bluuebunny/crossref_metadata_2025 huggingface dataset:
Disruption of self-tolerance in autoimmune diseases and therapeutic modulation to restore immune balance
Authors: Chakraborty Mousumi , Shashkova Elena , Cline-Smith Anna , Aurora Rajeev
Published: January 2023

Immune homeostasis is maintained by tolerogenic & immunogenic subsets of cells. They determine the intensity & type of response to infection & vaccination. Loss of tolerance results activation of self-reactive T-cells that attack self-tissues & develop pathologic conditions like arthritis. An important mechanism by which tolerance is maintained in periphery is by induction of regulatory T-cells (TREG).

Collagen induced arthritis (CIA) is a well-established model of rheumatoid arthritis. Immunization with collagen in presence of adjuvant in mice leads to production of proinflammatory cytokines, which leads to bone erosion by osteoclasts. Our laboratory has shown that osteoclasts function as antigen presenting cells (APCs), which induce FOXP3 in CD8+ T-cell (OC-iTcREG). Previous studies demonstrated that OC-iTcREG limit bone resorption in murine osteoporosis. We have also identified that, osteoclast express Notch ligand DLL4 & use Notch signaling to induce FOXP3 in CD8 T-cells. Expression of Notch ligand is also observed on other APCs, which we hypothesize are tolerogenic. Thus, we will characterize & examine whether these tolerogenic APCs can induce TREG & re-establish tolerance. We will present the results using adoptive transfer experiments, to measure immunomodulatory activity of DLL4 expressing APCs in CIA model. Efficacy of the treatment will be assessed by studying serum CTX, proinflammatory cytokines, immune-phenotypes of T-cell & APC subtypes from joint tissues of mice. Further, effect of these tolerogenic APCs will be compared with conventional APCs in CIA model to assess its therapeutic potential. This study will help to understand the complex interplay of tolerogenic cells in re-establishing immune homeostasis.
Quantitative screening of active renal SLE patients’ urine for protein biomarkers using kiloplexed glass slide arrays
Authors: Soomro Sanam , Attia Maria , Petri Michelle , Mohan Chandra
Published: January 2023

Background

Systemic Lupus Erythematous (SLE) is a chronic inflammatory autoimmune disease marked by flares in multiple organ systems. Leading causes of morbidity and mortality in this disease include lupus nephritis and renal failure. Because the diagnosis of lupus nephritis is currently done using kidney biopsy, noninvasive diagnostic biomarkers are crucially needed to limit the use of this costly and invasive procedure. In this effort, active renal SLE patients’ urine was interrogated for potential biomarkers using a novel quantitative antibody array based protein screen of 1000 protein targets.

Methods

The urine of 9 active renal (AR), 6 active non-renal (ANR) patients, and 9 healthy controls (HC) were subjected to a quantitative antibody based protein screen of 1000 unique human proteins. After creatinine normalization, dysregulated proteins were selected for further validation using group wise fold change (FC) and Mann Whitney tests.

Results

The quantitative array based screen of 1000 targets showed 336 proteins to be elevated in the urine of active SLE patients compared to HC. Of these, 8 proteins were elevated in the urine of AR patients compared to ANR (FC>2 P<0.05). Array results correlated well ELISA results (Spearman r > 0.6 in 60% of proteins validated thus far). Relationship of biomarkers to disease activity and renal pathology is under investigation.

Conclusions

We have successfully validated one of the largest targeted proteomic technologies currently available for biomarker identification. Although validation of hits as diagnostic biomarkers is still in progress, the identified proteins show promise in the noninvasive diagnosis of active renal disease in SLE patients.
The psoriasis therapeutic potential of a novel short laminin peptide C16
Authors: TSAO YEOU PING
Published: January 2023

Psoriasis is a chronic skin disorder characterized by cutaneous inflammation, keratinocyte hyperproliferation and desquamation formation. There are nearly 10 million patients around the world. Current remedies have potential serious side effects. There is an unmet need for new therapies with better efficacy and less adverse effects. Recently, we found that the C16 peptide derived from extracellular matrix component laminin 111 has the potential for psoriasis therapy. Topical treatment with C16 ameliorates imiquimod (IMQ)-induced psoriasis-like skin inflammation. BALB/c Mice (8-week-old) received a daily topical of IMQ cream (5%; Aldara; 3M Pharmaceuticals) on the shaved back for six consecutive days. The mice developed psoriasis-like skin inflammation and desquamation formation. Topical application of IMQ combining with C16 showed reduced skin inflammation and desquamation. Histology analysis confirmed obvious reduction of inflammatory cell infiltration and acanthosis. In cell culture, C16 also inhibited IMQ-induced keratiocyte proliferation. Since C16 peptide has been found that can bind to α5β1 and αvβ3 integrins, integrins α5β1 and αvβ3 may be involved in the mechanism of psoriasis therapy. Results of ongoing investigation will be presented in the meeting.
BTK inhibition ameliorates renal, skin, and brain disease in a spontaneous murine model of systemic lupus erythematosus
Authors: Chalmers Samantha A. , Wen Jing , Doerner Jessica L. , Stock Ariel D. , Webb Deborah , Herlitz Leal , Bosanac Todd , Nabozny Gerald H. , Fine Jay S. , Klein Elliott , Ramanujam Meera , Putterman Chaim
Published: January 2023

Systemic lupus erythematosus (SLE) can affect multiple different organ systems, including the kidneys (lupus nephritis, LN), brain (neuropsychiatric SLE, NPSLE), and skin (cutaneous lupus, CLE). B cells and macrophages are implicated in the pathogenesis of these disease manifestations. We have previously shown the importance of Bruton’s tyrosine kinase (BTK), an enzyme important in B cell and macrophage signaling, in reversing disease in a model of immune nephritis induced by the passive transfer of nephrotoxic antibodies.

To extend our findings to a more severe type of nephritis more similar to human disease and examine the effects of BTK inhibition on extra-renal lupus manifestations, we treated a spontaneous model of lupus, MRL/lpr mice, with the novel BTK inhibitor, BI-BTK-1. Early treatment with BI-BTK-1 normalized proteinuria (p<0.001) and BUN (p<0.001) levels, and significantly improved renal histopathology. Importantly, in separate studies BTK inhibition also significantly reversed established proteinuria. Finally, BI-BTK-1 treated MRL/lpr mice had significantly improved survival as compared to control treated age, sex, and background matched mice (p < 0.0001).

MRL/lpr mice develop brain and skin disease that model the pathology of NPSLE and CLE, respectively. Early BI-BTK-1 treatment significantly improved cognitive function and decreased inflammatory cell infiltration into the brain choroid plexus. Moreover, skin disease was also significantly improved as determined by attenuated macroscopic and histologic skin scores.

Our results further highlight the therapeutic potential of BI-BTK-1 in SLE, not only in treatment of LN, but potentially also for skin and brain disease as well.
Protein Kinase D1 deletion ameliorates collage-induced arthritis
Authors: Yoon Taewon , Cho Hongsik , Stuart John M. , Yi Ae-Kyung
Published: January 2023

Although the etiology of Rheumatoid arthritis (RA) is unknown, it is thought that TLR signaling may be involved in the initiation and progression of the disease. Disruption of the TLR signaling at early stages of RA could be a possible therapeutic strategy. We have previously identified protein kinase D1 (PKD1) as an indispensable component in the MyD88-dependent TLR signaling pathway that plays a critical role in acute and chronic inflammation. Although it is currently not known whether PKD1 plays a pathogenic role in RA in humans, we have previously found that daily treatments of a PKC/PKD inhibitor Gö6976 significantly ameliorated arthritic symptoms in collagen-induced arthritis (CIA). Here, we further investigated the role of PKD1 in pathogenesis of CIA in humanized HLA-DR1 transgenic mice using a tamoxifen-inducible PKD1-gene deletion system. We have found that PKD1 deletion resulted in reduced incidence and severity of arthritis in HLA-DR1 mice immunized with type II collagen. In addition, Gr-1+ myeloid lineage cell infiltration into the joint space was significantly reduced and joint destruction (assessed by joint histopathology study and mCT scanning) was ameliorated in the PKD1-deleted mice compared to wild-type. Our results provide evidences that PKD1 might play an important pathogenic role in inflammatory arthritis and that PKD1 could be a new therapeutic molecular target for arthritis.
Nrf2 mediated metabolic reprogramming of tolerogenic dendritic cells is protective against aplastic anemia
Authors: Wei Hsi-Ju , Pareek Tej K. , Gupta Ashis , Kao Weiming , Almudallal Omar , Letterio John J.
Published: January 2023

Aplastic anemia (AA) is a rare autoimmunity characterized by suppression of bone marrow (BM) function resulting in progressive pancytopenia. BM transplants or immunosuppressive therapies remain the major treatment choices with limited benefit and significant side effects. Here we report for the first time the therapeutic utility of Nrf2 induced metabolically reprogrammed tolerogenic dendritic cells (TolDCs) in AA treatment. First, CDDO-DFPA-induced Nrf2 activation resulted in a TolDC phenotype as evidenced by induction of IL-4, IL-10, and TGF-b and suppression of TNFa, IFN-g, and IL-12 levels in Nrf2+/+but not in Nrf2−/− DCs. Cellular metabolism holds the key to determining DC immunogenic or tolerogenic cell fate. We observed the similar pattern of oxidative phosphorylation (OXPHOS) and glycolysis in immature and LPS-induced (matured) Nrf2+/+ and Nrf2−/− DCs. However, only Nrf2+/+DCs partially restored OXPHOS and reduced glycolysis during CDDO-DFPA-induced Nrf2 activation. We also observed significantly enhanced HO-1 and reduced iNOS/NO production in Nrf2+/+compared to Nrf2−/−DCs, suggesting Nrf2-dependent TolDC induction is linked to suppression of the inhibitory effect of NO on OXPHOS. Besides, Nrf2−/−DCs demonstrated higher antigen-specific CD4 T cell proliferation. Lastly, administration of TolDCs improved hematopoiesis and survival rate in AA murine model, with fewer Th17 and more Treg cells. Concomitantly, AA patient BM biopsies displayed higher DCs, T cells, and iNOS expression accompanied with lower Nrf2 and HO-1 expression compared to normal subjects. These results provide a new insight into therapeutic utility of metabolically reprogramed TolDCs by CDDO-DFPA induced Nrf2 signaling in treatment of AA.
Functional reprogramming of regulatory T cells in the absence of Foxp3
Authors: Charbonnier Louis-Marie , Lopez David , Bleesing Jacob , Garcia-Lloret Maria , Chen Karin , Ozen Ahmet , Li Ming , Pellegrini Matteo , Chatila Talal A.
Published: January 2023

Foxp3-deficient regulatory T (Treg) cells lack suppressor function and manifest a T effector (Teff) cell-like phenotype. We demonstrate that Foxp3 deficiency dysregulates mTORC2 signaling and gives rise to augmented aerobic glycolysis and oxidative phosphorylation. Mutant Treg cell-specific deletion of the mTORC2 adaptor gene Rictor or expression of a Foxo1 transgene improved regulatory function and ameliorated disease. Rictor deficiency reestablished a subset of Treg cell genetic circuits and suppressed the Teff cell-like metabolic program. Treatment of mutant Treg cells of patients with FOXP3 deficiency with mTOR inhibitors similarly antagonized their Teff cell-like program and restored suppressive function. Thus, regulatory function can be reestablished in Foxp3-deficient Treg cells by targeting their metabolic pathways, providing opportunities to restore tolerance in Treg cell disorders.
Induction rate of regulatory T cells from conventional T cells is affected by substrate rigidity
Authors: Lee Jounghyun Helen , Nataraj Neha , Dang Alex , Kam Lance C.
Published: January 2023

The immune system maintains a balance between protection and tolerance. Regulatory T cells (Tregs) act as a tolerance mechanism to suppress effector immune cells. Recent studies suggest that the induction of Tregs from conventional T cells (Tconvs) has many practical and therapeutic advantages. Previously, both human and mouse T cells were reported to exhibit different responses to activating substrates of different rigidities as indicated in IL-2 secretion levels. In this work, we explore the previously unknown effect of substrate rigidity on the induction of Tregs from Tconvs. We used Sylgard 184 poly(dimethylsiloxane) (PDMS) to obtain rigidity ranging a few hundred kilopascals to megapascals. Mouse CD4+ T cells (Foxp3-GFP linked B6 mouse) were obtained from spleen and further isolated to CD25+ and CD25− cells using magnetic bead-based isolation kits. CD4+CD25− T cells (>99% Foxp3−) were then seeded onto the surfaces coated with antibodies to CD3 and CD28 in IL-2 and TGF-b-enriched media. Surprisingly, there was a significant increase in Treg induction rate at lower substrate rigidities (i.e., Young’s modulus, E ~ 100 kPa) compared to high rigidity (i.e., E ~ 3 MPa). To confirm that this significant difference in induction rate is truly related to T cell mechanosensing, we administered compound Y-27632 (cY) to inhibit myosin contractility. In the presence of cY, the difference in induction rate at varying rigidities was significantly reduced. This study furthers our understanding of mechanosensing properties of immune cells and raises questions about the underlying molecular mechanisms involved in this process of T cells choosing to proliferate or differentiate.
PD-L1-engineered pancreatic islet grafts overcome rejection in allogeneic recipients
Authors: Batra Lalit , Shrestha Pradeep , Yolcu Esma S. , Zhao Hong , Bowen William S. , Woodward Kyle B. , Coronel María M. , Tan Min , García Andrés J. , Shirwan Haval
Published: January 2023

The use of chronic immunosuppression is a major hurdle for clinical islet transplantation as a curative modality for the treatment of type 1 diabetes. T effector cells (Teffs) are a main culprit of allograft rejection, and their pathogenic response is kept in check by CD4+CD25+FoxP3+ T regulatory cells (Tregs). The PD-1/PD-L1 immune checkpoint pathway plays an important role in the Teff/Treg balance with demonstrated clinical efficacy in cancer immunotherapy. This study was designed to assess the immunoregulatory function of PD-L1 in preventing allogeneic islet graft rejection. A chimeric protein (SA-PD-L1) containing the extracellular portion of PD-L1 fused to a modified form of core streptavidin was generated. SA-PD-L1 protein was expressed in insect cells and purified. The protein was functional as it enhanced TGF-beta-induced conversion of Teffs into Tregs and suppressed the proliferation of Teffs in response to alloantigens. Importantly, SA-PD-L1 was transiently displayed on the surface of biotinylated islets, and such engineered islets survived for over 100 days in allogeneic recipients treated with a short course (15 days) of low dose rapamycin. In marked contrast, unmodified islets under the same rapamycin regimen were rejected within 20 days. SA-PD-L1-engineered islets without rapamycin, although showing prolonged survival, were eventually rejected within 40 days. These results provide strong proof-of-efficacy and feasibility for the use of SA-PD-L1 protein as an immunomodulator to promote allogeneic islet graft survival in the absence of continued immunosuppression.
Treatment with benign IgE reduces asthma symptomology in a mast cell dependent manner
Authors: Martin Rebecca , Zellner Matthew P , Lownik Joseph Cornelius , Luker Andrea J
Published: January 2023

Notoriously known for its involvement in type I hypersensitivities, Antigen (Ag)-specific IgE mediates a variety of mild to severe responses, including asthma. Ag-specific IgE is additionally important for immunity against gastrointestinal helminths, but these helminths also elicit production of low-affinity, poly-specific IgE. Helminth infection has been shown by several groups to be protective against asthma. The mechanisms behind these findings have included the induction of regulatory pathways, but the role of poly-specific or “benign” IgE has not been fully assessed. To test this in the absence of helminth infection, we utilized a four week model of murine asthma and injected IgE anti-DNP as our “benign” IgE twice a week. We found that treatment significantly reverses the eosinophil accumulation, airway hyperresponsiveness, and lung pathology seen with this model. Further analysis shows that the “benign” IgE treatment is only needed during the antigen challenge phase and not during sensitization phase. Using in vitro assays we were able to show that non-specific IgE can block Ag-specific IgE degranulation even after Ag-specific IgE pre-incubation. Overall, these data demonstrate a novel avenue for blockage of mast cell degranulation and a potential regulatory role for IgE.
Poly(ADP)Ribose Polymerase inhibition by gene knockout or pharmacologically by olaparib restores steroid sensitivity in murine model of steroid-resistant Asthma
Authors: Ghonim Mohamed Ahmed , Wang Jeffrey , Ibba Salome Valentina , Luu Hanh , Boulares Hamid
Published: January 2023

Background

Asthma is a serious health issue worldwide and a common treatment for the disease is a combination of steroids with β2-agonist; however, many patients are refractory to steroid treatment. Individuals with steroid-resistant asthma have limited therapeutic options; some can die by status asthmaticus. steroid-resistant asthma is intimately associated with neutrophilic inflammation. Our laboratory has established a critical role for PARP-1 in asthma and neutrophile-based inflammation. Thus we hypothesize that PARP-1 may restores steroid sensitivity in murine model of steroid-resistant asthma.

Objective

To investigate whether PARP-1 inhibition could restore steroid sensitivity in steroid-resistant asthma model.

Methods

Murine model of steroid-resistant asthma and cell culture system were used.

Results

OVA challenge promotes eosinophilic-Th2 mediated response that is blocked by dexamethasone treatment. OVA priming with LPS/IFNγ results in class switching into neutrophile with developing resistance to the treatment with dexamethasone. PARP inhibition by gene knockout or by olaparib reversed the established steroid-resistant asthma manifestation in mice. This is attributed to marked reduction in the inflammatory cells infiltrating the airways and the significant reduction in the Th2 cytokines production, AHR, and the OVA-specific IgE secretion in both BALF and serum of the mice. The reversal of steroid resistance is explained by marked increase in the expression of Glucocorticoid Receptors (GR).

Conclusion

Our findings demonstrate a critical role for PARP-1 in steroid resistance and support the potential of PARP inhibition as a viable therapeutic strategy for treatment of steroid-resistant asthma.
Suppressive role on bacterial resistance of CD10+Pax5+cells in alcohol use disorder patients
Authors: Tsuchimoto Yusuke , Asai Akira , OHAMA Hideko , Fukunishi Shinya , Higuchi Kazuhide
Published: January 2023

Alcohol use disorder (AUD) patients are the immunocompromised hosts and these patients have an increased risk of several opportunistic infections. CD10+Pax5+cells are included in lineage negative cells which have been described as suppressor cells for host antibacterial defenses. In this study, we examined the immunological role of CD10+Pax5+cells in AUD patients. CD10+Pax5+cells were isolated from peripheral blood of AUD patients and healthy donors (HD) and analyzed for retinoic acid receptor (RAR) expression by flow cytometry. These cells (1 x 106cells/ml) were cultured with 10 μg/ml of retinoic acid (RA) for 24 hours. After washing, culture fluids of these cells for 24 hours were harvested. Monocytes (1 x 106cells/ml) isolated from HD, were stimulated with culture fluids from CD10+Pax5+cells after the RA stimulation and the bactericidal activity against Enterococcus faecalis was assayed by colony counting methods. In the results, the number of CD10+Pax5+cells was same between AUD patients and HD. CD10+Pax5+cells from HD were expressed RAR (63.7 ± 2.19%), but those of AUD patients were not (12.4 ± 3.31%). When monocytes were stimulated with culture fluids of CD10+Pax5+cells after the RA stimulation from HD, the bactericidal activity of these cells was 62.75 ± 3.78%. However, the bactericidal activity of same monocytes which were stimulated with culture fluids of CD10+Pax5+cells after the RA stimulation from AUD patients, was suppressed(5.5 ± 3.15%). In conclusions, through the production of some soluble factors active against killing functions of monocytes, CD10+Pax5+cells increase the susceptibility of AUD patients to opportunistic infections.
Evaluation of T regulatory cells responses in acute and chronic phases of Chikungunya patients
Authors: Gois Bruna Macedo , Guerra-Gomes Isabel Cristina , Peixoto Rephany Fonseca , Dias Cinthia Sousa Nobrega , Palmeira Pedro Henrique Sousa , Araújo Josélio Maria Galvão , Keesen Tatjana
Published: January 2023

The aim of this work was to characterize T regulatory cells (Treg) from patients presenting both acute and chronic forms of Chikungunya disease. To reach this aim, peripheral blood samples from controls (n = 7), acute (n = 7) and chronic (n = 6) patients were collected and evaluated by flow cytometry regarding Treg markers such as CD39, CD73, Granzyme B, Perforin, PD-1, CTLA-4, INF-γ, IL-10 and TGF-β. It was observed: 1) Decrease in Treg cells frequency of acute and chronic patients compared to controls; 2) Decreased expression of extracellular ATP cleavage enzymes (CD39 / CD73) in Treg cells from both patients groups; 3) Treg cells from acute and chronic patients presented a decreased production of Granzyme B and Perforin compared to control group; 4) Treg cells of acute patients showed a decrease of 50% in CTLA-4 expression, which is reestablished in patients presenting the chronic form of the disease; 5) In addition, PD-1 receptor was decreased on the Treg cell surface of acute and chronic patients compared to healthy group; 6) Production of INF-γ by patientsTreg was similar to controlsTreg; 7) IL-10 Treg producing cells did not change compared to control group, however the chronic patients showed higher frequencies of IL-10 Treg producing cells compared to acute patients; 8) Treg cells from patients in both forms of Chikungunya disease decreased the production of the TGF-β, compared to controls. Thus, our results suggest that the decrease of regulatory T cells and markers may contribute to the favoring of viral clearance, however, the maintenance of inflammation can promote cellular damages that are related to severe clinical characteristics of the disease, suggesting that Treg cells play a key role in the development Chikungunya pathogenesis.
Interleukin-27 enhances the function of regulatory T cells to prevent Graft-versus-host diseases
Authors: Le Thi Hong Nga , Hamilton Betty K. , Min Booki
Published: January 2023

Graft-versus-host diseases (GvHD) remain life-threatening complications after allogeneic stem cell transplantation. Foxp3+Tregs can inhibit GvHD, while preserving graft-versus-tumor activity, making Tregs a promising candidate for GvHD treatment. Yet, the clinical application is hampered by the limited numbers and ineffective functions during treatment. Conventional CD4 T cells acquire Foxp3 expression and regulatory functions by stimulating them with IL-2 and TGFb (iTregs). Unlike thymus-derived Tregs (tTreg), ineffective regulatory functions and phenotypic instability of iTregs remain areas of improvement. We reported that IL-27 is able to enhance Treg functions to reduce autoimmune and allergic inflammation. Herein, we sought to test if IL-27-stimulation in Tregs improves therapeutic efficacy of Tregs in a murine model of MHC-mismatched allogeneic GvHD. IL-27-pretreated tTregs efficiently reduced acute GvHD-associated mortality, whereas the efficacy of IL-27-pretreated polyclonal iTregs to prevent GvHD was marginal. We hypothesized that elevating antigen specificity in iTregs would increase the therapeutic functions of iTregs. Indeed, IL-27-pretreated allogeneic iTregs exhibited highly improved therapeutic function, equivalent to that of tTregs. IL-27-induced iTreg functions were also tested using human cells. IL-27 prestimulated third party human iTregs partially rescued mice from xenogeneic GvHD. Our data suggest that IL-27 stimulation in Tregs may play an important role in regulating Treg functions during GvHD, raising the hypothesis that defects in Treg functions (possibly via IL-27) may underlie different susceptibility of allogeneic bone marrow transplant patients who develop GvHD or not.
Identification of new T Cell Epitopes in the mold aeroallergen Aspergillus fumigatus
Authors: Sternberg Luise , Seumois Gregory , Paul Sinu , Sidney John , Schulten Véronique MJ , Sette Alessandro , Vijayanand Pandurangan , Peters Bjoern
Published: January 2023

Exposure and sensitization to mold is frequently associated with severe asthma that does not respond to medication. The objective of our study is to identify a comprehensive set of T cell epitopes from the mold aeroallergen Aspergillus fumigatus (Asp), which will enable the isolation and study of T cells associated with severe asthma. To date, T cell epitopes have only been identified for three Asp allergens, all of which were originally identified based on their recognition by IgE antibodies. To derive a more comprehensive picture of T cell antigen recognition in Asp, we applied our validated 2D-gel immunoproteomics methods, and probed for IgE/IgG reactivity of Asp allergic patient plasma to proteins from two Aspergillus extracts. We also mined the literature for previously studied Asp allergens and antigens. From the proteins thus identified, we generated peptides and assessed them for HLA binding bioinformatically. This resulted in the identification of a total of 2,769 predicted HLA binding peptides. In order to identify which of these peptides can be recognized by T cells, we stimulated PBMCs of Asp-reactive donors with allergen extracts and screened for cytokine production in response to these peptides. The most reactive peptides were all from newly identified antigens, but also confirmed previously identified allergens Asp f 15 and Asp f 17 as sources of broadly reactive T cell epitopes. Thus, our work identifies a comprehensive picture of T cell antigens contained in Asp and commonly recognized T cell epitopes within them. This provides a tool for future studies of the immunopathology associated with mold allergy and asthma.
Functional impact of a cellular Long non-coding RNA on CCR5 expression and HIV infection
Authors: Kulkarni Smita , Lied Alexandra , Kulkarni viraj , singh sukhvinder , Rucevic Marijana , Martin Maureen P. , Chawdhury Fatema , Le Gall Sylvie , Yu Xu , Carrington Mary
Published: January 2023

An intergenic single nucleotide polymorphism (rs1015164 A/G) showed genome-wide significant association with HIV outcomes in previously published meta-analyses. We found that rs1015164 marks expression levels of an antisense long non-coding RNA, which we termed CCR5AS. CCR5AS interferes with the interaction between an RNA binding protein, RALY and the untranslated region of CCR5, and thus enhances CCR5 expression. Inhibition of CCR5AS decreased CCR5 mRNA as well as surface expression levels and reduced in vitro infection of CCR5-dependent HIV. Our data reveal a previously unknown mechanism by which a variation in a long non-coding RNA region affects HIV infection.
Chronic HCV infection, HCV/HIV coinfection and older age are independently associated with naïve CD4+CD31+ lymphopenia
Authors: Auma Ann , Kowal Corinne , Zebrowski Elizabeth , Damjanovska Sofi , Shive Carey , Osborne Melissa , Kalayjian Robert , Anthony Donald D
Published: January 2023

Background

Chronic Hepatitis C virus (HCV), HCV/HIV and aging are known to predispose to impaired immunity to vaccines, new infections and cancers. Naïve CD4+CD31+ lymphopenia is exhibited in all three conditions, and may be one contributor to impaired immunity. Whether age and chronic HCV or HCV/HIV infection status independently impact naïve CD4+CD31+ lymphopenia in older chronic infected individuals has not been described.

Methods

We enumerated naïve CD4+ and CD4+CD31+ absolute counts in chronic HCV (n=28) and ART controlled HCV/HIV (n=13) participants spanning a broad age range (HCV 27–87, HCV/HIV 27–70), and age distribution-matched uninfected controls (n=18, age 24–88) by flow cytometry. Kruskal-Wallis test (by SPSS) was used to compare variables across groups, and Spearmans rho test was used to evaluate correlations within individual groups.

Results

Naive CD4+ and CD4+CD31+ counts differed across groups (HCV, HCV/HIV and control groups, p=0.01 and p=0.03), while age did not differ across groups. Lower naive CD4+ and CD4+CD31+ counts were observed in the HCV infected participants compared to the controls (p=0.007 and p=0.01). Within the HCV group age negatively correlated with the naive CD4+ and CD4+CD31+ counts (r= −0.51 p=0.006, and r= −0.64 p<0.001).

Conclusions

Both the state of infection (HCV or ART controlled HCV/HIV) and age independently impact naive CD4+ and CD4+CD31+ counts. Further studies are needed to understand how the mechanisms resulting in CD4+CD31+ lymphopenia during infection and ageing differ, and whether there is immune restoration following HCV DAA therapy.
Vδ1+ and Vδ2+ gamma delta T cells express different marker signatures and are distinctly linked to plasma markers of inflammation with ART-suppressed HIV infection and normal aging
Authors: Snyder-Cappione Jennifer E , Pihl Riley M , Olson Alex J , Lin Nina , Belkina Anna C
Published: January 2023

Our previous work implicates γδ T cells as an inflammatory driver in ART-suppressed HIV infection and suggests that this unique T cell population serves distinct roles in the ‘inflamm-aging’ that occurs with and without virally controlled HIV. In this study, we honed our previous findings to determine the precise γδ T cell subsets that are driving inflammation with age +/− HIV infection. We performed in depth immunophenotyping of circulating γδ T cells via multi-color flow cytometry and analysis of plasma markers of inflammation, coagulation, and intestinal permeability from subjects of our HIV and Aging cohort, which includes ART-suppressed HIV+ individuals and matched uninfected controls stratified by age into younger (≤35yo) and older (≥50yo) groups. We found discrete differences in the combinational expression the inhibitory receptors (IRs) TIGIT, PD-1, and CD160 between the Vδ1+ and the Vδ2+ γδ T cell subsets with HIV infection and healthy aging. Also, we found that specific IR signatures of Vδ1+ and Vδ2+ γδ T cells correlated with plasma inflammatory markers; however, different connections between the plasma analytes were found for each of the two γδ T cell subsets. Taken together, these data indicate that Vδ1+ and Vδ2+ γδ T cells exist in distinct states and possess divergent roles in the ‘inflamm-aging’ found in aviremic HIV+ infection and with normal aging. Further investigation into the anatomical locations, ex vivo functions, and additional surface markers that define γδ T cell subsets may reveal novel therapeutic targets to abrogate the aberrant inflammation found with HIV infection, aging, and other inflammatory conditions.
Role of Mycobacterium tuberculosis expressing unique protein Rv000B in immune modulation of host cell machinery
Authors: Arora Simran Kaur , Alam Anwar , Ahmad Javeed , Naqvi Nilofer , Hasnain Seyed Ehtesham , Ehtesham Nasreen Zafar
Published: January 2023

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb.), an intracellular pathogen remains a major global health problem. M.tb., comprising more than 120 species has evolved by successive genomic reduction events. Comparative genomic and proteomic analysis of 13 Mycobacterium species classified into Strict pathogens (virulent), Opportunistic pathogens (Non-tuberculous Mycobacteria, NTM) and the Non-pathogenic bacteria revealed the presence of 25 unique protein. These 25 proteins share less than 20% homology with any other proteins of Mycobacteria species used in the study. Rv000B, one of the unique proteins is present in M.tb and M. bovis BCG only. The presence of Rv000B in M. tb and M.bovis BCG only points to its importance as “Signature proteins”, suggesting that Rv000B could be used for its diagnostic potential. Rv000B is a hypothetical protein and hence it will be interesting to study their functional role in the pathogenesis of M.tb. In-silico analysis of Rv000B revealed that this protein is highly disordered and unstable. Immunogenicity of Rv000B protein was evaluated by treating the splenocytes ex-vivo obtained from mice that were previously immunized with Rv000B protein. Our results showed that Rv000B is highly antigenic and elicited a pro-inflammatory immune response. High IgG reactivity of Rv000B is also seen in immunized mice and different categories of TB patients. Besides inducing Th1 responses, it also significantly induced apoptosis of macrophages. Therefore, these pro-host antigenic proteins are ideal candidates to produce recombinant BCG vaccine to enhances its vaccine potential.
Induction of T cell-independent hypermutated and protective IgG and IgA antibody responses by TLR4-BCR or TLR5-BCR co-engagement
Authors: Rivera Carlos Eduardo , Chupp Daniel , Yan Hui , John IM , Wang Jingwei , Sanchez Helia Nasrollahi , Wu Shuai , Taylor Julia , Zan Hong , Xu Zhenming , Casali Paolo
Published: January 2023

Somatic hypermutation (SHM) and class-switch DNA recombination (CSR) together with plasma cell and memory B cell differentiation are central to the maturation of the antibody response. Prompted by our demonstrations that CSR and plasma cell differentiation can be induced in vitro by co-engagement of TLR4 and BCR, we tested the hypothesis that co-engagement of a TLR and BCR can induce mature T cell-independent antibody responses in vivo. To this end, we injected Tcrβ−/−Tcrδ−/− mice, which are devoid of all T cells, with TLR4-dependent NP-LPS or TLR5-dependent Salmonella flagellin. These mice readily generated NP-specific IgG antibodies and class-switched B cells and plasma cells. The high-affinity NP-LPS-induced IgG response was accompanied by a high-frequency of V186.2 region DNA mutations in class-switched B cells and NP-specific memory B cells. The TLR4-dependence and T cell-independence of this class-switched, anti-NP response was emphasized by immunedeficient NSG mice grafted with purified B cells, and mimicked in Tcrβ−/−Tcrδ−/− mice injected with TLR5-dependent flagellin. These mice generated flagellin-specific IgG and IgA, which displayed strong bactericidal activity and protected mice against Salmonella challenge. Thus, co-engagement of TLR4-BCR or TLR5-BCR, respectively, can effectively induce a mature and protective T cell-independent antibody response, thereby providing the basis for new TLR/BCR co-engagement-based B cell-targeted vaccine strategies.
Dissecting the role of proinflammatory chemokine CCL3 in the selection of germinal center B cells and in secondary antibody responses
Authors: Grigorova Irina , Benet Zachary , Ke Fang
Published: January 2023

Expression of CCL3 and CCL4 chemokines is upregulated in B cells upon crosslinking of B cell receptors and in germinal center (GC) centrocytes. However, the role of these proinflammatory chemokines in B cell responses is still unclear. Single cell qPCR analysis suggests that only a small fraction of GC centrocytes upregulates expression of CCL3/4. While B cell-intrinsic production of CCL3 promotes B cell interactions and negative control by follicular regulatory T cells at the peak of GC response, at the later stages of GC response it promotes B cells selection. Consistent with that, secondary antibody responses in CCL3 knockout mice are reduced. Our findings suggest that CCL3 is involved into multifaceted regulation of GC responses. Analyses of the mechanisms promoting both the negative and positive regulation of GC B cells in CCL3-dependent fashion are underway. These studies should be important for dissecting the role of CCL3 in normal immune responses and in B cell-originated lymphomas, where CCL3/4 are often elevated.
T follicular regulatory cells (Tfr) reduce proliferation and downregulate induction of costimulatory molecules on human Tfh and B cells
Authors: Sayin Ismail , Smith Carson , Buggert Marcus , Betts Michael R , Canaday David H.
Published: January 2023

Recently defined specialized subset of Treg expressing CXCR5+PD1+FoxP3+ termed as T follicular regulatory (Tfr) cells regulates T follicular helper (Tfh) mediated B cell high-affinity and isotype-switched antibody production. To date, much of our understanding of the suppression mechanism of Tfr cells has been obtained from animal studies. The mechanism of Tfr regulation of humoral immunity in humans is still understudied. Here, we used cells from human mesenteric lymph nodes to study how Tfr affect Tfh and B cells and what specific Tfr-induced changes occur on Tfh and B cells in vitro. We report that Tfr suppress both Tfh and B cell function and proliferation in a dose dependent manner. We find that Tfr downregulate CD40, CD80/CD86 and CD38 on B cells and CD28, OX40, ICOS and CD38 on Tfh cells. We also show that blocking immune checkpoint receptors inhibits Tfr suppression of Tfh and B cells and partially restores the Tfh mediated B cell antibody production. These findings reveal that Tfr regulate activation of Tfh and B cells by downregulating key costimulatory receptors and ligands that help induce activation in both Tfh and B cells. These changes ultimately lead to decreased proliferation of both B cells and Tfh and reduced production of immunoglobulin by B cells.

This research was supported in part by NIH R01 and VA.
Metabolic reprogramming of human pro-inflammatory B cells in aging and obesity
Authors: Blomberg Bonnie , Diaz Alain , Romero Maria , Frasca Daniela
Published: January 2023

The subset of pro-inflammatory B cells, called late memory (LM), tissuelike or double negative (CD19+CD27-IgD-), accumulates in the blood of elderly individuals. Here we show that LM B cells do not proliferate and do not make antibodies to new antigens (influenza vaccine), but they spontaneously secrete antibodies with autoimmune reactivity, due to spontaneous expression of the T-bet transcription factor. These cells also increase in the blood of individuals with obesity and autoimmune diseases, but causative mechanisms and signaling pathways involved are known only in part. Here we compare frequencies and metabolic requirements of these cells in the blood of healthy individuals of different ages and in the blood and subcutaneous adipose tissue (SAT) of individuals with obesity. Results show a metabolic reprogramming of LM B cells in aging and obesity, with LM B cells from young individuals having minimal metabolic requirements, LM B cells from elderly and obese individuals use higher amounts of glucose, make autoimmune antibodies and have higher aerobic glycolysis to support their function. LM B cells from the SAT have the highest metabolic requirements as they have increased oxidative phosphorylation, aerobic glycolysis and fatty acid oxidation. This metabolic reprogramming of LM B cells occurs after activation of AMPK (5′-AMP activated kinase) and Sestrins, both able to mitigate stress and cell death. These results indicate that metabolic reprogramming maintains LM B cells alive and maintains their function (secretion of autoimmune antibodies).
The IL-12/STAT4 pathway associates with critical functions in neutrophils
Authors: Keeter William Coles , Moriarty Alina , Ma Kaiwen , Glenn Lindsey , Waseem Tayab , Lattanzio Frank , Edemobi Stefan , Nadler Jerry , Kaplan Mark H , Galkina Elena
Published: January 2023

Signal transducer and activator of transcription 4 (STAT4) becomes activated via phosphorylation by JAK/Tyk kinases in response to IL-12 and other cytokines, which results in downstream transcription of pro-inflammatory genes. Our preliminary data are the first to demonstrate that IL-12 induces phospho-STAT4 in neutrophils. STAT4, JAK2, and p38 MAPK, but not Tyk2, become rapidly phosphorylated in response to IL-12 treatment in a time dependent manner. Pharmacologic inhibition of JAK1/2, but not p38, resulted in a two-fold reduction in phospho-STAT4, confirming its role as a signalling intermediate during STAT4 activation. Although IL-12 does not induce ROS formation in the short term (<3 hours), we were surprised to discover a 4-fold increase in ROS production in response to long term IL-12 treatment (14 hrs), compared to only 1.5-fold increase in STAT4-deficient neutrophils. Additionally, we observed a dramatic 15-fold decrease in in vitro migration of STAT4-deficient neutrophils towards the chemoattractant GM-CSF compared to wild-type. Preliminary in vivo studies have revealed deficient migration to the peritoneal cavity of STAT4-deficient mice following intraperitoneal injection of GM-CSF. Finally, we demonstrate that in vitro NET formation in response to PMA treatment is impaired 5-fold in STAT4-deficient neutrophils compared to wild-type. These findings highlight the novel IL-12/STAT4 pro-inflammatory signalling pathway in murine neutrophils. Our data suggest a major contributory role of STAT4 in mediating neutrophil functions that have been previously associated with the progression of atherosclerosis. Future in vivo studies will determine the neutrophil-specific role of STAT4 in atherosclerotic mice.
Dermal Fibroblasts Control Mast Cell reactivity to commensal bacteria
Authors: Chang Yu-Ling , Sriram Krishna , Wang Zhenping , Igawa Satomi , Wu Chia-Chi , Insel Paul , Di Nardo Anna
Published: January 2023

Background

Our data suggests, and current literature supports, that when mast cells (MCs) leave the blood and interact with dermal fibroblasts (DFs), they assume a bacterial tolerant phenotype in order to prevent unnecessary inflammation during the encounter with the beneficial commensal microbiome. We hypothesize that the mechanisms that induce the MC tolerant phenotype in human skin is due to dermal fibroblasts interactions.

Methods

We mimicked the “in vivo” setting by conditioning human cord blood MC (hMC) with human dermal fibroblast (hDF) and, then exposed them to commensal bacteria supernatant. Human MC transcriptome data from the RNA-seq FANTOM5 data collection on MCs freshly isolated from human skin (FANTOM5 study Motakis_et_al_2013) was used to investigate the innate immune phenotype of MCs in the skin. We determined the genes that make hDFs unique by comparing their gene expression profiles with lung and cardiac fibroblasts using RNA-seq analysis and challenged hMCs with the products of these genes.

Results

We have discovered that hDF conditioned with hMCs, show a decreased expression of interleukins upon commensal stimulation; specifically, multiple cytokines/chemokine such as pro-inflammatory cytokine GM-SCF, IL-8, MCP-1 and anti-inflammatory cytokines IL-10, IL-13 are down-regulated by at least 95% and these cytokines can be controlled by the product of hDF unique genes.

Conclusion

The human dermal MC reactivity is controlled by hDF’s unique genes that play an important role in regulating the hMC NF-Kb pathway.
Allo-specific NK cells differentially regulate T cell allo-immunity depending on prenatal education
Authors: Kang Hee Kap , Turner Lucas , Redden Catherine , Shaaban Aimen F
Published: January 2023

Allospecific NK cell hyporesponsiveness develops after in utero hematopoietic cellular transplantation (IUHCT) in mice who exhibit stable long-term engraftment. Conversely, allospecific NK cell responses are normal in mice that reject their graft. We hypothesized that these alternate NK cell responses also differentially influence T cell responses after IUHCT.

To challenge this hypothesis, we compared the helper or suppression potential of prenatally educated NK cells between prenatal recipients with stable engraftment (engrafter mice) and littermates that rejected their graft (rejecter mice) in a Balb/c --- > a B6 model of IUHCT. Allospecific NK cells from engrafter, rejecter, or naïve mice were co-cultured with naïve responder T cells (B6) and irradiated allogeneic target cells (Balb/c) in an in vitro IFN-g assay. Intracellular production of IFN-g was measured by flow cytometry of the naïve responder T cells after 66 hrs of co-culture.

We found that responder T cells displayed increased IFN-gamma production when co-cultured with allospecific NK cells from rejecter mice compared to co-culture with allospecific NK cells from naive control mice. Conversely responder T cells displayed decreased IFN-g production when co-cultured with allospecific NK cells from engrafter mice compared to co-culture with NK cells from naïve control mice.

From these results, we conclude that allospecific NK cells can influence the allospecific response of T cells in vitro depending on the context of their prenatal education. Further studies are needed to determine if similar mechanisms support engraftment or rejection in vivo.
PD-1 signaling impacts T cell function during the early phase of infection with Toxoplasma gondii
Authors: Perry Joseph Allan , Delong Jonathan , Clark Joseph , Park Jeongho , Gullicksrud Jodi , Shallberg Lindsey , Konradt Christoph , Christian David , Hunter Christopher
Published: January 2023

Long term resistance to T. gondii is mediated via T cell production of IFN-γ, but during chronic infection CD8+ T cells show signs of reduced capacity to secrete IFN-γ associated with increased expression of multiple inhibitory receptors. Blockade of the inhibitory receptor ligand PD-L1 during the chronic phase of infection has been reported to increase the functionality of CD8+ T cell responses and result in reduced parasite burden. However, the impact of PD-1 signaling on the immune response to T. gondii during the early phase of infection is unknown. PD-L1 blockade during the acute phase of toxoplasmosis resulted in an increased proportion of activated CD8+ T cells (CD69+, LFA-1hi) accompanied by an increased proportion of polyfunctional CD8+ T cells (IFN-γ+, Granzyme B+). This effect did not result in increased immune pathology or reduce parasite burden. Unexpectedly, this intervention resulted in a significant increase in the expression of inhibitory receptors such as PD-1, TIM3, and CTLA-4 on the long-lived T-intermediate (Tint, CXCR3+/KLRG1+) CD8+ T cell population, while increasing the proportion of terminally differentiated T cells in specific sites of infection. These datasets suggest PD-1 signaling in the early phase of T. gondii infection constrains CD8+ T cell effector differentiation and may act to preserve pathogen specific Tint subsets that are needed for long term resistance to this persistent infection.