bluuebunny/crossref_metadata_2025 huggingface dataset:
Administration of Sparstolonin B- a TLR4 antagonist, attenuates hepatic fibrosis by inhibiting TLR4 mediated hepatic fibroblast proliferation.
Authors: Dattaroy Diptadip , Chandrashekaran Varun Chandrashekaran , Seth Ratanesh Kumar , Alhasson Firas , Fan Daping , Nagarkatti Mitzi , Nagarkatti Prakash , Diehl Anna Mae , Chatterjee Saurabh
Published: January 2023

Previous studies have shown that TLR4 activation accentuates hepatic inflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Here we report an anti-fibrotic activity of Sparstolonin B (SsnB)-a TLR4 antagonist, derived from Chinese herb Sparganium stoloniferum. We hypothesize that, SsnB decreases TLR4 induced inflammation and fibrosis in liver by inhibiting TLR4 signaling, TGFβ signaling and by inducing senescence in fibrogenic hepatic stellate cells. Here we used Bromodichloromethane (BDCM) induced liver fibrosis model where diet induced obese (DIO) mice were administered with BDCM (a CYP2E1 activator) for 4 weeks. A similar group were administered SsnB for 4 weeks along with BDCM. Picrosirius stain and IHC of fibronectin images showed a robust decrease of extracellular matrix deposition upon SsnB administration. Mechanistically, we found that SsnB decreased fibroblast proliferation by inhibiting TLR4-PI3K akt mediated fibroblast proliferation-as was shown by increased PTEN, p53, p21 and decreased cyclin D1, MDM2 protein expression in SsnB treated mice. Interestingly, we found a parallel mechanism in which SsnB increased BAMBI (a TGFβ pseudo-receptor) protein expression. Inhibition was possibly mediated by targeting the TLR4 dependent MYD88-NfkB pathway and TGFβ signaling was decreased following attenuated SMAD2/3-SMAD4 co-localization in the nucleus. SsnB increased gene expression of cell senescence markers (p53, p21), decreased alpha SMA gene expression and increased anti-fibrotic matrix metallo protease 2 (MMP2) gene expression in rat stellate cells in vitro. BAMBI gene and protein expression- a negative regulator of pro-fibrogenic TGFβ signaling, were also increased by SsnB treatment in vitro.
The transcriptional response of Kupffer Cells to fat and cholesterol is non-canonical and distinct from bone-marrow derived macrophages
Authors: McGettigan Brett M , McMahan Rachel H , Rosen Hugo R
Published: January 2023

Diets high in fat and cholesterol can cause hepatic lipid accumulation and inflammation known as non-alcoholic steatohepatitis (NASH). Liver macrophages putatively drive disease by responding to PAMPS/DAMPS and producing pro-inflammatory cytokines. However, recent studies show that there are two principle subsets of macrophages in a NASH liver: Tissue-resident macrophages that are embryonically derived (Kupffer Cells; KCs) and infiltrating macrophages that are adult-derived from bone marrow (IMs). Furthermore, the role that each subset plays in disease pathogenesis is unclear. We FACS sorted the KC (F4/80hiCD11blow) and IM subsets (F4/80lowCD11bhi) from mice fed a high fat/cholesterol (HFHC) diet as well as those from healthy controls. RNA was extracted and gene expression was analyzed using RNA sequencing techniques. HFHC mice had a prominent IM population that was absent in controls. IMs were the primary producers of cytokines and chemokines. KCs did not upregulate pro-inflammatory cytokines in response to dietary fat and cholesterol, yet upstream analysis revealed that lipid metabolism and bile acid signaling pathways were more active than IMs. 3,617 genes were differentially expressed KCs, while 3,421 genes were differentially expressed in IMs. Genes known to be expressed by restorative macrophages were enriched in KCs (CD163, MARCO, TIMD4, MMP12, MMP13), while pro-inflammatory and M1 genes predominated in the IM population (TNF, SPP1, CXCL2, CCL2, S100A8). In conclusion, bone marrow-derived macrophages have a transcriptional profile consistent with the previously reported role of liver macrophages in NASH pathogenesis. However, Kupffer Cells respond to dietary stress in a non-canonical manner.
Adipokine Leptin mediated NOX-2 promotes kidney inflammation in nonalcoholic fatty liver disease via miR21-dependent mesangial cell immune activation
Authors: ALHASSON FIRAS A , DAS SUVARTHI , Chandrashekaran Varun , Dattaroy Diptadip , Seth Ratanesh Kumar , Nagarkatti Mitzi , Nagarkatti Prakash , Chatterjee Saurabh
Published: January 2023

High insulin and leptin resistance followed by underlying inflammation is often ascribed to the ectopic manifestations in nonalcoholic fatty liver disease (NAFLD) but the exact molecular pathways remain unclear. We have shown previously that CYP2E1-mediated oxidative stress and circulating leptin in NAFLD is associated with renal disease severity. Extending the studies, we hypothesized that high circulatory leptin in NAFLD causes renal mesangial cell activation and tubular inflammation via a NOX2 dependent pathway that upregulates proinflammatorymiR21. High fat diet (60%kcal) was used to induce fatty liver phenotype with parallel insulin and leptin resistance in WT, Leptin knockout (ob/ob) and p47 phox knockout mice. Kidneys were collected after pathology showed NASH-like phenotype and accelerated oxidative stress in the liver. The kidneys were probed for mesangial cell activation and tubular inflammation. Results showed that NAFLD kidneys had significant increases in α-SMA, a marker of mesangial cell activation, miR21 levels, tyrosine nitration and renal inflammation while they were significantly decreased in leptin and p47 phox knockout mice. Micro RNA21 knockout mice showed decreased tubular immunotoxicity and proinflammatory mediator release. Mechanistically, use of apocynin or phenyl boronic acid (FBA) or DMPO or miR21 antagomir inhibited leptin primed-miR21-mediated mesangial cell activation in vitro suggesting a direct role of leptin-mediated NOX-2 in miR21-mediated mesangial cell activation. Finally JAK-STAT inhibitor completely abrogated the mesangial cell activation in leptin-primed cells suggesting that leptin signaling in the mesangial cells depended on the JAK-STAT pathway.
Characterisation of gene-modified CD3-enhanced CD4+ T cells for cancer immunotherapy
Authors: Piapi Alice , Nicholson Emma , Morris Emma C , Stauss Hans J
Published: January 2023

TCR gene transfer is used to redirect the antigen specificity of T lymphocytes towards known tumour antigens. TCR gene therapies in murine studies have shown promising results. However, in the clinic they often generate sub-optimal responses, when compared to treatments with tumour infiltrating lymphocytes. Previous work to improve TCR gene therapy has demonstrated that transferring additional CD3 genes increases TCR expression of both endogenous and introduced TCR in CD4+ and CD8+ T cells. In vivo experiments demonstrated that CD8+ T cells, transduced with TCR and additional CD3 were more effective in tumour protection than T cells transduced with the TCR alone. Whilst, CD4+ T cells transduced with TCR and additional CD3 initially showed improved tumour protection, lethal toxicity, unrelated to tumour burden, was later observed. To investigate the effects of CD3 overexpression, CD3 genes only (no TCR genes) were transferred into purified CD4+ and CD8+ T cells. Following adoptive transfer, CD3-enhanced CD4+ T cells survived for longer and were recovered in higher percentages in spleen, lymph nodes, bone marrow and liver, compared to CD3-enhanced CD8+ T cells and mock transduced CD4+ T cells. The same trend was also seen in competition experiments where mice received a 1:1 ratio of CD3-enhanced CD4+ T cells and mock-transduced CD4+ T cells. Interestingly, this was observed despite a twofold downregulation of TCR levels in the CD3-enhanced CD4+ T cells, compared to their pre-transfer TCR levels. Current experiments are aimed at dissecting the mechanisms responsible for, and the physiological implications of the observed TCR downregulation.
Ovarian cancer ascites induces a T cell suppressive phenotype in mature neutrophils: a potential barrier to anti-tumor immunity
Authors: Singel Kelly L , Khan ANM Nazmul H , Moysich Kirsten B , Odunsi Kunle , Segal Brahm H
Published: January 2023

Ovarian cancer (OC) is often diagnosed at advanced stages and presents with ascites. Necrosis is a hallmark of advanced cancer and releases DAMPs that activate innate immune responses (e.g., neutrophils). While neutrophils are critical in host defense, their role in the context of tumor is less understood. CTLs correlate with better outcomes in advanced OC, and immunotherapy for cancer is based on augmentation of CTL responses. Our prior studies showed that granulocytes from ascites of patients with newly diagnosed OC suppressed donor T cell proliferation ex vivo. The current work probes mechanisms for this suppression.

In ascites from patients with newly diagnosed OC, ~90% of cells were inflammatory (CD45+). Neutrophils comprised ~15% of CD45+ cells and the neutrophil:CD8+ T cell ratio was 1.5:1. In ex vivo studies, cell-free ascites (ASC; 500g) attracted normal donor neutrophils (NDN). While neither ASC nor NDN alone impaired CD3/CD28-stimulated normal donor T cell proliferation, ASC (10/15 patients) when combined with NDN (1:1) completely suppressed T cell proliferation. Suppression by ASC + NDN required cell contact and was lost if NDN were added after overnight CD3/CD28 stimulation of T cells. Finally, heat-inactivating ASC (56C, 1h) abrogated the ability of NDN to suppress T cell proliferation.

Here we provide evidence that ascites recruits neutrophils to the tumor microenvironment and induces a suppressive phenotype of neutrophils that requires cell contact and heat-labile cell-free constituents. Ongoing studies aim to identify the putative protein(s) in ascites and the signaling effects on neutrophils. Our long-term goal is to develop novel approaches to overcome this suppression and improve durable anti-tumor immunity.
Rosiglitazone, a peroxisome proliferator-activated receptor-gamma agonist, reduces but does not prevent, the pulmonary CD4+ effector T-cell response in a murine model of asthma
Authors: Maslanka Tomasz , Otrocka-Domagała Iwona , Zuśka-Prot Monika , Gesek Michał
Published: January 2023

Nuclear factor (NF)-κB is regarded as a promising target for the development of a novel therapeutic strategy in asthma. Peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists inhibit the activity of NF-κB. Therefore, these agents are potent therapeutic agents for the treatment of asthma. It was found that rosiglitazone (ROS), a PPAR-γ agonist, attenuated airway inflammation in a murine model of asthma. The present study attempts to shed light on the involvement of CD4+ effector (Teff) and regulatory (Treg) T cells in the anti-asthmatic action of ROS in ovalbumin (OVA)-induced airway allergic inflammation (AAI) in BALB/c mice. We studied the effect of a 10-day treatment with ROS on the development of AAI and the absolute counts of Treg (Foxp3+CD25+CD4+) cells and activated Teff (Foxp3−CD25+CD4+) cells in the mediastinal lymph nodes (MLNs) and lungs of OVA-sensitized/challenged mice. The severity of AAI was only reduced, but not abolished, by the treatment with ROS. The absolute number of CD4+ Treg and Teff cells was increased in the MLNs and lungs of OVA-sensitized/challenged mice treated with the vehicle as compared to healthy mice. No differences were found in the count of CD4+ Treg cells between vehicle- and ROS-treated OVA-sensitized/challenged mice. The treatment with ROS reduced, but did not fully prevent, the OVA-induced increase in the count of activated CD4+ Teff cells in the MLNs and lungs. The results suggest that the impairment of the activation and clonal expansion of CD4+ Teff cells in the MLNs is involved in the anti-asthmatic action of PPAR-γ agonists. This effect does not seem to be mediated through increased recruitment of CD4+ Treg cells into the MLNs and lungs and/or local generation of Treg cells.
T cell mediated mechanisms underlying HSC dysfunction and bone marrow failure
Authors: Hoyer Katrina K , Gravano David M
Published: January 2023

Bone marrow failure occurs when the bone marrow cannot produce sufficient numbers of red blood cells, white blood cells or platelets. Bone marrow failure disorders are rare, yet serious conditions, and are often thought to be the result of immune destruction within the bone marrow. IL-2-deficient mice, which have a deficit in functional regulatory T cells, develop spontaneous bone marrow failure. We show that disease occurs in these mice with a specific accumulation of T cells in the bone marrow, a shift in the CD4/CD8 ratio, IFNγ-mediated hematopoietic stem cell dysfunction, and antibodies directed against bone marrow and bone tissue, recapitulating human disease, and suggesting that this disease may be caused by self-reactive immune responses. Disease onset is rapid at 12 days of age with death by 27 days, and T cell responses are essential for disease progression, providing a novel model for defining the immune cell dysregulation in this autoimmune disease. We use this model to define the mechanisms underlying the deficit in myelopoiesis observed during bone marrow failure, including the contribution of activated lymphocyte subsets to disease induction. Our study describes the influence of IFNγ, CD4 and CD8 T cell subsets, and local bone marrow proliferation and migration on the kinetics and severity of disease.
S100a9-deficiency accelerates lupus disease and renal failure in male (NZB x NZW)F1 mice
Authors: Dand Hardik A , Davison Laura M , Alberto Andres , Vogl Thomas , Jorgensen Trine N
Published: January 2023

All (NZB x NZW)F1 (BWF1) female mice develop lupus-like disease, compared to ~30% of male BWF1 mice. Our previous research described an immunosuppressive effect of Gr1hiCD11bhi cells in male BWF1 mice. Specifically, as BWF1 mice age, female Gr1hi cells lost their ability to suppress altogether, while antibody-mediated depletion of Gr1hi cells accelerated disease development in male mice only. Reverse transcriptase-PCR studies revealed upregulated S100a9 and S100a8 mRNA levels in male flow-sorted Gr1hi cells as compared to cells from female BWF1 mice and non-autoimmune stains, leading us to hypothesize that S100a8/a9 produced by male Gr1hi cells is immunosuppressive. To study this, we created S100a9-deficient BWF1 mice. Our results showed spleen weight and splenocyte count, indicators of lupus advancement, were significantly higher in S100a9−/−male mice. Additionally, increased IgG-IC deposition in kidney glomeruli of S100a9−/−male mice was observed along with a significant increase in serum anti-chromatin IgG levels. Deficiency of S100a9 also resulted in increased frequencies of memory B cells and plasma cells in male mice, but not in female mice. Interestingly, S100a9-deficiency caused an accumulation of Gr1hiCD11b+ splenocytes in male mice. We speculated that this is due to a lack of trafficking of Gr1hi cells into the glomerulus of the kidney due to S100a9-deficiency. Subsequent IHC confirmed the lack of S100a9 expression in kidneys from S100a9-deficient mice. Finally, H/E and Trichrome stainings indicated accelerated renal failure in S100a9−/−mice. In conclusion, S100a9 may function as a chemoattractant of Gr1hi cells exerting immunosuppression and protection against lupus-like renal disease in male BWF1 mice.
Functionality of lung type II NKT cells developing in CD1d−/− animals
Authors: Pasquet Lise , Kato Shingo , Adams Tony , Sharrow Susan O , Davies-Hill Theresa , Jaffe Elaine S , Xia Zheng , Suzuki Motoshi , Kovalovski Damian , Berzofsky Jay A , Terabe Masaki
Published: January 2023

NKT cells are lymphocytes that recognize lipid antigens presented by CD1d. Type I NKT cells with semi-invariant TCR recognize α-galactosylceramide while many type II NKT cells with a more diverse TCR repertoire recognize sulfatide. We developed a stable sulfatide-loaded CD1d tetramer that allows the specific identification of type II NKT cells and found that they are enriched in the lung, a major site of tumor metastasis. An in-depth phenotypical analysis revealed that lung type II NKT cells were CD4 or CD8 single positive cells, like conventional T cells, whereas type I NKT cells were either CD4+CD8- or CD4-CD8-. We showed that type II NKT cells expressed markers of myeloid cells, c-Kit, CD11b and Ly6C, even though morphological analysis revealed lymphocytic morphology. In contrast to type I NKT cells, type II NKT existed in PLZF−/− and CD1d−/− mice. Thus, we asked the importance of CD1d in the in vivo and in vitro activation of type II NKT cells. In the steady state, lung type II NKT cells expressed granzyme A but not granzyme B or perforin. By injecting sulfatide in vivo, we observed that WT but not CD1d−/− lung type II NKT cells upregulated granzyme A and Ki67 expression. Using syngeneic WT bone marrow-derived dendritic cells (BMDC) as APCs, ex-vivo stimulation of WT but not CD1d−/− lung type II NKT cells with sulfatide induced the secretion of IFN-γ, IL-13 and IL-17. This was consistent with the expression of specific transcription factors, T-bet and RORγt, by lung type II NKT cells. In conclusion type II NKT cells still develop in CD1d−/− mice but are not functional. The ability to study responses of type II NKT cells in vitro can now help elucidate the mechanism by which these regulatory cells interact with other cells and modulate tumor immunity in the lung.
Immune microenvironment modulation via the interaction of human invariant natural killer T cell (iNKT cells) and Kupffer cells (KC), dendritic cells (DC) in colorectal liver metastasis
Authors: Feng Hao , Thasler Wolfgang
Published: January 2023

Aims

To investigate the immune microenvironment modulation via the interaction between human iNKT cells and KC, DC in colorectal liver metastasis.

Methods

Different subtypes of iNKT cells, KC and DC from the core of tumor (CT), tumor invasive margin (IM), and adjacent normal liver tissue (NL) were analyzed by BD LSRFortessa™, TMA, and immunofluorescence(n=138). Ex vivo 3D co-culture, Quansys Multiplex array, and Elisa array were performed.

Results

In metastasis, CXCR6+iNKT positively correlated with better prognosis. In addition, iNKT cells were enriched at IM, especially iNKT2. Intriguingly, although there were few iNKT cells in CT, the iNKT/Lymphocytes rate increased to ~5 folds and expressed elevated IL9, IFNγ, and FasL compared to NL and IM. This implied the critical role of iNKT cells in tumor control. Both DC and KC could drive iNKT activation, the proportion of BDCA-3+DCs significantly increased from NL to CT; though they expressed higher CXCL16 compared to other subtypes, the capability of activating iNKT cells via CD1d was less strong than plasmacytoid DC(pDC). pDCs showed significantly elevated α-GalCer: CD1d complex formation in an ex vivo iNKT cells activation assay, which implied its critical role of activating capability of pDC. However, pDCs, as well as BDCA-1+DCs, decreased from NL to CT. In this study, KC was found also activating iNKT cells. Interestingly, iNKT expressed Th2 cytokines when activated by KC, however, Th1 biased response could be found when activated with DC. In addition, KC subsets also differed with location. Furthermore, transmembrane CD1d density and intra/tmCXCL16 expression, Th1/2/17 biased immune responses of KC and DC were associated with the lipid metabolism preferences of these cells.
Group B Streptococcus infection induces M1-like activation in human placental and mouse decidual macrophages.
Authors: Sutton Jessica A , Rogers Lisa M. , Doster Ryan Steven , Gaddy Jennifer Angeline , Aronoff David M
Published: January 2023

Group B Streptococcus (GBS), a vaginal colonizing gram-positive bacterium, is a leading cause of neonatal death and sepsis. How GBS evades macrophage defenses in the gravid uterus is not yet known. In response to stimuli, macrophages are activated along a spectrum known as polarization ranging from classically-activated, pro-inflammatory “M1” to alternatively-activated anti-inflammatory “M2” macrophages. These activation states can contribute to disease pathogenesis. This study aims to define changes in macrophage activation in response to GBS infection. Human monocyte derived macrophages (MDMs) were infected with GBS or polarized in vitro with M1 (IFNγ/LPS) or M2 (IL-4 or IL-10) stimuli. Placental macrophages (PMs) were infected with GBS or treated with IFNγ/LPS. To mimic human ascending vaginal infection in vivo, C57BL/6 mice were inoculated vaginally with GBS at day E13.5 for 48h. Subsequently, decidual macrophages were isolated and analyzed for polarization markers via flow cytometry and tissues were used for histology. Resulting phenotypes were characterized using qRT-PCR, ELISA, and flow cytometry. In MDMs, GBS upregulated M1 genes (CCR7, CD80) and downregulated M2 genes (CD209, CD163). GBS increased the release of M1 cytokines (TNFα, IL-1β) in MDMs and PMs. PM surface expression of M1 markers (CCR7, CD80) increased while M2 markers (CD206, CD163, CD209) decreased. Mouse decidual macrophage surface expression of CD206 was downregulated while CD11c exhibited little change. Our results suggest that GBS may induce M1 activation in both human and mouse models. These studies provide new insights into the role of macrophage activation in GBS pathogenesis.

Studies supported by NIH NIAID Training Grant 5T32AI007281-27.
Cross-dressing and antigen transfer after DC targeting induce robust T cell response
Authors: Yao Chen , Hirai Toshiro , Igyarto Botond , Zurawski Sandy , Zurawski Gerard , Kaplan Daniel H
Published: January 2023

Cutaneous dendritic cells (DC) are critical in protecting host from environmental insults as well as maintaining skin homeostasis. Using mouse models that are lack certain subset of cutaneous DC, studies have showed the distinct function of different subsets of skin DC, However, the interaction between skin DC subsets is less known. By targeting antigen to Langerhans cells (LC), we showed that endogenous CD4+ T cell response is independent of MHC II expression on LC. We also showed that LC handed over antigen to CD11b+ dDC and DN dDC. At the same time, MHC II deficient LC acquired MHC-II in the lymph node through cross-dressing. We demonstrated unexpected collaboration between cutaneous DC subsets, and provided insight on DC targeted vaccine design.
STAT4 regulates CD8+Treg/Tfh cell axis and promotes atherogenesis in insulin-resistant Ldlr−/− mice
Authors: Galkina Elena , Taghavie-Moghadam Paresa L , Waseem Tayab C , Gleen Lindsey , Kaplan Mark H , Dobrian Anca D , Yang Yi , Nurieva Roza , Nadler Jerry L
Published: January 2023

The metabolic syndrome support atherosclerosis, but the exact mechanisms for accelerated atherogenesis remain unclear. While the pro-inflammatory role of signal transducer and activator of transcription 4 (STAT4) in atherosclerosis and diet-induced insulin resistance (IR) was recently established, an impact of STAT4 on atherogenesis in conditions of IR is not known. To study the role of STAT4 in IR-accelerated atherosclerosis we generated Stat4−/−Ldlr−/− mice that were fed a diabetogenic diet with added cholesterol (DDC). DDC fed Stat4−/− Ldlr−/−mice demonstrated improved glucose tolerance, insulin sensitivity, and a 36% reduction in atherosclerosis compared with Ldlr−/−controls. Interestingly, we detected a reduction in T follicular helper (Tfh) and plasma B cells, but a sharp elevation in CD8+ Tregs in spleens and aortas of Stat4−/−Ldlr−/−versus Ldlr−/−mice. Similarly, STAT4 deficiency supported CD8+ Treg differentiation in vitro. Additionally, Stat4-deficient CD8+Tregs suppressed Tfh and germinal center B cell development upon immunization with KLH indicating an important role for STAT4 in CD8+ Treg functions in vivo. Effects of STAT4 deficiency were not only restricted to T cells, but had a significant impact on macrophage phenotype. Stat4−/−Ldlr−/− macrophages displayed decreased IFNγ and MHC-II expression. Conditioned media from Stat4−/−Ldlr−/−MFs supported CD8+ Treg but not Tfh cell differentiation in TGFβ-dependent manner, suggesting a role for MF-specific STAT4 in Th cell differentiation. These new findings suggest a novel mechanism by which STAT4 supports atherosclerosis in obese, IR Ldlr−/− mice via increases in MF activation, and modulation of the Tfh/ CD8+ Treg axis systemically and within the aorta.
Comparative analysis of stress on faculty and students based on salivary IgA
Authors: Redmond Sarah , Biggs Erick , Blevins Kasey , Brimer Kelsey , Brown Cameron , Bryan Sandra , Burger Haley , George Anna , Hunt Mckenzie , Jannise Caitlin , Kesterson Melissa , Kwan Anthony , Marshall Jacob , Miller Kenzie , Mohamed Attia , Rajashekara Arpitha Mysore , Pagan Rachael , Peters Mary , Saenz Allison , Steeves Gregory , Stowers Samuel , Welch Nate , Williams Alexis , Williams Devonn , Wingfield Kyle , Zimmermann Krista
Published: January 2023

The goal of this project is to test students and faculties stress levels in their saliva compared between the beginning of this semester and the end of the semester. IgA levels are influenced by many physiological factors, including the hormones associated with stress. The findings conclude that there is significance between IgA levels in a student or faculty based on hours since they had left class and hours until they have class. Students and faculty showed a correlation between IgA and time since being in class or work (p-value = 0.03), however no significance of the duration of the expected rest period (p-value = 0.71). The significance of this finding is that students and faculty tend to be more stressed after being in class/work based on IgA levels in their saliva.
The expressions of 164 histone modification enzymes in seven groups are modulated in respond to pathogenic processes of metabolic diseases, tumors and regulatory T cell differentiation
Authors: SHAO YING , Chernaya Valeria , Wang Hong , Yang Xiao-Feng
Published: January 2023

To determine whether the expression of histone modification enzymes is regulated in physiological and pathological conditions, we took an experimental database mining approach pioneered in our labs to determine a panoramic expression profile of 164 enzymes in 19 human and 17 murine tissues. We have made the following significant findings: 1) Histone enzymes are differentially expressed in cardiovascular, immune and other tissues; 2) Our new “pyramid model” showed that heart and T cells are among a few tissues/cell types, in which enzymes mediating histone acetylation/deacetylation, histone methylation/demethylation are in the highest varieties; and 3) Histone enzymes are more downregulated than upregulated in metabolic diseases, and regulatory T cell polarization/ differentiation, but not in tumors. These results have demonstrated that histone modification enzyme expressions are modulated in different pathophysiological conditions as well as in the processes of T cell subset conversion, differentiation and polarization, which makes upregulated histone enzymes being potential novel therapeutic targets in suppressing disease activities and Treg functions.
Cytotoxic CD8+ interleukin (IL)-17+ T-cells play a role in hypertension-specific inflammatory responses associated with ovariectomy in Dahl rats
Authors: Pai Amrita V , West Crystal A , Souza Aline , Ji Hong , Wu Xie , Sandberg Kathryn
Published: January 2023

Background and Objective

Oophorectomy is associated with increased body weight (BW) gain and incidence of hypertension (HT) compared with age-matched women. Increased BW gain and HT coincide with increased inflammatory biomarkers. To separate immune responses due to HT from those due to BW gain, we compared T-cell subpopulations in normotensive Dahl (DR) and hypertensive Dahl (DS) rats after ovariectomy.

Methods

Six week old DS and DR rats were ovariectomized (Ovx) or had sham surgery (Sham). Mean arterial pressure (MAP) was measured in anesthetized rats after 4 and 10 months (mo) acutely by indwelling vascular catheter. Splenic T-cells were assessed by flow cytometry at 10 mo.

Results

Ovariectomy increased the MAP in DS but not DR rats at 4 mo [MAP (mmHg): DS-Ovx, 143±1.0 vs DS-Sham, 129±2.9; p=0.0002 & DR-Ovx, 123±4.4 vs DR-Sham, 126±1.3; ns n=10–12/group]. At 10 mo, the MAP remained higher in the ovariectomized DS compared to DR rats [MAP (mmHg): DS-Ovx, 190±12 vs DR-Ovx, 114±1.2; n=5–8/group; p=0.002]. At 10 mo, ovariectomy increased BW to a similar extent in DS and DR rats [BW gain (g): DS, 179±13 vs DR, 202±14; ns]. The frequency of CD8+IL17a+ T-cells increased in the ovariectomized DS but not DR rats [Frequency (%) at 10 mo: DS-Ovx, 9.6±2 vs DR-Ovx, 1.3±0.4; n=5–8/group; p<0.0001].

Conclusion

Cytotoxic CD8+ IL-17a+ T-cells may play a role in the HT-specific inflammatory response in ovariectomized DS rats.
T Lymphocyte Immunophenotyping: 14-Color Flow Cytometry Panel Design Using the Attune NxT Flow Cytometer and Super Bright Fluorescent Dyes
Authors: Bradford Jolene A , Zhang Elizabeth Yan , Smith Wesley , Punj Manik , Buller Gayle , Hoenes Candice , Sardina Patricia , Melquist Penny , Oram Shane
Published: January 2023

The Invitrogen™ eBioscience™ Super Bright polymer dyes represent a suite of bright fluorophores excited by the violet laser (405 nm). Optimized for use in flow cytometry, the Super Bright dyes allow for expanded use of violet laser excitation and promote streamlined multicolor panel design. Due to their inherent brightness, detection of cell populations with low abundance targets is possible. A 14-color panel used to characterize human T-cell lymphocytes is presented, with information on differentiation profiles, activation or exhaustion status, and co-stimulation activity. Both cryopreserved human peripheral blood mononuclear cells (PBMC) and fresh blood collected from a normal donor was used. Acquisition and analysis was performed using the Attune™ NxT Flow Cytometer four laser system, which has the ability to detect multiple violet-excited fluorophores. The results show well characterized cell populations, with expected changes between unstimulated and stimulated PBMC ex vivo. Correction for spectral overlap was performed using standard instrument auto-compensation procedures. The unique acoustic focusing technology of Attune NxT Flow Cytometer enables rapid and accurate detection with sensitivity at high flow rates without compromise of data quality, as demonstrated with this 14-color data set. The Attune NxT cytometer is designed to accommodate a wide variety of experimental conditions and combined with the Super Bright dyes provides expanded choice for panel design. For research use only. Not for use in diagnostic procedures.
T Cell Activation and the Role of Ubiquitin Ligases
Authors: Lu Xiaolong , Singh Rajesh , Song Changcheng , Mason Mark
Published: January 2023

Protein ubiquitylation has emerged as a key regulatory mechanism in innate and adaptive immune systems. E3 Ligases such as Cbl play fundamental roles in immune regulation. These enzymes are expressed in all leukocytes and are key checkpoints in maintaining the balance between activating and inhibitory signaling to immune cells, including T cells, NK cells, B cells and different types of myeloid cells. Thus, targeting ubiquitin ligases is a novel strategy to treat human diseases such as cancer and autoimmunity. However, the detailed mechanisms and functions of ubiquitin ligases in human cell is still largely unclear. Several ubiquitylation substrates of Cbl have been identifed in immune cells, e.g., Tyro3, Zap70, p85 and LAT. Therapeutic modulation of ligases is expected to alter the ubiquitylation of their substrate proteins, and documentation of such changes is critical to establishing the mechanism of action of therapeutic agents. Current methods to study poly-ubiquitin on substrates are cumbersome and inefficient. Here we describe a powerful technology, UbiQuant S, that allows selective and quantitative analysis of poly-ubiquitylated proteins in a cellular, high throughput format. We are able to track the changes in ubiquitylation status of Cbl targets upon T-cell activation, which provides valuable information on how Cbl and other ligases regulate T cell signaling elements. This technology also provides a powerful cellular platform for validating small molecule cancer immunotherapy agents and other checkpoint inhibitors.
Vaccinia virus preferentially infects primary human memory B cells
Authors: Shepherd Nicole , Lan Jie , Li Wei , Yu Qigui
Published: January 2023

Vaccinia virus (VACV) has shown an extreme preference for binding to and infection of primary human antigen-presenting cells including monocytes, macrophages, and dendritic cells in the peripheral blood. Although VACV infection in monocytes, macrophages, and dendritic cells has been examined, few studies have evaluated VACV infection in B cells. In this study, we characterized VACV binding to and infection of primary human B cells. Flow cytometric analysis revealed that B cells isolated from human peripheral blood mononuclear cells exhibited two distinct populations of B cells, referred to as Bsmall and Blarge. Phenotypic analysis demonstrated that the Bsmall compartment was more quiescent compared to the Blarge population and enriched with CD138-positive plasma cells. VACV preferentially entered into and infected memory B cells. Especially, VACV preferentially infected ex vivo CXCR5+ B cells, particularly CXCR5+HLA-DRhigh B cells. Assessment of VACV gene expression by RT-PCR in total B cells to evaluate permissiveness of VACV infection in B cells revealed early and intermediate viral gene expression, but little to no late viral gene expression, suggesting that VACV infection was aborted at the late viral gene expression stage in unstimulated B cells. Additionally, preliminary data demonstrated enhanced VACV binding to and infection of B cells stimulated by B cell receptor crosslinking and TLR-9 agonist treatment. With the risk of smallpox or monkeypox being used as bioterrorism agents and the multitude of adverse reactions to the current vaccine, understanding VACV infection in B cells is important for developing a better smallpox vaccine.
Suppression of IFN-β production by Epstein-Barr virus lytic transactivator Zta
Authors: Wong Nok Hei Mickey , Yuen Kit-San , Chan Chi-Ping , Jin Dong-Yan
Published: January 2023

Interferon β (IFN-β) production is an innate antiviral immune response against virus infection and lytic virus production. Several molecular mechanisms, including the sensing of cytosolic double-stranded DNA (dsDNA) by cyclic GMP-AMP synthase (cGAS), have been described to induce IFN-β production. Epstein-Barr virus (EBV) is a γ-herpesvirus that infects most of the population in the world and is associated with the development of various lymphoid and epithelial malignancies in a subset of people. In this study, we identified and characterized EBV lytic transactivator Zta as a potent suppressor of IFN-β production. EBV lytic infection was shown to suppress IFN-β production. To identify the viral proteins that mediate this suppression, a functional screen was performed using a dual-luciferase reporter system and an expression library of all EBV proteins. In this screen EBV lytic transactivator Zta was found to be capable of potently suppressing IFN-β production induced by different stimuli of interferon regulatory factor 3 (IRF3) activation. Zta is previously known to directly interact with and suppress IRF7, but its impact on IRF3 and the mode of action have not been demonstrated. We showed that Zta prevented the activation of IRF3 and IFN-β promoter by cGAS and STING (stimulator of interferon genes). Mechanistically, Zta suppressed the phosphorylation of IRF3 and its upstream interacting partner TANK-binding kinase 1 (TBK1), leading to the inhibition of IRF3 activity and nuclear translocation. Therefore, Zta may help EBV to evade host immunity during lytic infection by circumventing IRF3 activation and thus IFN-β production. Our findings revealed a new mechanism for EBV evasion of innate immunity.
Roles of Reactive Oxygen Species in Modulation of Thymocyte Subpopulations during Thymic Atrophy
Authors: Majumdar Shamik , Raghavan Abinaya , Nandi Dipankar
Published: January 2023

The thymus is the site of T cell development, selection and maturation. Precursor cells migrate from the bone marrow to the thymus, where they undergo sequential development followed by selection to give rise to mature and functional T cells. The cell surface expressions of various markers define distinct stages of development and maturation of thymocytes. With the help of these markers and multicolour flow cytometry, we have standardized staining procedures to study different thymocyte subsets during atrophy. We have also standardized an acute and lethal model of oral Salmonella Typhimurium infection in BALB/c mice, wherein drastic reduction of thymic cellularity is observed. Using multicolour flow cytometry, we investigated the changes occurring in thymocyte subsets in this model of infection-induced thymic atrophy. In addition, thymic atrophy was also induced by administration of LPS, the chemotherapeutic drug etoposide and the synthetic glucocorticoid, dexamethasone. Extensive thymic atrophy occurred across the different groups and thymocyte subsets were studied in a comprehensive manner. CD4 and CD8 profile on total thymocytes revealed CD4+CD8+ cells to be the most sensitive across all the modes of thymic atrophy studied. Subsequent detailed analysis of the thymic subpopulations revealed distinct changes during thymic atrophy. The roles of reactive oxygen species (ROS) in modulation of thymocyte subsets in the above mentioned conditions were studied using the ROS quencher, N-acetyl cysteine. We further intend to understand the mechanisms involved in changes occurring in the thymocyte subsets that may aid in development of candidate therapeutic molecules with thymopoietic potential.
Epigenetic regulator CXXC5 recruits DNA demethylase Tet2 to regulate TLR7/9-elicited IFN response in pDCs
Authors: Xiao Hui , Ma Shixin
Published: January 2023

TLR7/9 signals are capable of mounting massive IFN response in pDCs immediately after viral infection, yet the involvement of epigenetic regulation in this process has not been documented. Through immune gene profiling, we found that the zinc finger CXXC family epigenetic regulator CXXC5 is specifically expressed in pDCs, but not in cDCs or macrophages. Interestingly, CXXC5-deficient pDCs exhibited impaired IFN-I response to TLR7/9 ligands and RNA/DNA viruses. Furthermore, we uncovered that CXXC5-deficiency resulted in aberrant methylation of CpG DNA islands (CGIs) of a subset of genes including Irf7 in the steady-state pDCs, leading to diminished expression of this group of genes in pDCs. Mechanistically, CXXC5 seemed to be responsible for the recruitment of DNA demethylase Tet2 to maintain the hypomethylation of these CGIs in pDCs, a process coincided with histone remodeling and active transcription in these regions. Upon challenged by viruses, CXXC5-deficient mice were unable to launch robust IFN-I response at the early stage, and became highly susceptible to HSV and VSV infection. Together, our results not only identified CXXC5 as a novel regulator of TLR7/9-induced IFN-I response in host defense, but also provided new insight into the understanding of epigenetic regulation of pDC function.
Identification of a conserved regulatory network underlying the differentiation of immunosuppressive human macrophage
Authors: Bayik Defne , Tross Debra , Klinman Dennis M
Published: January 2023

There is considerable interest in generating human macrophage capable of down-modulating over-exuberant immune responses that characterize inflammatory and autoimmune diseases. Historically referred to as ‘M2-like’, macrophage with suppressive activity are conventionally generated by treating human monocytes with macrophage colony-stimulating factor (M-CSF). Unfortunately, the pleiotropic effects of M-CSF prevent it from being used therapeutically. This work identifies the TLR2/1 agonist Pam3CSK4 (hereafter PAM3) as being unique among TLR agonists in replicating the ability of M-CSF to induce monocytes to differentiate into immunosuppressive macrophage. The macrophage generated by PAM3 or M-CSF suppressed the proliferation of activated T cells and endocytosed particles with high efficiency. Consistent with their functional activity, these macrophage up-regulated expression of the surface markers DC-SIGN, SR-A and PD-L2, and secreted the anti-inflammatory mediators IL-1RA and IL-10. To clarify the pathway(s) by which macrophage with these properties arise, the regulatory networks activated by PAM3 and M-CSF were examined. NF-κB and Akt were central to the general process by which monocytes differentiate into macrophage, as their blockade prevented such maturation. By comparison, the Erk1/2 and p38 MAPK pathways were specifically involved in the differentiation of monocytes into macrophage with immunosuppressive activity. These findings identify targetable pathways to modulate the generation of immunosuppressive macrophage and demonstrate the potential utility of PAM3 for the treatment of chronic inflammation and autoimmunity.
Modulation of autophagy via lentivirus-derived shAtg5 ameliorates lupus-like disease in mice
Authors: Liu Chi-Jui , Sun Kuang-Hui
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of anti-nuclear antibodies. Traditionally, immunosuppressive and cytotoxic agents were widely used to treat lupus. However, the side effect and toxicities are inevitable. Autophagy is a process which can degrade the aged organelles and unfolded proteins to maintain the cell homeostasis. Previous studies have shown that autophagy levels were positively correlated with disease severity and elevated in B and T cells of lupus-prone mice and lupus patients. Furthermore, autophagy is essential for plasma cell differentiation, survival and antibody production. These results implicate that autophagy may promote lupus progression through regulating the survival of autoreactive immune cells. Therefore, we aim to modulate the autophagy of lupus-prone mice by lentivirus-derived shRNA targeting Atg5, an essential protein for autophagy. First, we found that levels of autophagy in splenocytes and lymphocytes of peripheral blood (PB) were elevated and positively correlated with disease severity in lupus-prone mice. The shAtg5-lentivirus, which can effectively inhibit autophagy in vitro, were then i.p. injected into TREM-1−/−. lpr mice. Mice treated with shAtg5-lentivirus exhibited lower levels of proteinuria, serum anti-dsDNA antibody and B-cell activating factor (BAFF). Lymphadenopathy as well as autophagy levels in lymph node cells and PB lymphocytes were decreased following Atg5 suppression. We also found numbers of plasma cells, CD4−CD8−, and CD4+T cells were decreased in mice treated with shAtg5-lentivirus. Thus, inhibition of autophagy may suppress immune cell expansion in lupus-prone mice and provide a novel target therapy for SLE.
Apoptotic cell driven ROS burst drives AhR dependent immunologic tolerance and suppression of lupus
Authors: Shinde Rahul Suresh , Hezaveh Kebria , Utsch Lara , Lamorte Sara , Ravishankar Buvana , Liu Haiyun , Chaudhary Kapil , Medina Tiago , Kloetgen Andreas , Halaby Marie Jo , Madaio Michael , Wither Joan , Tsirigos Aristotelis , De Carvalho Daniel , Munn David , McGaha Tracy
Published: January 2023

Tissue-resident macrophages (MΦ) are crucial in driving tolerance and preventing systemic autoimmunity. We have previously shown that exposure to apoptotic cells triggers a regulatory circuit dependent on IL-10 production in resident MΦ. However, key molecular mechanisms driving the regulatory response to apoptosis are not clear. RNA transcriptome analysis of MΦs after exposure to apoptotic cells identified strong transcript association with the aryl hydrocarbon receptor (AhR) signaling pathway, an association that was confirmed by phenotypic and biochemical analysis. When AhR activity was blocked, apoptotic cells induced an alteration in the mRNA signature enhancing proinflammatory effector expression. Functional analysis revealed that the DNA from apoptotic cells activated AhR in a reactive oxygen species (ROS) dependent mechanism and AhR is required for IL-10 production. Consequently, inhibition or deletion of AhR signals fundamentally altered immune responses to apoptotic cells in vivo resulting in proinflammatory cytokine production, increased effector T cell responses, and failure of long-term tolerance to apoptotic cell-associated antigens. Surprisingly, mice lacking AhR developed progressive systemic autoimmunity characterized by excessive MΦ and lymphocyte activation and renal pathology. Similarly, SLE-prone mice treated with AhR antagonist exhibited poor survival, while agonist treatment ablated disease pathology. Finally, an AhR transcriptional signature was significantly associated with active SLE flare in SLE patients. Thus, the data demonstrates the AhR pathway is a key molecular circuit responsible for apoptotic cell driven tolerance and suppression of inflammatory autoimmunity.
Kallikrein-kinin system and Type I Interferons in Neurolupus
Authors: Sriram Uma , Zuluaga-Ramirez Viviana , Merkel Steven Franklin , Winfield Malika , Fernandes Nicole C , Gajghate Sachin , Gallucci Stefania , Persidsky Yuri
Published: January 2023

Neuropsychiatric lupus is one of the most common manifestations of human systemic lupus erythematosus (SLE), causing depression in many. SLE is an autoimmune disease characterized by multi-organ damage including the brain and Interferon-α (IFN-α) is a central mediator in disease pathogenesis. Excessive production/response to Type I IFNs in SLE (called the “IFN signature”) is a hallmark of the disease. The mechanism of how IFN causes depression remain poorly understood; however, it is known that administration of IFN-α in certain chronic viral infections and some cancers causes the development of depressive symptoms in a high percentage of patients. The Kallikrein-Kinin System (KKS) that is comprised of kallikreins (klks), bradykinins (bk), angiotensin converting enzyme (ACE), and many other molecules, has classically known to be involved in a variety of physiological processes, including coagulation, angiogenesis and control of blood pressure. The KKS has been explored recently for their regulation of brain functions. We hypothesize that IFN-α may cause some of the symptoms in neurolupus patients and the KKS can ameliorate these effects. In this study, we used the MRL/lpr lupus-prone mouse model and showed that exposing MRL mice to IFN-α (a) increased depressive-like behavior, (b) decreased klk expression, and (c) enhanced ACE expression in the brain. Administering captopril (a commonly prescribed ACE inhibitor) decreased IFN-induced gene expression in the brain. It is also known that ACE activity decreases BK levels. The role of key KKS effectors and their interactions with the IFN pathway may provide a rationale for therapeutic use of KKS molecules for treatment of neurolupus.