yet well understood. In naïve murine lungs, we detected a small population of Lin−CD34+c-Kit+ common myeloid progenitors (CMPs) by flow cytometry and single cell RNA-seq analysis. Males had 0.087±0.06% CMPs, while females had a larger population of 0.18±0.08%, out of CD45+ cells. In a 3 challenge in vivo ovalbumin model of allergic inflammation, we observed a six-fold expansion of CMPs in the lung tissue and the appearance of Siglec-F+CD34+IL-5Rα+ eosinophil progenitors (EoPs) (1.505±0.6% of CD45+). To illustrate the hematopoietic potential of the lung independent of bone marrow recruitment, we cultured naïve lung single cell suspensions ex vivo in presence of SCF, FLT3 and IL-5. This was sufficient to yield up to 20% mature eosinophils, which is comparable to tissue eosinophil levels in models of allergic inflammation. We further tested whether Tenascin-C (TNC), a known component of bone marrow hematopoiesis, may equally support the hematopoietic niche in the lung. TNC is a provisional matrix glycoprotein that strongly associates with epithelial remodeling and eosinophil accumulation in asthma. TNC−/− mice had a significant defect in CMP and EoP expansion in a murine model of asthma and ex vivo IL-5 lung cultures. Administration of recombinant TNC rescued normal lung eosinophilopoiesis. These results suggest the existence and significant potential of the lung hematopoietic niche to supply effector cells during inflammation.
Resistin-like molecule-β negatively regulates chronic features of fungal asthma
Authors: Samarasinghe Amali , LeMessurier Kim , Palipane Maneesha , Tiwary Meenakshi , Gavin Brian
Published: January 2023

Severe asthma with fungal sensitization (SAFS) is a subtype of asthma with high incidence in which patients are often therapy-resistant and have chronic disease features such as airway wall remodeling. Shared embryonic, immunologic, and microbiomic lineages of the gut-lung axis should be considered in disease pathogenesis investigations. A resistin family hormone, RELM-β, has been implicated in regulation of mucus production, immune responses, and bacterial colonization in the gut. RELM-β has been previously shown to increase macrophagic inflammation, goblet cell (GC) metaplasia, and fibrosis in mouse models. Since fungal asthma has unique early- and late-phase responses, we investigated the functions of RELM-β in our well-established model of SAFS utilizing Retnlb-null (KO) mice. Characteristics of SAFS developed in KO mice with some differences from wild-type (WT) controls. Kinetics of airway inflammation were delayed in KO although tissue inflammation was similar to WT. GCs interspersing the bronchial epithelia were similar, however, hyperexpression of Muc5ac and Muc5b in KO mice suggests of an altered mucin composition. Subepithelial fibrosis and airway resistance were markedly increased at late timepoints in KO mice compared to WT, indicating that RELM-β inhibits lung fibrosis. RELM-β may function as a homeostatic regulator of B cells since naïve KO mice had elevated systemic IgA. Related molecule, RELM-α, did not increase in KO mice although antimicrobial peptide Camp, Il13, and Tlr4 were hyperexpressed in lungs of these mice. Cumulatively, these data suggest that RELM-β is a regulator of allergic responses and that these effects may occur through direct/indirect mechanisms.
Therapeutic blockade of CD3-mediated signaling ameliorates memory Th2-driven allergic asthma
Authors: Gracias Donald Tom , Mehta Amit K. , Croft Michael
Published: January 2023

Allergic asthma is an inflammatory disease of the lungs characterized by Type 2 helper T cell (Th2)-driven immune responses to inhaled allergens. Memory Th2 cells are a major driving force behind asthma, secreting cytokines like IL-4, IL-5, and IL-13 as well as persisting in the host long after allergen encounter. The selective removal of pre-existing memory Th2 cells could be a key process resulting in enhanced tolerance. One such method could involve targeting CD3 through the use of monoclonal anti-CD3 antibodies, which have been shown to successfully induce tolerance in animal models of autoimmune diseases and have also shown promise in clinical trials. We tested blocking CD3 complex signaling in a HDM model of allergic inflammation, to determine if we could induce airway tolerance. Importantly, pre-sensitized C57BL/6J mice displayed strongly ablated lung inflammation and Th2 responses when treated at the time of memory T cell reactivation with F(ab′)2 anti-CD3 antibody. This treatment was found to specifically inhibit HDM-specific CD4+ T cell proliferation and Th2 cytokine production. Interestingly, mice treated with anti-CD3 also showed an increased frequency of Treg cells, improving the Treg:Th2 cell ratio in favor of airway tolerance. Additionally, a significant reduction in airway inflammation and Th2 responses was maintained with therapeutic anti-CD3 antibody even when given several months later during a further memory T cell recall response to allergen. These data suggest that blocking signaling mediated by the CD3 complex could be employed to inhibit memory recall responses to complex allergens thus promoting airway tolerance.
Response Gene to Complement-32 Expression Is Upregulated in Lupus T cells and Promotes IL-17A Expression
Authors: Rus Violeta , Tatomir Alexandru , Nguyen Vinh , Rus Horea G.
Published: January 2023

RGC (Response Gene to Complement)-32 is a cell cycle regulator expressed in normal tissues, tumors and a variety of cell lines. RGC-32 is preferentially upregulated in murine Th17 cells and promotes their differentiation. Patients with Systemic Lupus Erythematosus (SLE) display increased serum levels, expanded frequency of IL-17 producing cells. Whether RGC-32 plays a role in human Th17 differentiation pathway and in Th17 abnormalities in lupus patients has not yet been investigated. RGC-32 mRNA expression was determined in CD4+ T cells stimulated under Th0, Th1, Th2, Th17 and Treg conditions and the effect of RGC-32 overexpression and silencing under Th17 conditions was determined by nucleofection. RGC-32 expression was evaluated by RT-PCR, flow cytometry and cDNA Array in PBMC from lupus patients vs controls. RGC-32 mRNA expression was upregulated by TCR stimulation and TGFβ, and was more robust under Th17 (3.2 ± fold) and Treg (2.6 ± 0.8 fold) vs. Th1 (1.3 ± 0.4 fold) and Th2 (1.8 ± 0.1 fold) conditions. Overexpression or silencing of RGC-32 in CD4+ T cells upregulated, respectively downregulated IL-17A transcript levels and protein secretion. RGC-32 mRNA and protein level was significantly increased in CD19+ B cells and CD3+ T from lupus patients compared to controls. These results suggest that RGC-32 promotes the differentiation of human Th17 cells. Furthermore, T cells from patients with SLE exhibit increased expression of RGC-32 compared to controls. These data support the idea that RGC-32 signaling may enhance disease expression in SLE by promoting abnormalities in the Th17 pathway and provide a compelling rationale for further investigating the therapeutic potential of blocking RGC-32 in SLE.
Enhanced interferon gamma response contributes to disease remission in Rheumatoid Arthritis.
Authors: Sharma Vishal , Pope Brandon J. , Bolland Molly , Reynolds Richard , Sun Dongmei , Bridges S. Louis , Raman Chander
Published: January 2023

Rheumatoid arthritis (RA) is an autoimmune disease with debilitating bilateral inflammation in multiple joints. Although newer biologics are very effective in managing RA, critical gaps in immunopathogenic mechanism remain. We recently showed that increased expression of the interferon gamma (IFN-γ) receptor in PBMC associated with RA and increased radiographic severity. We hypothesized that enhanced IFN-γ signaling in specific subpopulations of mononuclear cells from patients with RA contributes to disease severity. To test this, we collected PBMC from healthy controls (HC) and RA patients in remission, low disease and moderate disease severity as per American College Criteria. As an autoimmune disease control, we compared PBMC from treatment naïve multiple sclerosis (MS) patients. We quantitated classical (JAK/STAT) and non-classical (ERK/MTOR) IFN-γ induced signaling in subpopulations of T and B cells within PBMC using phospho-flow cytometry. For analysis we utilized t-SNE and SPADE data visualization and clustering tools to interrogate changes in activation and marker expression within and between T and B cell populations. We observed IFN-γ-induced activation of STAT1 in CD4+ naïve and central memory T-cells from was greatest in RA patients in remission and lowest in individuals with moderate disease. IFN-γ induced activation of STAT1 in all CD8+ T-cell sub-populations of RA were similar to that from HC. Furthermore, the activation of STAT1 in all CD4+ T-cell sub-populations in PBMC from MS patients very generally greater than from RA patients and HC. From our results we infer that that while expression of IFNGR correlates with disease in RA, gain in response to IFN-γ is indicative of treatment response and RA remission.
Natural killer cell dysregulation underlies atopic dermatitis
Authors: Mack Madison R. , Brestoff Jonathan R. , Niu Hiaxia , Whelan Timothy M. , Oetjen Landon K. , Bodet Nancy D. , Wang Fang , Xu Amy Z. , Park Eugene , Margolis David J. , Yokoyama Wayne M. , Kim Brian S.
Published: January 2023

Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease characterized by dry, itchy rashes that affects 5–10% of the US adults. AD commonly presents in childhood and is known to be complicated by reduced antiviral skin immunity. Although classically thought to be driven by adaptive TH2 cells, recent work has demonstrated that dysregulation of innate immune cells such as group 2 innate lymphoid cells (ILC2s) and basophils can promote its pathogenesis. However, how these innate immune cells become dysregulated remains unclear. Using paired RNA-Seq of lesional and non-lesional skin in patients, we identified an enrichment for natural killer (NK) cell-associated gene sets in AD skin lesions. NK cells are well-recognized innate effector cells for antiviral and anticancer immunity. However, their role in AD has not been established. We utilized unbiased multidimensional analysis of gene expression and flow cytometry data to identify underlying NK cell dysregulation in AD patients. Further, we demonstrate that NK cells negatively regulate type 2 inflammation in a mouse model of AD. Collectively, these studies reveal previously unrecognized functions of NK cells in cutaneous immune homeostasis.
The expression of CD22 and CD72 on B cells negatively correlate with IgE level in eosinophilic chronic rhinosinusitis with nasal polyps
Authors: Wang Hai , Song Jia , Wang Zhe-zheng , Ruan Jian-wen , Liu Zheng
Published: January 2023

Background

CD22 and CD72 have been demonstrated to act as B cell inhibitory receptors, which suppress B cells activation. However, the roles of CD22 and CD72 on B cells in nasal polyps (NPs) are largely unknown.

Objective

To explore the expression of CD22 and CD72 on B cells and the association with immunoglobulins (Igs) in NPs.

Methods

The expression levels of CD22 and CD72 was measured by RT-PCR, immunohistochemistry, immunofluorescence and flow cytometry. Igs levels in NPs were quantified by Bio-Plex assay or the UniCAP system. Computed tomography scan and symptom severity were graded.

Results

The mRNA expression of CD22 and CD72 were elevated in eosinophilic chronic rhinosinusitis with nasal polyps (Eos CRSwNP) patients compared with non-eosinophilic chronic rhinosinusitis with nasal polyps (non-Eos CRSwNP) and controls. CD22 and CD72 were mainly expressed on CD19+ B cells. The numbers of CD22+ and CD72+ B cells and the percentage of CD22+ B cells and CD72+ B cells in B cells were increased in Eos CRSwNP compared with non-Eos CRSwNP and controls. IgA, IgG and IgM were elevated in both Eos CRSwNP and non-Eos CRSwNP, while IgD and IgE were increased in Eos CRSwNP. CD22 mRNA and CD72 mRNA expression level were negatively correlated with IgE, but not IgA, IgG, IgM and IgD in Eos CRSwNP. The percentage of CD22+ B cells and CD72+ B cells in B cells negatively correlate with the total visual analog scale symptom scores and total endoscope scores.

Conclusion

CD22 and CD72 were significantly increased in Eos CRSwNP patients and negatively correlated with IgE level, indicating the down-regulation role of CD22 and CD72 in B cell activation and a suppressive role in the synthesis of IgE in Eos CRSwNP.
Gut and Oral Microbiota, Immunity and Clinical Features in Taiwanese Children with Atopic Dermatitis
Authors: Yen Hung-Rong , Yang Pei-Shan , Wei Chang-Ching , Wu Po-Yuan
Published: January 2023

Atopic dermatitis (AD) is a skin inflammatory disease, characterized by itch, redness, swelling, oozing, thickening or dryness of skin. The condition typically starts from infancy with changing clinical severity in adolescence and adulthood. It bothers not only the health but also quality of life. The interactions between skin microbes and the skin conditions have been implicated in atopic dermatitis. However, the interplay between host immunity, gut and oral microbiota and clinical features in children remains unclear. In this study, we recruited 30 subjects (17 boys and 13 girls) with atopic dermatitis in a medical center in Taiwan. The mean age was 7.11±5.20 years old. The presentation of skin lesions included redness (73.3%), itch (66.7%), dryness (56.7%) and oozing (36.7%). Some of the patients have comorbidities of allergic rhinitis (30%) and asthma (10%). The mean SCORAD index was 55.33±16.96, with an involvement of 52.58±19.35% of body surface area. The mean eosinophil count was 12.01% and the total IgE was 5040.17 IU/mL. The gut and oral microbiomes were analyzed by using 16S rRNA gene and metagenome sequence analyses. Sterptococcal species are the major oral species and Bacteroides species are the major gut species associated with AD in children. Our data suggest that oral and gut microbiota may underlie the clinical features of AD. Further investigations on the interplay of microbiota, immunity and clinical features are warranted.
Live or let die: Pyroptosis and inflammasome activation in glial cells during multiple sclerosis and experimental autoimmune encephalomyelitis
Authors: McKenzie Brienne , Mamik Manmeet , Saito Leina , Boghozian Roobina , Monaco Maria Chiara , Major Gene , Lu Jian-Qiang , Branton William , Power Christopher
Published: January 2023

Inflammasome-associated caspases mediate the maturation and release of the proinflammatory cytokines IL-1β and IL-18, and activate the pore-forming protein gasdermin-D (GSDMD). Recently, GSDMD was shown to be the primary executioner of pyroptosis, a lytic form of programmed cell death. Excessive GSDMD pore formation causes local osmotic swelling and eventual membrane lysis. Although evidence has emerged for inflammasome activation and pyroptosis in neurological diseases, the role of GSDMD in neuroinflammation remains uncharacterized. Multiple sclerosis (MS) is the prototypic inflammatory demyelinating disease of the central nervous system (CNS). We provide molecular evidence for GSDMD-mediated inflammasome activation and pyroptosis in both macrophages/microglia and, unexpectedly, in myelin-forming oligodendrocytes (ODCs) in the CNS of patients with MS and in the associated animal model, experimental autoimmune encephalomyelitis (EAE). We observe inflammasome activation, GSDMD expression, and pyroptosis in human microglia and ODCs in vitro inflammatory stimulation. Further, we demonstrate that both caspase-1 inhibition by the small molecule inhibitor VX-765 and GSDMD inhibition via siRNAs suppress pyroptosis in human microglia. VX-765 treatment of EAE animals reduced expression of inflammasome- and pyroptosis-associated proteins in the CNS, reduced neuroinflammation, prevented axonal injury, and improved neurobehavioral performance. Thus, GSDMD-mediated pyroptosis in multiple glial populations represents a previously unrecognized mechanism of inflammatory demyelination and represents a unique therapeutic opportunity for mitigating neuroinflammation.
IRF5 hyper-activation is a driver of systemic lupus erythematosus (SLE) onset and severity
Authors: Barnes Betsy J. , De Saurav , Song Su , Nelson Victoria
Published: January 2023

The transcription factor interferon regulatory factor 5 (IRF5) is a central mediator of innate and adaptive immunity. Genetic variations within IRF5 associate with risk for systemic lupus erythematosus (SLE) and mice lacking Irf5 are protected from lupus onset and severity. Here we show that IRF5 genetic variants confer risk by increasing basal IRF5 activation in plasmacytoid dendritic cells resulting in elevated IFNα production. IRF5 hyperactivation in SLE immune cells correlates with SLEDAI and dsDNA titers. Upon characterization of immune cells from NZB/W F1 lupus-prone mice, we found that Irf5 was hyperactivated at 19 weeks-old, which precedes clinical onset. To test whether IRF5 hyperactivation is a driver of SLE, we developed novel therapeutics targeting IRF5 activation. Inhibitors are cell permeable, non-toxic and selectively bind to full-length inactive IRF5 monomer. In vivo analysis revealed functional mimicry with Irf5 knockout mice and treatment of NZB/W F1 mice for a two-week period prior to clinical onset gave significantly reduced dsDNA titers, anti-nuclear antibody (ANA) staining, and improved survival. The IRF5 inhibitor also blocked ex vivo SLE serum-induced IRF5 activation and reversed IRF5 hyperactivation detected in SLE immune cells. Results implicate IRF5 hyperactivation as a risk factor that drives SLE and provide the first pre-clinical support for treating SLE patients or those at risk of SLE with IRF5 inhibitors.
MCP-1 plays a critical role in neutrophil function and pyroptosis during Carbapenemf-Resistant Klebsiella Pneumoniae
Authors: Jin Liliang , Ghimire Laxman , Paudel Sagar , Cai Shanshan , Rangasamy Tirumalai , Jeyaseelan Samithamby
Published: January 2023

Monocyte chemoattractant protein-1 (MCP-1) is important for monocyte recruitment to the lungs in response to bacterial infection. MCP-1 is also essential for protective neutrophil recruitment to the lungs during Escherichia coli pneumoniae infection. Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection has been rapidly growing as a life-threatening nosocomial disease in many countries. Little is known regarding the role of chemokines and early cellular immune responses in protective immunity to pulmonary infection with CRKP. In the current study, we investigated the role of MCP-1 in pulmonary innate immunity against CRKP infection in C57Bl/6 mice and MCP-1−/− mice. MCP-1 appears to be essential for restricting bacterial outgrowth and death in mice. Compared to the C57Bl/6 mice, MCP-1−/− mice showed reduced influx of neutrophils in the airways and lung parenchyma, as assessed by nucleated cell concentrations in BALF and myeloperoxidase activity (MPO) in lung tissue. Moreover, the neutrophil extracellular traps (NETs) induced by CRKP were reduced in MCP-1−/− mice in both in vitro and in vivo experiments. Intriguingly, subsequent to CRKP infection, MCP-1−/− mice demonstrated increased pyroptosis through higher expression of IL-1β and cleaved gasdermin-d in the lung. These findings enhance our understanding of the critical role of MCP-1 in modulating neutrophil function and pyroptosis in neutrophils and suggest that therapies targeting modulation of NETs and pyroptosis during CRKP infection should be explored.
Combining Drop Array Laminar Flow Processing and Image-based Flow Cytometry to Evaluate Changes in Monocyte Intracellular Cytokine Production
Authors: Gary Melody A. , Tanner Elizabeth A. , Stelly Steven , Davis Asheal , Bowman Erin M. , McFarlin Brian Keith
Published: January 2023

The use of image-based flow cytometry has exponentially expanded the knowledge that can be derived regarding cellular changes compared to traditional PMT-based flow cytometry. Other recent advances in cell processing (i.e. Drop Array laminar flow processing) have further increased robustness by protecting cells from loss and damage during flow cytometry processing and labelling. Despite the advances in these respective fields to our knowledge, no study has attempted to combine both technologies to further increase sensitivity and throughput. Thus, the purpose of this report was to compare and evaluate a traditional cell prep workflow to a Drop Array workflow in the preparation of cells for image-based flow cytometry analysis. In order to complete this testing, we selected monocyte LPS-stimulated intracellular monocyte cytokine expression (i.e. IL-6 and TNF-alpha). Monocytes are an ideal cellular target to study because they are commonly lost during traditional centrifuge processing methods. Blood samples were collected from subjects according to IRB approved procedures, the latest Declaration of Helsinki, and a signed consent form. We found that processing with the Drop Array workflow resulted in reduced monocyte loss during staining, reduced cellular debris, and improved noise-to-signal ratio for intracellular IL-6 and TNF-alpha. More research is needed to evaluate the Drop Array workflow for other cell types, but it appears to be an effective technology to processing monocytes for image-based flow cytometry analysis.
Advanced age does not diminish phagocytic activity or NET production in zebrafish (Danio rerio) kidney leukocytes
Authors: Hannum Lynn G. , Chen Tianzeng
Published: January 2023

The pace of growth in populations over the age of 65 is increasing in the U.S. and worldwide, posing serious challenges for health care systems. Studies in both humans and mice have revealed evidence of dysregulation of innate immunity in older individuals, although experimental systems and results of those studies vary widely. To explore whether the zebrafish (Danio rerio) model could be valuable in understanding the effects of aging on innate immune processes, we assessed phagocytic activity, respiratory burst response and neutrophil extracellular trap formation (NETosis) by anterior kidney phagocytes from healthy young (7–8 months old) and aged (>2.5 year old) zebrafish. Exposing leukocytes from transgenic mpx:GFP fish to opsonized pHrodo-labeled killed E. coli for 30 or 60 minutes revealed is no difference in the mean percentage of neutrophils that phagocytosed bacteria between the two ages at either time point. Interestingly, older fish displayed a wider range of responses and a higher level of individual variability than the younger population. Active neutrophils from the aged fish also exhibited a higher mean fluorescence intensity (MFI) than young fish at 30 min incubation, but fluorescence levels were similar in the two populations after 60 minutes of incubation. Leukocytes from older and younger wild type AB fish showed identical respiratory burst responses to PMA. When NET production was stimulated by calcium ionophore, neutrophils from aged AB zebrafish exhibited a higher NET index than the younger cohort. These initial results indicate that older zebrafish preserve a similar level of activity, if not hints of higher activity, in tested neutrophil functions compared to younger fish.
Aging-associated alterations in lymphatic vessels and mast cells in perilymphatic tissues
Authors: Pal Sarit , Meininger Cynthia J. , Gasheva Olga , Griffith William , Zawieja David C. , Gashev Anatoliy A.
Published: January 2023

Aging alters lymphatic vessel structure (by increasing the size of low muscle cell investiture zones), ultrastructure (through loss of the glycocalyx), and proteome composition causing a concomitant increase in lymphatic permeability. Our recent studies have shown that the contractile function of aged lymphatic vessels is depleted with abolished role of nitric oxide and an increased role of lymphatic-born histamine in flow-dependent regulation of lymphatic phasic contractions and tone. Thus, aging-related reduced vessel functionality and increased oxidative stress of the lymphatic vasculature facilitate the spread of pathogens into perilymphatic tissues. In addition, aging causes the basal activation of the perilymphatic mast cells, which, in turn, restricts the recruitment/activation of MHCII+ -positive cells and eosinophils in perilymphatic tissues. This aging-associated basal activation of perilymphatic mast cells limits proper functioning of the mast cell/histamine/NF-kB axis that is essential for the regulation of lymphatic vessel transport and barrier functions as well as for both the interaction and trafficking of immune cells near and within lymphatic collecting vessels. Cumulatively these alterations play crucial roles in the progression of aging-related pathogenesis in inflammation and immunity.
The role of Runx1 in the regulation of T cell tolerance
Authors: Wilfahrt Drew Nicholas , Hsu Fan-Chi , Chung Julia , Arocha Sinibaldo Romero , Schwab Aaron , Shapiro Virginia Smith
Published: January 2023

The transcription factor Runx1 is critical for T cell development and maturation. CD4-Cre Runx1 conditional knockout mice have a block in T cell maturation, leading to complement deposition on CD4+ recent thymic emigrants, leaving very few CD4+ T cells in peripheral lymphoid organs. Consistent with a block in maturation, peripheral Runx1-deficient CD4+ T cells fail to produce normal levels of TNF-a upon stimulation. These CD4+ T cells that persist in CD4-Cre Runx1 cKO mice possess a phenotype not found in other genetically modified mice with a block in T cell maturation. Thymic- and splenic-derived T cells from the CD4-Cre Runx1 cKO exhibit altered expression of markers indicative of anergy including FR4 and CD73. This suggests Runx1 plays a role in regulating the development of T cell tolerance which is disrupted upon deletion of Runx1 in CD4+ T Cells. To test this, we have generated a mixed bone marrow chimera (BMC) system with Estrogen Receptor-Cre Runx1 knockout and B6.SJL bone marrow to knock out Runx1 in mature T cells. Surprisingly, in the mixed BMC both the ER-Cre Runx1 cKO and the B6.SJL peripheral T cells acquire a functionally hyporesponsive phenotype, suggesting that Runx1 regulates an extrinsic signaling mechanism controlling tolerance. Moving forward, this BMC model allows us to investigate the development of this anergic phenotype in mature T cells, and tests the hypothesis that Runx1 plays a critical role in regulating T cell tolerance.
Mechanism underlying the induction of Foxp3+ regulatory T cells by lactoferrin
Authors: Kim Pyeung-Hyeun , Jang Young-Saeng , Song Ha-Eon , Seo Goo-Young , Kang Seung-Goo , Lee Jeong Hyun , Kwon Bo-Eun , Ko Hyun-Jeong
Published: January 2023

Lactoferrin (LF) is multifunctional in the immune response. We have previously demonstrated that LF acts like TGF-β in IgA B cell differentiation. Therein, we explored whether LF affects peripheral regulatory T cell (Treg) differentiation. Indeed, LF induced Foxp3+ Treg differentiation by itself and in combination with TGF-β1 synergized to express Foxp3. It was conceivable that LF may increase Foxp3 expression through secretion of active TGF-β or facilitating latent TGF-β to active form. There was little active TGF-β in the supernatant from LF-stimulated T cells. Surprisingly, however, pan anti-TGFβ Ab completely abolished the LF-induced Foxp3 expression, suggesting that membrane-bound TGF-β may be involved. In this, we found that both LF and TGF-β1 increase latency-associated peptide negative (LAP−)TGF-β on the surface of Foxp3+T cells, and this increase was more dramatic when treated with LF plus TGF-β1. As was the case in B cells, LF-induced Foxp3 expression was virtually disappeared by pretreatment with soluble TβRIII. Collectively, these results suggest that LF induces Foxp3+Treg through TβRIII and subsequent expression of membrane-bound/LAP-negative TGF-β.
Prostaglandin I2 promotes Treg function in vitro
Authors: Norlander Allison Elizabeth , Bloodworth Melissa H. , Peebles R. Stokes
Published: January 2023

Our laboratory has shown in recent years that prostaglandin I2 (PGI2) promotes immune tolerance through inhibition of T cell and dendritic cell function. Specifically, we have shown that PGI2 is able to restrain Th1 and Th2 immune responses, suggesting that it has immunosuppressive properties. However, PGI2’s impact on the functionality of T regulatory (Treg) cells, a T cell subtype important for the suppression/resolution of immune responses which express the canonical transcription factor forkhead box p3 (Foxp3), is unknown. Thus, we hypothesized that PGI2 promotes the function of Tregs. To test this hypothesis, we utilized mice deficient in IP (IP KO), the receptor for PGI2.. We found that splenic Tregs from IP KO mice have a significantly lower mean fluorescence intensity (MFI) of Foxp3 compared to wild-type (WT) Tregs. Furthermore, the percent suppression of CFSE-stained WT effector T cells by IP KO Tregs was significantly decreased compared to the percent suppression of CFSE-stained WT effector T cells by WT Tregs. Moreover, the percentage of induced Tregs (iTregs) generated from naïve T cells isolated from IP KO mice was significantly reduced compared to WT mice. As a whole, our preliminary data demonstrate the ability of PGI2 to promote Treg function, and thus suggest that PGI2 may serve as a novel treatment strategy for T cell mediated diseases.
The generation of memory CD8 T cells is regulated by intracellular Galectin-3
Authors: Farhad Mohammad , Redmond William L.
Published: January 2023

The ability of tumors to induce immunosuppression and reduce the cytolytic function of tumor-infiltrating lymphocytes (TIL) is a major obstacle to creating effective therapies for cancer patients. The levels of Galectin-3 (Gal3) protein are increased during tumor progression in many cancers. Gal3 induces the polarization of tumor-associated macrophages and plays a role in the survival and metastasis of cancer cells. Gal3 is functional within cells as well as in the extracellular space and recent studies suggest that it has opposing functions in these two compartments. The biology of extracellular Gal3 has been extensively studied, however, the function and mechanisms by which intracellular Gal3 regulates CD8 T cells responses are poorly understood. CD8+ T cells are a critical component of anti-tumor immunity and enhancing their function can promote tumor elimination. Intracellular Gal3 has been suggested to positively regulate CD8+ T cell activation and survival. In the current study, we demonstrate that Gal3-deficient CD8+ T cells has no defect in early activation in vitro. However, Gal3−/− OT-I CD8+ T cells exhibit decreased proliferation, activation, survival and transition to memory in response to cognate antigen in vivo. Together, these data implicate Gal3 as a critical mediator of CD8 T cell survival and memory formation following antigen challenge in vivo.
PSGL-1 regulates the extent of TCR-dependent activation in T cells and prevents their suppression by Tregs
Authors: Otero Dennis C. , Takahashi Amy , Chao Grace , Leung Donmienne , Bradley Linda
Published: January 2023

We recently demonstrated that PSGL-1 (P-selectin glycoprotein-1) could act as a T cell-intrinsic checkpoint inhibitor during chronic viral infection. As such, PSGL-1 may be a viable target to control T cell responses in the setting of autoimmune disease. In this study, we investigated the role of PSGL-1 on the activation of T cells and in mouse models of autoimmune disease. Here we find that the lack of PSGL-1 on T cells results in clinically worse disease in both EAE and adoptive transfer colitis, which supports its function as a negative regulator of T cell activation. However, in vivo treatment with an anti-PSGL-1 antibody known to block binding of the ligand, P-selectin, from the time of disease initiation had little effect on disease severity in the transfer colitis model and led to exacerbated disease in the EAE model. Crosslinking PSGL-1 on naïve T cells in vitro in the presence of TCR signals results in up-regulation of PD-1 but also up-regulation of other activation markers, suggesting that PSGL-1 exerts regulatory effects during the early activation program in T cells. Surprisingly, however, in an in vitro assay, crosslinking PSGL-1 on effector T cells resulted in resistance to regulatory T cell suppression. Therefore, anti-PSGL-1 treatment results in enhanced T cell activation and resistance to Treg suppression, thereby leading to more severe autoimmune disease by altering/blocking PSGL-1 signaling to phenocopy PSGL-1-deficiency.
A mechanism involving sibling transcription factor assistance preserves the integrity of an active Foxp3 locus in peripherally induced regulatory T cells.
Authors: Ghosh Sayantani , Rubtsov Yuri P. , Jung Haejin , Roy-Chowdhuri Sinchita , kang Keunso , Im Sin-hyeog , Surh Charles D. , Rudensky Alexander Y. , Rudra Dipayan
Published: January 2023

Regulatory T (Treg) cell mediated dominant negative regulation is critical for suppressing hyper-immune responses. Broadly classified as thymus derived (tTreg) and peripherally induced (iTreg), these cells share a stringent dependence on the transcription factor Foxp3. In spite of significant advances, precise understanding of the molecular events that promote and preserve unceasing expression of Foxp3 in Treg cells is still incompletely understood. Here we show that Foxp1, a forkhead transcription factor and thus a sibling family member of Foxp3, is essential for sustained high level expression of Foxp3 specifically in iTreg cells. By being readily associated with the Foxp3 locus in a TGFβ dependent manner, Foxp1 critically participates during multiple phases of iTreg development and maturity. By employing an inducible model of temporal deletion of Foxp1 in precursor CD4+ T cells, we found that it is required for optimum expression of Foxp3 during the onset of iTreg induction. Even a conditional ablation of Foxp1 in iTreg cells at a later developmental time point, when high level transcription of Foxp3 was already established, resulted in dramatic instability of the differentiated cell state. Accordingly, Treg specific deletion of Foxp1 renders cells gradually lose Foxp3, resulting in dramatically reduced Nrp1−Helios− iTreg compartment in mice, ensuing significantly augmented intestinal inflammation. Interestingly, the stability of Foxp3 expression in tTreg cells remained unaffected in the absence of Foxp1. Thus, our study unravels a mechanistic module of evolutionarily akin members of a transcription factor subfamily in establishing a novel molecular program indispensable to ensure efficient immune homeostasis.
VSIG-8 is a co-inhibitory ligand and an immune checkpoint molecule for human T cells
Authors: Wang Jinghua , Manick Brian , Renelt Mark , Hansen Louis , Wu Guoping , Kalabokis Vassilios
Published: January 2023

Cell surface-localized and secreted immunoglobulin superfamily (IgSF) proteins play central roles in regulating adaptive and innate immune responses, and are primary targets for the development of new immunotherapeutics. In this work we provide biologic-functional insight for VISG-8, one member of this important class of proteins. VSIG-8 inhibits the production of cytokines (IL-2, IFN-gamma, IL-17, IL-6, and IL-19), chemokines (MCP-1, MCP-10, and IP-10) and other proteins (IGFBP3 and RBP4) on anti-CD3 activated human CD3 T cells. Furthermore, VSIG-8 significantly reduces the production of IFN-gamma and IL-2 on both CD4 and CD8 T cells in the presence of T-cell receptor signaling. In addition, VSIG-8 markedly suppresses anti-CD3-induced human T cell proliferation. Moreover, VSIG-8 profoundly decreases the conversion of naïve CD4+ T cells into Th1 cells. Thus, we have identified VSIG-8 as a new immune checkpoint molecule that is able to inhibit human T-cell activation. This novel human T-cell co-inhibitory ligand may be a unique target for developing new immunotherapy strategies for the treatment of human cancers, autoimmune disorders, and infection diseases.
Id protein function in adipose resident regulatory T cells
Authors: Frias Adolfo B. , Ji Junyi , Buechel Heather Marie , Hand Timothy , D’Cruz Louise M.
Published: January 2023

Immune cells in the visceral adipose tissue play critical roles in modulating chronic system inflammation associated with obesity and type II diabetes. Studies have shown that CD4+ FoxP3+ adipose resident regulatory T cells (aTregs) restrict inflammation through their anti-inflammatory properties, such as production of high levels of IL-10. This population of aTregs is severely decreased in mice fed a high-fat-diet (HFD), indicating a critical role for these cells in regulating obesity-associated inflammation. However, the transcriptional programs that sustain the differentiation and survival of these cells are poorly understood. Using reporter mice, we show that Id2, a negative regulator of the E protein transcription factors, is highly expressed in aTregs while lymphoid Tregs have an inverse expression profile with low Id2 and high Id3 expression. Our preliminary data using Id2 and Id3 Treg specific conditional deficient mice show that Id2 deficiency results in a severe reduction in aTregs. Moreover, Id2-deficient aTregs displayed low expression of key aTreg markers such as ST-2, KLRG1, and CCR2. Loss of Id3, in contrast, resulted in increased aTreg frequency, suggesting the reciprocal expression of these two transcriptional regulators is essential for maintaining the aTreg transcriptional program. We postulate that Id2 is required by aTregs to support aTreg survival and function. Understanding the transcriptional regulation of aTregs will allow for better understanding and treatment of chronic inflammation associated with obesity and type II diabetes.
Stem cell-like T-bet+ IgM memory cells generate multi-lineage effector B cells
Authors: Levack Russell C. , Kenderes Kevin J. , Cabrera-Martinez Berenice , Winslow Gary
Published: January 2023

Our laboratory has been studying the differentiation of CD11c+ T-bet+ IgM memory B cells during secondary ehrlichial infection.

These cells are closely related to B cells that have been described in a range of other contexts, including hepatitis, AIDs, malaria, SLE, and age-related autoimmunity.

Following secondary infection, IgM memory cells, as a population, undergo self-renewal, and differentiate into effector cells, including splenic and bone marrow antibody secreting cells (ASC).

Moreover, IgM memory cells enter germinal centers, where they undergo class-switch recombination and give rise to class-switched memory and effector cells.

Although these data suggest that a single memory cell has multi-lineage potential, we sought to formally address this question by searching for shared clones among IgM memory cell-derived effector cells.

Among the IgM memory cell-derived subsets, we identified several clones common to all effector cell populations. The number of common clones varied for each pairwise comparison of effector cells and suggested lineal relationships.

IgM memory cells accumulated mutations following rechallenge; although all memory cell-derived subsets displayed similar numbers of V region mutations.

Lineage analysis demonstrated that the effector cell subsets underwent varying degrees of clonal diversification, although this was clone-dependent.

These studies reveal that a single IgM memory cell clone can give rise to different memory and effector cell subsets, that is, they exhibit stem cell properties. This property distinguishes these T-bet+ IgM memory cells as a unique memory cell subset.