bluuebunny/crossref_metadata_2025 huggingface dataset:
Loss of IL-4Rα signaling is partially able to restore anti-Mycoplasma pneumoniae IgG titers during allergic airway disease
Authors: Totten Arthur H , Xiao Li , Luo Danlin , Crabb Donna M , Ratliff Amy E , Waites Ken B , Atkinson T. Prescott
Published: January 2023

Rationale

Mycoplasma pneumoniae (Mpn) is the leading bacterial agent of pediatric community acquired pneumonia. Epidemiologic evidence has suggested increased susceptibility to Mpn infection among asthmatics. We hypothesized that allergic airway sensitization would impair host immune responses to Mpn infection, but that loss of Th2 cytokine signals (IL-4, IL-13) would overcome this impairment in a murine model.

Methods

Wild type (Wt) and IL-4Rα−/− BALB/cJ mice were sensitized and challenged with ovalbumin (OVA) to induce allergic airway inflammation, and then infected with Mpn. Immune parameters were studied by analysis of cellular profiles in bronchoalveolar lavage fluid (BALF), serum IgG and IgE antibody levels to a whole bacterial antigen preparation, recombinant Mpn P1 adhesin (rP1), and OVA. Total lung RNA was used to examine cytokine transcript levels for Th1, Th2 and Th17 biasing transcripts.

Results

Anti-Mpn and P1-specific total IgG responses were decreased in allergen-sensitized animals compared to unsensitized controls (p < 0.01). Decreased titers of IgG1, IgG2a, IgG2b and IgG3 were present in rP1-specific, but not OVA-specific IgG subclass levels. Loss of IL-4Rα signaling partially restores total and subclass-specific responses. Th2 cytokine mRNA levels were increased in Wt, but not IL-4Rα−/− OVA sensitized groups compared to controls. IL-4Rα−/− animals had increased Interferon-γ(IFN-γ) and IL-17A mRNA levels compared to Wt animals.

Conclusions

Anti-Mpn IgG antibody titers were decreased in Wt allergic animals compared to unsensitized controls and the impairment in immune response to the pathogen was partially restored by inhibition of Th2 signaling in IL-4Rα−/− animals.
The distinct role of Tfh and Th17 cells and their interplay in the pathogenesis of rheumatoid arthritis
Authors: Zhang Xin , Migliore Frank , Budziakowska Magdalena , Quresh Quretul , Zakem Jerald , Webb-Detiege Tamika , Davis Willam , You Zongbing , Quinet Robert
Published: January 2023

Objectives

Rheumatoid Arthritis (RA) is an autoimmune disease characterized by the presence of autoantibodies and chronic inflammation in synovium leading to joint destruction. Recently Tfh and Th17 cells have emerged as novel T cell subsets controlling autoimmunity. Our previous data showed that circulating Tfh (cTfh) and circulating Th17 (cTh17) cells were increased in RA patients. Here, we further investigated their role and interplay in the pathogenesis in RA.

Design and Methods

Peripheral blood was collected from 61 RA patients and 61 healthy donors. RA patients were divided into remission (<2.6) and active groups (>2.6) based on disease activity score. Clinic parameters including RF, anti-CCP, ESR and CRP were obtained. The frequencies of cTfh and cTh17 cells were measured by flow cytometry. Serum IL-21 and IL-17 were detected by ELISA. Correlations of the frequency of cTfh/IL-21 with cTh17/IL-17 and clinic parameters were statistically determined.

Results

The increased frequency of cTfh cells significantly correlated with the level of anti-CCP antibody (p<0.01) in active RA patients, whereas increased frequency of cTh17 cells correlated with CRP level (p<0.05). Furthermore, the level of cTfh cells and serum IL-21 level were significantly correlated with the percentage of cTh17 cells in RA patients (P<0.05). The serum IL-17 level only closely correlated with CRP in RA patients.

Conclusion

Tfh cells may be involved in RA pathogenesis by inducing generation of autoantibody producing plasma cells, while cTh17 cells largely related to inflammation. Tfh cells and its cytokine IL-21 may also steer T cell differentiation into Th17 cells which secrete the pro-inflammatory cytokine IL-17 and contribute to inflammation in RA.
Role of SIRT1 in C5b-9 mediated oligodendrocyte cell cycle activation
Authors: Tatomir Alexandru , Rao Gautam , Boodhoo Dallas , Ciriello Jonathan , Rus Violeta , Rus Horea
Published: January 2023

SIRT1 is a NAD-dependent histone deacetylase involved in the regulation of transcription, apoptosis, metabolism and differentiation. We have previously demonstrated that sublytic levels of complement C5b-9 terminal complex increased the survival of oligodendrocytes (OLGs) and induced their dedifferentiation. In this study we investigated the role of SIRT1 in OLGs differentiation and the effect of sublytic levels of C5b-9 on SIRT1 expression. We also investigated the downstream effects of SIRT1 by measuring histone H3 Lysine 9 trimethylation (H3K9me3) and expression of cyclin D1. OLG progenitor cells purified from the brain of rat pups were differentiated in vitro and stimulated with sublytic C5b-9 or C5b6 for 3, 6 and 18 h. The level of SIRT1 mRNA was measured using real-time PCR and SIRT1, cyclin D1 and H3K9me3 protein expression were measured using western blotting. We found a decreased expression of SIRT1 mRNA and protein, a decreased level of H3K9me3 and an increased expression of cyclin D1 during OLG differentiation. Stimulation of OLGs with sublytic C5b-9 for 3h resulted in a significant decrease in SIRT1 mRNA and protein levels while stimulation with C5b6 had no effect. SIRT1 protein level in OLGs after 8h of exposure to C5b-9 were significantly lower than in C5b6 treated cells. H3K9me3 levels also decreased significantly after stimulation with C5b-9 as compared with unstimulated or C5b6-treated OLGs. Cyclin D1 expression increased after stimulation with C5b-9, indicating cell cycle activation. Our data show that C5b-9 stimulation of OLGs reduces SIRT1 expression, contributing to cell cycle activation by decreasing repressive trimethylation of histone H3 lysine 9.
Expression of the TNF-family cytokine TL1A (TNFSF15) by renal CX3CR1+ resident mononuclear phagocytic cells and role of TL1A in autoimmune nephritis
Authors: Richoz Nathan , Farley Taylor K. , Chalmers Samantha A. , Putterman Chaim , Meylan Francoise , Clatworthy Menna R. , Siegel Richard M.
Published: January 2023

Systemic lupus erythematosus is a polygenic autoimmune disease with a broad spectrum of clinical manifestations including organ-threatening kidney inflammation associated with immune complex (IC) deposition. The kidney is populated by a rich network of tissue-resident mononuclear phagocytes that express Fc receptors (FcRs) and internalize ICs. TL1A (TNFSF15) is a tumor necrosis factor (TNF) superfamily cytokine that can be induced in myeloid cells following ligation of TLR’s and FcRs by IgG IC’s and co-stimulates T cells through its receptor DR3. We have found that DR3 deficiency protects MRL/lpr lupus-prone mice from the renal inflammation but not systemic autoimmunity that develops in these mice, suggesting that TL1A/DR3 interactions could play a role in activating autoimmune T cells and antigen presentation in the kidney. We identified four distinct populations of renal phagocytes based on the expression of F4/80 and CD11b. One population of cells constitutively expressed high levels of surface class II and the CD28 ligand B7-2(CD86), suggesting that they would be potent antigen presenting cells. A fifth population expressing similar markers was present only in inflamed kidneys, both in the MRL/lpr mouse model of lupus and nephrotoxic serum-induced nephritis. Both of these populations constitutively expressed surface TL1A. The new population increases with the severity of nephritis and is unequally distributed between the cortex and the medulla. We will further analyze these populations via transcriptomics and functional assays after immune complex stimulation. This work identifies CX3CR1+ MNP’s as possible antigen-presenting cells in the kidney and a source of TL1A for costimulation of autoreactive T cells in nephritis.
CXCL17 is a stomach-expressed chemokine and plays a role in diet-induced obesity
Authors: Hernandez Marcela , Herrera Carolina , Nguyen Hong-Tam , Zlotnik Albert
Published: January 2023

CXCL17 is a mucosal chemokine that attracts dendritic cells and monocytes to specific tissues. CXCL17 is mainly expressed in the respiratory and gastrointestinal tracts and has been shown to have anti-inflammatory effects. Its recently described receptor, GPR35/CXCR8, is a GPCR that has been involved in metabolic functions. Given that macrophages are known to be involved in fat tissue homeostasis, we decided to study a possible role of the CXCL17/CXCR8 axis in obesity. To this end, we used a mouse model of diet-induced obesity to compare Cxcl17−/− mice and wild type (WT) C57BL/6 mice. Mice were fed either a 10% or 60% fat diet for four months. Their development and weight gain as well as several metabolism tests were monitored for two or four months. Plasma and fat tissue samples were used in adipokine assays and to measure levels of obesity-related cytokines and hormones. Gastrointestinal tract tissue, adipose and liver tissues were obtained to perform further RNA or protein assays.

The results indicate that Cxcl17−/− mice fed a 60% fat diet gained significantly more weight than WT mice. We also determined that under homeostatic conditions CXCL17 is highly expressed in the upper gastrointestinal tract (oral cavity, esophagus and stomach) in WT mice. Obese Cxcl17−/− mice also exhibit impaired response to glucose in an insulin tolerance test. Adipokine assay results revealed differences in FGF-21 levels and in other obesity-associated molecules between WT and Cxcl17−/− mice. We conclude that CXCL17 influences development of obesity and systemic metabolic dysfunction. Our results suggest that CXCL17 may be a therapeutic target to treat obesity-related conditions.
Structures of gasdermin-D C-terminal domain reveal mechanisms of autoinhibition
Authors: Liu Zhonghua , Wang Chuanping , Yang Jie , Rathkey Joseph , Abbott Derek W , Xiao Tsan Sam
Published: January 2023

Gasdermin-D (GSDMD) is a member of the gasdermin family, and is a critical mediator for host defense against microbial infection and danger signals. Inflammatory caspases (caspases-1, 4, 5, and 11) cleave GSDMD to generate an N-terminal cleavage fragment (GSDMD-N) that oligomerizes in cell membrane to form ring-like pores during pyroptosis, an informatory form of programmed cell death. In addition, the GSDMD membrane pores facilitate the release of inflammatory cytokines such as IL-1b and IL-18. However, how GSDMD-N is inhibited by the C-terminal domain of GSDMD (GSDMD-C) in the rest state is unknown. In addition, the mechanisms for the release of GSDMD-N from GSDMD-C upon caspase cleavage and the formation of membrane pores have not been established. Here we report the crystal structures of the C-terminal domains of human and murine GSDMD protein. Even though the overall structures of the two GSDMD-C structures are similar to that from mGSDMA3, two regions of the helical structures in GSDMD-C are significantly different from that in mGSDMA3. Overexpression of GSDMD-N induces lysis of the bacterial expression host, while concomitant expression of GSDMD-C reduces such bactericidal activities. Mutation of GSDMD-C residues involved in autoinhibition attenuates the intramolecular domain-domain interactions, as well as enhances pyroptosis in 293T cells transiently expressing GSDMD. Our study thus reveals mechanisms of autoinhibition of GSDMD, and suggests potential therapeutic application of GSDMD as a bactericidal agent.
Attenuation of Osteoclast Differentiation by the Knockdown of Nicotinamide PhosphoribosylTransferase
Authors: Islam M Shamima , Heruth Daniel P , Zhang Li Qin , Ye Shui Qing
Published: January 2023

Rheumatoid arthritis (RA) is characterized by chronic inflammation and progressive bone destruction. Nicotinamide phosphoribosyltransferase (Nampt) functions in NAD synthesis, inflammation and apoptosis. Our recent finding that Nampt+/− mice were protected against bone erosion in a Collagen Induced Arthritis mouse model led us to hypothesize that Nampt may play a critical role in osteoclast differentiation and knockdown of Nampt could attenuate osteoclast formation.

We studied the effects of decreased Nampt expression in osteoclastogenesis in both Nampt siRNA transfected RAW macrophages and bone marrow macrophages (BMM) from wild-type (Nampt+/+) and Nampt+/− mice. Number of differentiated osteoclasts were decreased from Nampt deficient RAW cells (86±2.8% vs 100%, N=3, p<0.001) and Nampt+/− BMMs (13.4±3 vs 17±4.8%, N=3, p=0.02 by paired T-Test) vs Nampt+/+ controls. Acp5 (0.1±0.035, N=4, p<0.0001), and Nuclear factor of activated T-cells (Nfatc1) mRNA were all decreased (0.29±0.08, N=4, p<0.0001) in Nampt deficient cells relative to the scrambled control. Luciferase reporter activity was decreased in RAW cells co-transfected with Nfatc1 luciferase reporter and Nampt siRNA relative to the scrambled control (0.33±0.18, N=3, p<0.005). ChIP analysis demonstrated that the epigenetic structure of the Nfatc1 promoter was altered in response to decreased Nampt with decreased histone acetylation.

In summary, our data suggest that knockdown of Nampt transcriptionally inhibited Nfatc1 expression via decreasing histone acetylation, resulting in an attenuated osteoclastogenesis. Nampt could be a potential new risk factor and therapeutic target for arthritis.
IFIT1 exerts opposing regulatory effects on the inflammatory and interferon gene programs in LPS-activated human macrophages
Authors: John Sinu P , Sun Jing , Carlson Rebecca , Cao Binh , Fraser Iain D
Published: January 2023

Activation of the TLR4 signaling pathway by LPS leads to activation of both inflammatory and interferon stimulated genes, however the mechanisms through which these coordinately activated transcriptional programs are balanced to promote an optimal innate immune response remains poorly understood. In a genome-wide siRNA screen of the LPS-induced TNF-a response in macrophages, we identified the interferon-stimulated protein IFIT1 as a negative regulator of the inflammatory gene program. Transcriptional profiling further identified an unexpected positive regulatory role for IFIT1 in type I interferon expression, implicating IFIT1 as a reciprocal modulator of LPS-induced inflammatory and interferon gene expression. We demonstrate that these effects of IFIT1 are mediated through modulation of a Sin3a-HDAC2 transcriptional regulatory complex at LPS-induced gene loci. Beyond the well-studied role of cytosolic IFIT1 in restricting viral replication, our data demonstrate an unappreciated function for nuclear IFIT1 in differential transcriptional regulation of separate branches of the LPS-induced gene program. IFIT1 depletion led to a significant increase in the replication of B. cenocepacia. Prior treatment of IFIT1 depleted cells with recombinant IFN-β reversed the enhanced replication of B. cenocepacia, indicating a role for autocrine IFN-β in controlling B. cenocepacia infection and a requirement for IFIT1 in interferon induction in response to bacterial infection. We also observed reduced expression of IFNβ1 in response to viral and bacterial infection in IFIT1 depleted cells, suggesting a broad role for IFIT1 in supporting IFNβ1 expression.
CD36 regulates malaria-induced innate immune responses
Authors: Puttalingaiah Ramesh Thylur , Wu Xianzhu , Gowda Nagaraj M , Punnath Kishore , Gowda Channe D
Published: January 2023

Malaria causes enormous morbidity and mortality worldwide. However, an effective vaccine is not yet available due to the knowledge gap in various molecular and cellular processes involved in protective immunity development. CD36, a multifunctional scavenger receptor, plays several important roles in malaria, including sequestration of parasites in host organs, parasitemia control, and protective immunity. Although the role of CD36 in the former two processes has been well understood, less is known about the function of CD36 in the latter. We studied the role of CD36 in host survival and immune responses in non-lethal Plasmodium yoelii infected WT and CD36−/− mice. WT mice showed lower parasitemia and higher survival rate compared to the CD36−/− mice. At early but not at later stages of infection, the circulatory pro-inflammatory cytokines were higher and anti-inflammatory cytokines were lower in WT mice than in CD36−/− mice. DCs, NK and T cells of WT mice had higher frequencies of pro-inflammatory and lower frequencies of anti-inflammatory cytokine-producing cells than CD36−/− mice. Additionally, Th1 response was decreased, whereas Th2 response was increased in CD36−/− mice. Compared to CD36−/− mice, macrophages and neutrophils of WT mice showed higher phagocytic activity and phagocytic receptors expression in an IFN-g dependent manner. However no difference in the malaria-induced humoral responses between WT and CD36−/− mice was observed. Our data demonstrated that CD36 plays an important role in parasitemia control and protective immunity by upregulating pro-inflammatory cytokine responses by DCs and NK cells, promoting Th1 response, and contributing to phagocytic receptor expression and phagocytic activity.
Recruitment of inflammatory monocytes during influenza infection predisposes host to secondary S. aureus infection
Authors: Sun Keer , Fischer Karl Jacob , Yajjala Vijaya Kumar , Bauer Christopher , Bansal Shruti
Published: January 2023

Staphylococcus aureus has emerged as a leading contributor to morbidity and mortality during recent influenza pandemics and epidemics. The mechanism for this influenza-induced susceptibility to secondary bacterial infection is incompletely understood. We have previously reported that innate antibacterial immunity is suppressed during the recovery stage of influenza infection. This impairment of bacterial control coincides with airway accumulation of inflammatory monocytes, and therefore, we investigated in this study a novel hypothesis of influenza-induced suppression of antibacterial immunity: recruitment of monocytes. C-C chemokine receptor type 2 (CCR2) is responsible for the recruitment of Ly6C+ monocytes into lungs after influenza infection. Here we demonstrate that CCR2−/− mice were more resistant than wild-type mice to influenza and S. aureus coinfection. CCR2 deficiency not only resulted in diminished monocyte recruitment but also reduced proinflammatory cytokine response to coinfection. Importantly, despite a delay in viral clearance, CCR2−/− mice exhibited significantly improved lung bacterial control, in agreement with their increased survivals from influenza and S. aureus coinfection. Furthermore, we show that CCR2-recruited monocytes served as reservoirs to protect bacteria from killing by antibiotics and more potent neutrophils. Collectively, our findings indicate that inflammatory monocytes constitute an important and hitherto underappreciated mechanism of the conflicting immune requirements for viral versus bacterial control by hosts, which subsequently leads to the worsen outcome of influenza and S. aureus coinfection.
Suppression of NFAT5-mediated inflammation and chronic arthritis by novel κB-binding inhibitors
Authors: Lee Naeun , Yoo Seung-Ah , Choi Susanna , Kim Yumi , Kim Wan-Uk
Published: January 2023

Nuclear factor of activated T cells 5 (NFAT5), also known as the tonicity-responsive enhancer-binding protein, is a transcription factor whose DNA binding domain shares structural homology with NF-kB and other members of the NFAT family. Evidence is emerging that NFAT5 is crucial to the pathogenesis of various human diseases, including cancer and arthritis. However, therapeutic agents specifically inhibiting NFAT5 activity are currently unavailable. To discover NFAT5 inhibitors, we screened a library of >40,000 chemicals for the suppression of nitric oxide, a NFAT5 target, through high-throughput screening. We validated anti-NFAT5 activity of the primary hit compounds using an NFAT5-specific reporter and identified the novel NFAT5 inhibitor KRN2, 13-(2-fluoro)-benzyl-berberine and its oral derivative, KRN5. KRN2 suppressed the transcriptional activation of NFAT5 by blocking NF-κB binding to the NFAT5 promoter region, inhibiting NFAT5 mRNA and protein expression in macrophages stimulated with lipopolysaccharide. Interestingly, KRN2 selectively inhibited the expression of pro-inflammatory genes, including Nos2, Tnf, and Il6 without hampering high-salt-induced NFAT5 and its target gene expressions. Moreover, KRN2 and KRN5 ameliorated experimentally induced arthritis in mice, decreasing pro-inflammatory cytokine production. Particularly, orally administered KRN5 was more potent in suppressing arthritis than methotrexate, a commonly used anti-rheumatic drug. Therefore, KRN2 and KRN5 can be potential therapeutic agents in the treatment of chronic arthritis.
Inducing Immunological Tolerance with Synthetic Nanoparticles
Authors: Maldonado Roberto A , LaMothe Robert , Ferrari Dan , Griset Aaron , O’Neil Conlin , Johnston Lloyd , von Andrian Ulrich H , Kishimoto Takashi Kei
Published: January 2023

Here we describe the use of synthetic, biodegradable tolerogenic nanoparticles carrying rapamycin, to induce durable and antigen-specific immune tolerance, even in the presence of potent Toll-like receptor (TLR) agonists. Treatment with these synthetic vaccine particles (SVP) and antigen (inside the particles or admixed) results in the inhibition of CD4+ and CD8+ T cell activation, differentiation of T and B regulatory cells, durable B cell tolerance resistant to multiple immunogenic challenges, the inhibition of antigen-specific hypersensitivity reactions, relapsing experimental autoimmune encephalomyelitis (R-EAE) and tolerance to biologic drugs such as the coagulation factor VIII (in hemophilia A animals), adalimumab (Humira in TNFa-transgenic animals), pegylated uricase (in uricase deficient animals), even in animals previously sensitized to antigen. Only encapsulated rapamycin, not the free form, could induce immunological tolerance. SVP therapy represents a potential novel approach for the treatment of treatment of allergies, autoimmune diseases, and prevention of anti-drug antibodies (ADA) against biologic therapies. Tolerogenic nanoparticle therapy for the prevention of ADAs against pegylated uricase in the treatment of hyperuricemia is currently being evaluated in Phase 2 clinical trials. Data from our Phase 1 clinical trial will be presented.
IRAK1 attenuates TLR signaling and activates the inflammasome in response to dual-TLR stimulation
Authors: Vayttaden Sharat Jacob , Smelkinson Margery , Ernst Orna Rabinovich , Carlson Rebecca Joy , Sun Jing , Gottschalk Rachel A , Oh Kyu Seon , Pegoraro Gianluca , Meier-Schellersheim Martin , Germain Ronald N , Varma Rajat , Fraser Iain D.C.
Published: January 2023

Macrophages encounter many kinds of pathogen-associated molecules and a coherent immune response involves crosstalk among various immune pathways. How can a cogent response form in the face of crosstalk between various pathways? We address this in the context of combinatorial stimulation of multiple TLRs to assess TLR pathway crosstalk.

We find that IRAK1 forms cytoplasmic clusters that sequester downstream signaling proteins upon dual TLR stimulation. This could explain the less-than additive gene response on dual TLR activation. Accordingly, IRAK1 deficient IMMs and BMDMs have higher phosphoprotein, cytokine and transcriptional responses to dual TLR ligands than WT cells, but their single TLR ligand responses are comparable to WT cells. This suggests that IRAK1-dependent sequestration of key TLR signaling proteins might be a mechanism of modulating the innate immune response to high pathogen load.

We also find that IRAK1 cluster-enriched cells exhibited higher ASC nucleation and increased IL1α/β secretion, which are hallmarks of an active inflammasome. The colocalization of ASC suggests a possible role for dual TLR-induced IRAK1 clustering in diversifying signaling to multiple innate immune signaling pathways.

We conclude that IRAK1 aids in tuning the TLR arm of signaling in response to multi-TLR ligands, allowing the engagement of other PRR pathways like the inflammasome. Thus, the rerouting of immune signals by IRAK1 clusters allows the cell to diversify the immune response to complex microbial signals through activation of multiple host response pathways, and improve the quality of the immune response during an infection.

This work was supported by the Intramural Research Program of NIAID, NIH.
Evaluation of Serum Cancer Antigens (CA15-3 and CA27.29) and Circulating Immune Complexes as Important Tools in the Management of Breast Cancer in Nigeria
Authors: Emele Felix Emele , Chukwurah Ejike Felix
Published: January 2023

Background

Increasing reports suggest a world-wide increase in the incidence of breast cancer. Breast cancer can be affected by racial/ethnic, environmental, and other variables. Serum bio-markers, such as cancer antigens (CA 15-3 and CA 27.29) and circulating immune complexes (CIC), are recognized as important tools in the evaluation and management of breast cancer, although conflicting reports and opinions exist regarding the clinical utility of these markers. In our locality, there is paucity of information in this regard, despite increasing incidence of the disease. We, therefore, saw the need for this study.

Methodology

A total of 42 treatment naïve females with clinically and pathologically confirmed breast tumour were recruited, along with control subjects. Patients with malignant tumours were further grouped into early stage and advanced stage. Serum from patients and controls were investigated for CA 15-3, CA 27.29, and CIC. The patients were subsequently subjected to standard treatment modalities. Follow-up investigations for the markers were carried out at intervals after treatment. Assays for serum tumour markers were by immunoenzymatic methods.

Results

Tumour of all forms recorded significantly higher pre-treatment mean values for CA 15-3 and CA 27.29, compared with the control group (P<0.05). Therapeutic interventions significantly reduced the values for CA 15-3 and CA 27.29 (P< 0.05) but had no consistent effect on the level of CIC.

Conclusion

Tumour markers CA 15-3 and CA 27.29 showed promising diagnostic and prognostic potentials, while CIC appears to be of limited utility in the management of breast cancer in our environment.
Quadruple Negative Breast Cancers in African American women have an enriched Basal and Immune signature.
Authors: Tripathi Shweta , Davis Melissa B. , Hughley Raymond , He Qinghua , Bae Sejong , Grizzle William E. , Karanam Balasubramanyam , Marini Rachel , Colomb Windy D. , Yates Clayton
Published: January 2023

There is increasing evidence that Androgen Receptor (AR) expression has prognostic usefulness in breast cancer suggesting that Triple negative breast cancer (TNBC) which lack AR expression are Quadruple Negative Breast Cancers (QNBC). However, a comprehensive analysis of AR expression within all breast cancer subtypes or stratified by race has not been reported. We assessed AR RNA expression in 925 tumors from The Cancer Genome Atlas, and 136 tumors in 2 confirmation sets. AR protein expression was determined by immunohistochemistry in 197 tumors from a multi-institutional cohort, for a total of 1258 patient analyzed. We used Cox hazard ratios to determine correlations to PAM50 breast cancer subtypes, and TNBC-QNBC subtypes. K-means clustering distincted immune-related gene expression signature in QNBC tumors. AA tumors express the AR at both the RNA and protein levels at lower rates than tumors of White women which is independent of ER and PR expression (p<0.0001). TNBC tumors from both racial groups demonstrate the lowest expression of AR. AR-negative tumors have odds ratio (66.60; 95% CI, 32–146) of being basal-like. QNBC tumors have odds ratio (4.01; 95% CI, 0.88–38.60) of being BL1 subtype. K-means clustering demonstrated that AA patients had a distinctive gene signature that could be subdivided into five signatures; AR pathway, T cell >response pathway, Cancer B cell response, stem cell pathway, and hormone receptors enriched pathways. These observations indicate that AR expression should be included with traditionally used markers and that QNBC should be used as a prognostic marker for breast cancer patients. Further, AR-negative tumors have a characteristic immune signature that could be exploited by targeted immune therapy.
The role of interferon lambda during influenza, Staphylococcus aureus super-infection.
Authors: Rich Helen , Robinson Keven M , McHugh Kevin J. , Clay Michelle E , Alcorn John F
Published: January 2023

While pandemic influenza A H1N1 infection causes significant morbidity and mortality on its own, it also renders an individual more susceptible to secondary infection with bacterial pathogens such as Staphylococcus aureus. We have previously shown that type I interferons produced by influenza impair bacterial defense six to seven days post-influenza infection. However, the role of the predominant type III interferons (interferon lambda/IFNL, IL-28A/B) in bacterial super-infection is less clear. Thus, we explored the role of IFNL in influenza, S. aureus super-infection. First, we examined the impact of attenuating IFNL signaling directly before bacterial challenge. We found that treatment with the IFNLR1 Fc protein significantly increased bacterial burden in the lung compared with controls. However, when we challenged mice with super-infection that have constitutively attenuated IFNL, we found reduced bacterial burden compared to controls, which was significantly correlated with the degree of IFNL expression. While cellular inflammation was not altered, preliminary results suggest that higher bacterial burden is significantly correlated with higher weight loss, suggesting that mice with constitutively attenuated IFNL are protected from both influenza infection and bacterial super-infection. It has been published that mice lacking IFNL receptor 1 have lower bacterial burden during super-infection, which is corroborated by data from our constitutively attenuated IFNL mice. However, attenuation of IFNL signaling directly prior to bacterial challenge increases bacterial burden, suggesting that the timing of IFNL production regulates susceptibility to secondary bacterial infection.
HIV-induced modifications of TIGIT expression impair CD8 T cell polyfunctionality
Authors: Scharf Lydia , Tauriainen Johanna , Frederiksen Juliet , Naji Ali , Ljunggren Hans-Gustaf , Sönnerborg Anders , Lund Ole , Reyes-Terán Gustavo , Hecht Frederick M , Deeks Steven G , Betts Michael R , Buggert Marcus , Karlsson Annika C
Published: January 2023

HIV-specific CD8 T cells display an accumulation of inhibitory receptors that are associated with poor effector functions. Expression of the inhibitory receptor, T cell immunoglobulin and ITIM domain (TIGIT), its co-stimulatory receptor CD226 and their ligand poliovirus receptor (PVR) are altered during chronic viral infections and cancer. Here we show that the TIGIT/CD226/PVR axis is dysregulated during HIV infection and linked to poor polyfunctional activity, increased expression of inhibitory receptors and an altered effector transcriptional programming. We report that TIGIT expression is increased on CD8 T cells in untreated HIV infection. Additionally, a longitudinal increase in TIGIT expression was demonstrated despite early initiation of antiretroviral therapy during acute HIV infection. The HIV-specific TIGIT+ CD8 T cells co-expressed PD-1, CD160 and 2B4 and had an inverse expression profile of the T-box transcription factors T-bet and Eomes. The HIV-specific CD8 T cells were almost exclusively TIGIThiCD226neg/dim and the frequency of TIGIThi cells was inversely correlated with polyfunctionality. Furthermore, the TIGIT/CD226 ligand PVR was increased on CD4 T cells, especially T follicular helper (Tfh) cells in HIV-infected lymph nodes. These results demonstrate a preferential skewing of the TIGIT/CD226 axis towards the inhibitory receptor TIGIT on CD8 T cells during HIV-1 infection, which is linked to severe T cell dysfunction. The findings highlight the importance of the TIGIT/CD226/PVR axis as an immune checkpoint barrier that potentially could hinder future therapeutic “cure” strategies that require potent HIV-specific CD8 T cells and/or the clearance of HIV-1 infected Tfh cells.
Harnessing innate anti-tumour immunity using a Klebsiella-derived therapeutic to reduce tumour burden and improve outcomes in mouse models of lung cancer
Authors: Bazett Mark , Costa Amanda L , Bosiljcic Momir , Alexander Matthew P , Anderson Rebecca M , Wong Stephanie WY , Gunn Hal , Kalyan Shirin , Mullins David W
Published: January 2023

Tumour regression and increased survival has been associated with certain acute microbial infections. Immune dysfunction contributes to the development and progression of lung cancer, and therapies that re-constitute anti-tumor immune responses provide an important means to effectively treat malignancies and improve health outcomes. We hypothesized that stimulating the innate immune system with bacterial-derived immunomodulators could induce protective anti-cancer immune responses. A Klebsiella-derived drug product, QBKPN (Qu Biologics), was used to specifically stimulate the innate immune niche in the lungs in established mouse models of lung cancer. Repeated subcutaneous administration with QBKPN significantly reduced lung tumor burden and increased survival. The protective action of QBKPN required prior exposure to Klebsiella through either environmental exposure or lung infection. However, this QBKPN-mediated anti-tumour response was independent of adaptive immunity, as the protective effect remained in RAG2-knockout mice. QBKPN intervention was characterized by a rapid, acute-like systemic immune response, including increased circulatory inflammatory cytokines and innate immune cells, leading to recruitment of immune effector cells into the lung tissue, including macrophages and natural killer (NK) cells. In addition to recruitment of innate immune cells, QBKPN increased markers of classically activated macrophages and increased production of NK cell effector molecules. Together, these data suggest that QBKPN, a Klebsiella-derived immunomodulator, causes activation and recruitment of macrophages and NK cells into the lungs, reducing cancer tumour burden and improving survival outcomes.
Improving immunotherapeutic efficacy against metastatic tumors in a pre-clinical model of murine renal cell carcinoma.
Authors: Boi Shannon Kathryn , Orlandella Rachael M , Norian Lyse A
Published: January 2023

Renal and pelvic cancers are the ninth most common cancer in the U.S., and although five-year survival rates for localized renal tumors are >90%, those for metastasized tumors are <12%. Metastatic renal cell carcinoma (RCC), the most common type of renal neoplasm, is resistant to radio- and chemotherapies. Immune-based therapies, including checkpoint blockade, are being pursued in an attempt to amplify protective adaptive immunity but overall response rates remain at 20–30%. We hypothesized that a therapeutic strategy employing T cell priming plus checkpoint blockade would result in improved clearance of renal tumors verses checkpoint alone. Utilizing our previously described in vivo T cell priming therapy, adenovirus-encoded tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (AdTRAIL) with class B oligonucleotides (CpG), we evaluated therapeutic effects on primary tumor outgrowth and metastatic burden in a pre-clinical model of orthotopic murine RCC. We report here that administration of intra-renal AdTRAIL/CpG in combination with α-CTLA-4, but not α-PD-1, causes significant decreases in primary tumor burdens. Strikingly, we observed that this therapeutic strategy caused significant reductions in metastatic lung burdens – equating to a 98% reduction over no therapy controls. Flow cytometric analysis revealed increases in CD8+ T cells in AdTRAIL-treated groups over no therapy/checkpoint alone; results were further supported by immunosuppressive CD4+ to CD8+ T cell ratios that were in favor of anti-tumoral responses. Our results suggest AdTRAIL/CpG can be used as an effective upstream therapeutic approach to enhance the efficacy of α-CTLA-4 checkpoint blockade in a pre-clinical model of murine RCC.
Inhibition of activation-induced autophagy induces hyporesponsiveness in effector T helper cells
Authors: Dowling Samuel Dae , Mocholi Enric , Gruber Ross , Ray Alex , Shafit-Zagardo Bridget , Macian Fernando
Published: January 2023

T cell activation requires the regulation of signaling pathways and metabolic programs downstream of T cell receptor and CD28 engagement. Upon activation, effector CD4+ T helper cells induce autophagy, a lysosomal-mediated protein degradation mechanism. The controlled degradation and recycling of cell components allow effector T helper cells to respond to various types of stimuli. However, it is unknown how autophagy may regulate the antigenic responses and functional capacity of CD4+T cells. We found that inhibiting activation-induced autophagy in CD4+T helper type 1 (TH1) cells induces a hyporesponsive state. This hyporesponsiveness is associated with a reduction in ATP generation and decreased glycolysis and mitochondrial respiration. Moreover, T cells that are unable to upregulate autophagy accumulate a protein tyrosine phosphatase that negatively regulates mitogen-activated protein kinase (MAPK) signaling. In vivo deletion of an essential autophagy-related gene in T cells delayed the onset of and decreased the severity of experimental autoimmune encephalomyelitis. Our data support that autophagy may be necessary for T helper cell activation by regulating signaling pathways and metabolic programs, and the inhibition of this catabolic process may induce tolerance.
New Flow-Based, Multiplex Assays Allow Simultaneous Quantification of 13 Soluble Factors Related to Macrophage/Microglia or B Cell Functions
Authors: Sun Binggang , Rughwani Priyanka , Cohane Kenya , Ji Shaoquan , Taylor Peggy , Jiang Weiping
Published: January 2023

Immune responses are mediated by innate immune cells (e.g., macrophages) and subsequently, adaptive immune cells (e.g., B cells). Macrophages secrete an array of cytokines to effect their functions in host defense, tissue repair and immunoregulation. Pro-inflammatory macrophages (M1) produce cytokines, such as IL-12p70, TNF-α, IL-6, IL-1β, IL-12p40, IL-23, IFN-γ, and IP-10, while anti-inflammatory and tissue repairing macrophages (M2) release a different set of factors, such as IL-4, IL-10, IL-6, Arginase, TARC and IL-1RA. B cells, in addition to antibody production, also secrete an array of cytokines that mediate Th1- and Th2-like immune responses. These cytokines are produced by regulatory B cells (e.g., IL-10, TGF-β1) and B effector cells, namely Be1 (e.g., TNF-α, TNF-β, IFN-γ, and IL-12p70) and Be2 cells (e.g., IL-2, IL-4, IL-6, TNF-α, and IL-13). Other cytokines associated with activation and survival of B cells such as APRIL, BAFF, and CD40L are also important targets in B cell related processes.

We have developed new assay panels targeting macrophage/microglia and B cells, using fluorescence–encoded beads that are suitable for use on general lab flow cytometers. Each panel allows simultaneous quantification of 13 related analytes as shown below.

PanelTargetMacrophage/MicrogliaIL-1β, 1RA, 4, 6, 10, 12p40, 12p70, 23, IFN-γ, TARC, TNF-α, Arginase, IP-10B cellIL-2, 4, 6, 10, 12p70, 13, 17A, IFN-γ, TNF-α, TNF-β, BAFF, APRIL, CD40L

These panels have been validated by detecting expected changes in biological samples. These high quality, low cost and ease of use panels provide an alternative multiplex solution to the biomedical research community.
Developing polymer-drug complex based toll-like receptor (TLR-2/TLR-4) antagonist for modulating gut innate immune system to prevent/treat IBD and colon cancer
Authors: Kesharwani Siddharth Shivkumar , Rajput Mrigendra , Tummala Hemachand
Published: January 2023

Continuous inflammation in the colon often leads to chronic diseases such as inflammatory bowel disease (IBD) and colorectal cancer. Recent evidence strongly suggests the role of Toll-Like Receptors 2 and 4 in the etiology of aforementioned diseases. Therefore, pharmacological inhibition of TLR-2/TLR-4 has been a new promising strategy for preventing inflammation in IBD and colorectal cancer. In the current study, we have developed a novel polymer-drug complex (Ora-Curcumin) as a TLR-2/TLR-4 antagonist to modulate the gut innate immune system. Ora-Curcumin is a molecular complex of curcumin with a hydrophilic polymer Eudrgit® S100 prepared by nano-precipitation. Ora-curcumin is more water soluble (~1000 times) and stable than curcumin in aqueous buffers. It is water soluble only at pHs above 6.8. Therefore, once consumed orally, it is expected to be soluble and functionally available only at the luminal side of the colon where the pH reaches 6.8. In addition, Ora-Curcumin was potent TLR-4 antagonist that significantly inhibited the release of IL-8 and TNF-a from TLR-4 expressing human kidney epithelial cells (HEK293TLR4YFP-MD2) and mouse dendritic cells, respectively, in response to TLR stimulation with Monophosphoryl Lipid A (MPLA) or dead E. coli. The in-vivo potential of Ora-Curcumin in modulation of acute inflammation and intestinal microbiome is currently in progress. To this end, we have developed a unique polymer based water soluble TLR-2/TLR-4 antagonist which also provides enhanced local delivery to colon to prevent or treat IBD and colorectal cancer. As an anti-bacterial and by altering host immune system to control inflammation, Ora-Curcumin is also expected to modulate colon microbiome and improve GI health.
New Lipid Metabolic Targets for Treatment of Inflammation
Authors: Hsu Ku-Lung
Published: January 2023

The human genome contains a vast number of enzymes that lack functional annotation. As a result, our basic understanding of enzymatic pathways that underlie human physiology and pathology remains largely incomplete. Molecular profiling technologies have emerged as an unbiased means to globally examine the biochemical makeup of cells and tissues and identify genes and proteins associated with pathophysiological states. In contrast, global methods for the functional characterization of enzymatic pathways in native biological systems are still lacking. I will describe our efforts to integrate activity-based protein profiling (ABPP) with functional proteomic/metabolomics methods to map novel bioactive lipid networks involved in inflammation. I will describe the development of the first selective and in vivo-active small-molecule inhibitors for the 2-arachidonoylglycerol (2-AG) biosynthetic enzyme, diacylglycerol lipase-beta (DAGLB), along with paired negative-control and tailored activity-based probes for the functional analysis of DAGLB in living systems. We utilize our newly developed chemical probes to discover and show that DAGLB inactivation lowers 2-AG production in mouse peritoneal macrophages, as well as arachidonic acid and eicosanoids in a manner that is distinct and complementary to cytosolic phospholipase-A2. A corresponding reduction in lipopolysaccharide (LPS)-induced TNF-alpha release was observed, indicating that DAGLB serves as a key metabolic hub within a lipid signaling network that regulates proinflammatory responses in macrophages. We will present recent findings that identify DAGLB as a promising anti-inflammatory target for treatment of inflammatory and neuropathic pain.
Host skin T cells survive stem cell transplant conditioning and are functional during acute GVHD
Authors: Divito Sherrie J , Elco Christopher , Guleria Indira , Milford Edgar , Cutler Corey , Kupper Thomas S
Published: January 2023

Graft-versus-host-disease (GVHD) is a major cause of morbidity and mortality in stem cell transplantation (SCT). Acute GVHD is thought to be mediated by donor T cells that target and destroy host tissues. Studies into the host immune system are limited, as it is assumed that host T cells in peripheral tissues such as skin are depleted by conditioning regimens in parallel to T cells in lymphoid organs and blood. However, it is recently appreciated that a high number of memory T cells reside long-term within the organs most affected by GVHD. We investigated whether host skin T cells survive SCT conditioning and are present during acute GVHD. To do so, excess skin biopsy tissue was obtained from patients with acute skin GVHD who had undergone myeloablative or non-myeloablative conditioning and allogeneic SCT with sex-mismatched donor cells. Tissue was stained for T cell markers via immunofluorescence concurrently with in situ hybridization for the X and Y chromosomes. All seventeen patients studied demonstrated persistence of host T cells in skin, often in equal or greater numbers than donor T cells. Host skin T cells survived regardless of myeloablative or non-myeloablative conditioning, of patient age, and of day post-SCT (samples ranged from 12–213 days post-SCT). Comparatively, blood T cell chimerism was 100% donor in nearly all samples, indicating diverging effects of conditioning on T cell compartments. Skin host T cells demonstrated pro-inflammatory cytokine production. These results provide a novel avenue of research into GVHD pathobiology and suggest that alternative conditioning regimens should be employed if host skin T cells are to be targeted.
Obesity predisposes to rapid gastrointestinal graft-versus-host disease lethality after allogeneic hematopoietic stem cell transplantation in mice
Authors: Khuat Lam T , Pai Chien-Chun Steven , Chen Mingyi , Aguilar Ethan George , Dunai Cordelia , Le Catherine T , Raybould Helen E , Abedi Mehrdad , Murphy William J
Published: January 2023

Obesity is a form of chronic inflammation, termed “meta-inflammation”, resulting in the development of many chronic diseases such as diabetes and cancer. In this study, we assessed the impact of obesity on recipient graft-versus-host disease (GVHD) outcome post allogeneic HSCT using mouse models. In a model of sclerodermatous chronic GVHD, lean or diet-induced obese (DIO) BALB/c mice received 800 cGy total body irradiation (TBI), bone marrow cells, and splenocytes from H2-identical but minor MHC-mismatched B10.D2 mice. To mimic major MHC mismatch transplant which results in acute GVHD impacting gut, skin and liver, lean or DIO C57BL/6 mice received 1050 cGy TBI, bone marrow cells, and different doses of purified T cells from BALB/c mice. Strikingly, after the minor mismatch HSCT, all DIO mice succumbed to gut pathology while all lean mice survived and developed sclerodermatous GVHD at week 3 post transplant. In both models, pathological assessment indicated marked gut pathology in DIO mice correlating with lethality. Lean mice in the major MHC mismatch strain combination had significantly increased survival and less severe gut pathology compared to the DIO mice. The DIO mice had higher expression of pro-inflammatory cytokines in the gut, liver, visceral fat, and serum post-HSCT. Flow cytometric analysis demonstrated increased numbers of donor CD8+ T cells in the gut of DIO mice compared to lean mice. In an initial evaluation, untreated DIO mice displayed increased gut permeability as well as having limited diversity in their microbiome which would contribute to severe GVHD post-HSCT. Taken together, these results indicate that obesity exacerbates GVHD after allogeneic HSCT primarily affecting the gastrointestinal tract.