bluuebunny/crossref_metadata_2025 huggingface dataset:
Discovery of huanglongbing (HLB) pre-symptomatic RNA biomarkers (INM3P.415)
Authors: Wren Melinda , Pardington Paige , Stover Ed , Gupta Goutam
Published: January 2023

HLB is the most devastating citrus disease and is associated with vector-borne Liberibacter. Currently there is no cure for HLB. Visual disease symptoms appear in only a few leaves months after initial Liberibacter exposure compromising disease management by tree removal. Since infected but non-symptomatic trees are inoculum sources, a robust platform for early diagnosis of HLB is urgently needed. Here, we describe our studies on the discovery of HLB pre-symptomatic RNA biomarkers. For this, we infected greenhouse citrus using Liberibacter inoculum delivered by psyllid vectors confined to branch cages for a single week. Total RNA was extracted from leaves at three distances from the point of inoculation and for a range of early post-exposure times. Gene expression data by RNA-seq was collected for all leaf samples. We expect citrus genes belonging to pattern recognition, plant hormone, and stress pathways to be expressed early during infection. The RNA-seq data will be analyzed to identify which citrus genes are altered in leaves during early infection. It is our hypothesis that pre-symptomatic citrus biomarkers will be systemically expressed during the early stages of infection even in leaves distant from the site of Liberibacter inoculation. We will validate the expression of the candidate pre-symptomatic biomarkers by qPCR. The candidate biomarkers will reflect citrus genes that are systemically expressed and the Liberibacter genes that are indicative of bacterial growth.
Cyclo(L-phe-pro), Vibrio vulnificus produced metabolite, suppresses innate immune response (INM3P.418)
Authors: Jang Sung , Kim Kiwan , Lee Gap
Published: January 2023

Cyclo(Phe-Pro)(cFP), known as a secondary metabolite of some bacteria and fungi, is also produced by Vibrio vulnificus. It has been suggested that cFP has a role in bacterial cell-to-cell communication. However, the role of cFP in host immune response has not been reported yet. In this study, we investigated whether cFP modulates host innate immune responses. cFP suppressed production of pro-inflammatory cytokines, TNFa and IL6, nitric oxide, and reactive oxygen species in LPS-induced macrophages and bone marrow-derived macrophages. cFP also inhibited IKK phosphorylation, IkBa degradation, and NF-kB translocation to the nucleus, indicating that cFP affects the NF-kB pathway. These results provide evidences that cFP has immune suppressive effects that enhance the pathogen’s ability to infect and survive in host environment. Moreover, it may contribute to reveal the role of cFP in host-pathogen interaction and establish potential therapeutic strategies for the diseases related to Vibrio vulnificus.
Identification of an E3 ubiquitin ligase that regulates cellular resistance to influenza virus infection by controlling levels of the antiviral protein IFITM3 (INM3P.363)
Authors: Chesarino Nicholas , McMichael Temet , Hach Jocelyn , Yount Jacob
Published: January 2023

Interferon (IFN)-induced transmembrane protein 3 (IFITM3) is a cellular antiviral restriction factor that is able to inhibit infection by a wide variety of viruses, including influenza and Ebola. We previously showed that IFITM3 turnover is promoted by its ubiquitination, though the ubiquitin ligase responsible for this modification was not identified. Here we demonstrate that the E3 ubiquitin ligase NEDD4 ubiquitinates IFITM3 in vitro and in cells. Accordingly, we found that NEDD4 knockout mouse fibroblasts accumulate IFITM3, and that this accumulation is lost upon reconstitution of these cells with NEDD4. Importantly, IFITM3 accumulation results in enhanced resistance of NEDD4 knockout cells to influenza virus strains. Similarly, knockdown of NEDD4 in several human lung cell lines resulted in a buildup of IFITM3 and resistance to infection, demonstrating an evolutionary conservation of this regulatory mechanism in mice and humans. Further supporting a role for NEDD4 as a regulator of IFITM3 turnover, overexpression of the NEDD4 inhibitory protein, interferon stimulated gene 15 (ISG15), led to increased IFITM3 levels in human lung cells but decreased IFITM3 ubiquitination, demonstrating a new synergistic link between ISG15 and IFITM3. This work implicates NEDD4 as a therapeutic target for the prevention or treatment of influenza virus infections, and introduces a new paradigm for up-regulating the cellular level of an IFN-inducible antiviral protein independently of IFNs.
IL-33 promotes innate IFN-γ production and controls multi-functional T cell responses in viral hepatitis (IRC7P.429)
Authors: Liang Yuejin , Jie Zuliang , Soong Lynn , Sun Jiaren
Published: January 2023

Recently, experimental vaccine based on replication-defective lymphocytic choriomeningitis virus (rLCMV) has been shown to induce effective protection in several models, suggesting that rLCMV may show utility as a vaccine vector against infectious diseases and cancer. However, the key molecules in regulating the LCMV-induced immunity and controlling tissue damage are not clear. Here, we report that IL-33 promotes the innate and adaptive immune responses in LCMV-infected mice. Deficiency of IL-33 resulted in fewer IFN-γ+ innate immune cells (e.g. γδ T and NK cells) at 16 hours post-infection and lower levels of IFN-γ and IL-12 in the liver at 3 days post-infection (dpi). Impaired anti-viral T cell responses were observed in IL-33-/- mice at 7 dpi, as evidenced by fewer multi-funtional CD4 and CD8 T cells and lower level of anti-viral cytokines (e.g. IFN-γ, IL-12 and IL-2). In contrast, recombinant IL-33 (rIL-33) facilitated IFN-γ-producing gd T and NK cells, but inhibited proliferation of IL-17+ γδ T cells. rIL-33 treatment also promoted anti-viral T cell responses in both wild-type (WT) and IL-33-/- mice, and attenuated liver injury at 6 dpi. Moreover, we found that type 2 innate lymphoid cells (ILC2), induced by rIL-33, were dispensable for anti-viral T cell response in LCMV-induced acute hepatitis. Our data revealed the key role of IL-33 in regulating innate immune cells, and subsequently enhancing the plurifunctionality of anti-viral T cell responses in LCMV infection.
Mature dendritic cells secrete functionally active CTLA-4 in microvesicles (IRC7P.430)
Authors: Halpert Matthew , Konduri Vanaja , Liang Dan , Chen Yunyu , Wing James , Paust Silke , Levitt Jonathan , Decker William
Published: January 2023

Cytotoxic T-Lymphocyte Associated protein-4 (CTLA-4), the target of the blockbuster cancer immunotherapeutic ipilimumab, is a regulatory molecule of the B7 superfamily known to inhibit T-cell responses by a variety of different mechanisms. While CTLA-4 expression and function have been well-characterized in T-cells, very little is known about the presence and/or role of CTLA-4 in other hematopoietic lineages. Previous reports have indicated that myeloid lineage dendritic cells (DC) might express the CTLA-4 mRNA transcript and that transcript levels could be regulated by external stimuli. In the present study, we substantially build upon these critical observations, definitively demonstrating that mature DC express significant levels of intracellular CTLA-4 which are constitutively secreted in Rab5+Rab11- microvesicular structures. We further demonstrate that CTLA-4-containing microvesicles are capable of competitively binding B7 costimulatory molecules with significant functional consequences for downstream T-cell responses both in vitro and in vivo. The data indicate a previously-unknown role for DC-derived CTLA-4 in the physiologic regulation of T-cell responses and have significant implication for the design and implementation of immunomodulatory strategies intended to treat cancer and infectious disease.
Extracellular matrix protein 1 plays a critical role in driving Th2 cell migration and controlling Th17 cell differentiation (IRC7P.434)
Authors: Sun Bing
Published: January 2023

Abstract: Extra-Cellular Matrix Protein 1 (ECM1), a secreted protein, is highly and selectively expressed in Th2 cells. ECM1 deficiency caused impaired Th2 responses and reduced allergic airway inflammation in vivo. Functional analysis demonstrated that ECM1 deficient Th2 cells exhibited a defect in migration and were retained in peripheral lymphoid organs. Based on the cross-inhibition between Th2 and Th1/Th17 cells, we discovered a novel function of ECM1 in ameliorating Experimental autoimmune encephalomyelitis (EAE) which is mediated by Th1/Th17 cells. After the recombinant ECM1 were administrated i.v during EAE induction, the development of Th17 cells was decreased. In mechanism study we found that ECM1 could interact with integrin αv and block the interaction of integrin αv and latent TGF-β, which is critically for activating latent TGF-β maturation and then consequently to determine Th17 cell differentiation. To confirm the novel function of endogenous ECM1 in vivo, ecm1 transgenic mice was used to induce EAE. The data showed that EAE induction was suppressed significantly as compared with control group, which was associated with the failure of Th17 cell development. Our clinical observation indicated that ECM1 function is correlated with the pathogenesis of MS. This discovery offers new concept that ECM1 has potential in clinical application for understanding the pathogenesis of MS and its diagnosis.
Control of T cell proliferation, differentiation, and effector function by the adaptor protein BCAP (IRC8P.449)
Authors: Singh Mark , Srivastava Shivani , Duggan Jeffrey , Hamerman Jessica , Campbell Daniel
Published: January 2023

The PI3K network is required for a robust effector T cell response. However, the molecular mechanisms involved in regulating PI3K function during T cell activation are poorly understood. The adaptor protein B Cell Adaptor for PI3K (BCAP) has previously been detected in B cells and myeloid cells, and there is evidence suggesting that BCAP is involved in the regulation of PI3K signaling pathways in these cell populations. We have generated novel data demonstrating the role of BCAP in ensuring optimal T cell activation, proliferation and differentiation. BCAP is not expressed in naïve T cells but is detected in effector memory T cell populations, and can be rapidly induced in naïve T cells upon T cell activation in vitro. BCAP deficient T cells are hypo-proliferative, and have attenuated signaling cascades downstream of PI3K in vitro. To study the role of BCAP in T cells in vivo, we infected wild type and BCAP deficient mice with the pathogen Lymphocytic Choriomeningitis Virus (LCMV). At 7 days post infection, notable skewing in the effector CD8+ T cell populations was observed, with an increased frequency of KLRG1loCD127hi memory precursor cells (MPCs) at the expense of KLRG1hiCD127lo terminal effector cells (TECs). Finally, use of mixed bone marrow chimeras showed that wild type T cells have a significant competitive advantage over BCAP deficient T cells in vivo. Collectively, these data suggest that BCAP may have a non-redundant role in determining T cell fate in vivo.
Enhanced IL-7 responsiveness provides competitive advantage to recent thymic emigrants in peripheral T cell survival and homeostasis (IRC8P.451)
Authors: kim hye kyung , Castro Ehydel , Voong Nga , Telford William , Gress Ronald
Published: January 2023

Recent thymic emigrants (RTE) are newly generated T cells that are necessary to replenish T cell antigen receptor diversity and rejuvenate the peripheral naïve T cell pool. Both naïve T cells and RTE require IL-7 to survive in the periphery, but it is not known how RTE successfully compete with resident T cells for IL-7 signaling. To address this question, we assessed IL-7 receptor (IL-7Ra) expression on RTE, and found that RTE expresse substantially lower levels of IL-7Ra than naïve T cells. Because IL-7 signaling downregulates expression of its own receptor, lower IL-7Ra expression suggest that RTE are potentially more responsive to IL-7 than naïve T cells. Indeed, in vitro IL-7 stimulation showed significantly increased STAT5 phosphorylation in RTE compared to naïve T cells. To understand the molecular basis for increased IL-7 responsiveness, we assessed STAT5 phosphorylation and IL-7R expression upon IL-7 re-stimulation, and found that RTE are significantly more responsive to IL-7 re-stimulation. Contrary, adoptive transfer of RTE cells into lymphopenic host mice resulted in less efficient proliferation and expansion than of naïve donor T cells, which was not rescued by increasing in vivo IL-7 levels by implanting IL-7 osmotic pumps. Collectively, these data indicate that increased IL-7 responsiveness in RTE maximizes their survival while decreasing their proliferative capacity such as under lymphopenic conditions.
Plasmacytoid dendritic cell activation is dependent upon type I IFN-driven fatty acid oxidation (IRM9P.456)
Authors: Wu Duojiao , Everts Bart , Qiu Jing , Pearce Erika , Cella Marina , Pearce Edward
Published: January 2023

Plasmacytoid dendritic cells (pDCs) are specialized for the secretion of type I interferons (IFN-I) and thus are considered mediators of antiviral responses. pDCs selectively express endosomal Toll-like receptor (TLR) 7 and TLR9 and the ligation of TLR7/9 leads to rapid cellular activation. Recently, we found that stimulation of pDCs with the TLR9 agonist CpG resulted in a marked increase in mitochondrial oxygen consumption rates (OCR) dependent on increased fatty acid oxidation (FAO). FAO is essential for full activation of CpG-stimulated pDCs because etomoxir or carnitine palmitoyltransferase 1A shRNA (short-hairpin RNA) severely attenuated IFN-alpha production by these cells. Using RNA-seq, we identified the expression of many interferon-stimulated genes (ISG) in CpG-stimulated pDCs, amongst which were genes encoding FAO pathway components. We found that IFNAR -/- pDCs failed to increase mitochondrial OCR in response to TLR9 agonists. Taken together, our data indicate that IFN-alpha-induces FAO which is essential for full pDC activation in response to CpG.
Neuropeptides stimulate pro-inflammatory mediator secretion from human microglia through mammalian target of rapamycin signaling, which is inhibited by the flavonoids luteolin and tetramethoxyluteolin (IRM9P.457)
Authors: Patel Arti , Tsilioni Irene , Theoharides Theoharis
Published: January 2023

Despite the rise in diagnosis of Autism Spectrum Disorders (ASD, 1:68 children), the pathogenesis and treatment remain unknown. Mounting evidence supports the involvement of brain inflammation and immune dysfunction. Microglia, the brain-resident immune cells are critical gatekeepers of neuronal function show aberrant growth and activation in ASD brains. Some ASD patients also have elevated serum levels of neurotensin (NT) and corticotropin-releasing hormone (CRH), which are stress-related neuropeptides. Moreover, 1-5% of ASD cases have mutations in proteins that inhibit signaling through the mammalian target of rapamycin complex 1 (mTORC1), which regulates immune cell proliferation and mediator production. We hypothesized that NT and CRH act synergistically to stimulate human microglia through activation of mTORC1 leading to pro-inflammatory mediator release. We show that unstimulated human microglia express the NT receptor-3, but not the CRH receptor-1 (CRHR-1). Stimulation with NT and CRH significantly increases the gene expression of CRHR-1, as well as expression and secretion of the pro-inflammatory interleukin IL-1beta, CXCL8, CCL2, and GM-CSF. Pretreatment with mTORC1 inhibitors and the natural flavonoids luteolin and tetramethoxyluteolin, significantly decreases mediator secretion. Our results implicate the involvement of mTORC1 signaling for microglia activation, which is inhibited by novel flavonoids that could be developed into effective ASD treatments.
B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses (IRM10P.608)
Authors: Lam Tonika , Pone Egest , Lou Zheng , Wang Rui , Greenberg Milton , Xu Zhenming , Casali Paolo
Published: January 2023

CSR in B cells is crucial to the maturation of antibody responses. It is induced by engagement of CD40 or dual engagement of a TLR and BCR. Here, we have addressed cross-regulation between two different TLRs or between a TLR and CD40 in CSR induction by using a B cell stimulation system involving LPS. LPS mediated long-term primary class-switched antibody responses and induced generation of class-switched B cells and plasma cells. Consistent with the requirement for dual TLR and BCR engagement in CSR induction, LPS, which engages TLR4 through its lipid A moiety, triggered cytosolic Ca2+ flux in B cells through its BCR-engaging polysaccharidic moiety. In the presence of BCR crosslinking, LPS synergized with a TLR1/2 ligand (Pam3CSK4) in CSR induction, but much less efficiently with a TLR7 (R-848) or TLR9 (CpG) ligand. In the absence of BCR crosslinking, R-848 and CpG, virtually abrogated CSR, as induced by LPS or CD154, and reduced secretion of class-switched Igs, without affecting B cell proliferation or IgM expression. The CSR inhibition by TLR9 was associated with reduced AID expression and/or IgH germline IH-S-CH transcription, and required co-stimulation of B cells by CpG with LPS or CD154. Unexpectedly, B cells also failed to undergo CSR when co-stimulated by LPS and CD154. Our study suggests the complexity of how different stimuli cross-regulate an important B cell differentiation process and an important role of TLRs in inducing T-independent antibody responses.
5-methoxytryptophan is a circulating anti-inflammatory molecule that controls lipopolysaccharide-induced systemic inflammation and sepsis (IRM11P.622)
Authors: Wang YiFu , Wu Hsu-Feng , Hsu Yu-Juei , Lee Guan-Lin , Wu Jing-Yiing , Huang Shih-Ming , Yet Shaw-Fang , Wu Kenneth , Kuo Cheng-Chin
Published: January 2023

We identified in the conditioned medium of human fibroblasts a tryptophan metabolite, 5-methoxytryptophan (5-MTP) which controls cyclooxgenase-2 expression and inhibits NF-κB binding and p300 activity. We postulated that as with fibroblasts, human endothelial cells produce 5-MTP which plays an important role in controlling systemic inflammatory responses. Human endothelial cells produced and released 5-MTP into the conditioned medium. Lipopolysaccharide (LPS) suppressed 5-MTP production in endothelial cells and induced 5-MTP deficiency in the murine sepsis model. Intraperitoneal infusion of 5-MTP restored serum 5-MTP accompanied by inhibition of systemic pro-inflammatory cytokines, chemokines, and pro-inflammatory mediators. 5-MTP administration rescued organ failure and prevented mortality. The beneficial effect of 5-MTP was correlated with inhibition of cytokine production and COX-2 expression in macrophages. 5-MTP inhibited LPS-induced p38 MAPK, p300 HAT and NF-κB activation in macrophages and endotoxemic lung tissues in vivo. Importantly, a considerable amount of 5-MTP was detected in 30 healthy subjects (1.05 ± 0.39 μM) which was significantly reduced in 50 sepsis patients (0.37 ± 0.15 μM, p<0.0001). 5-MTP is a novel circulating anti-inflammatory molecule which protects against excessive systemic inflammatory responses. It ameliorates LPS-induced sepsis and is a valuable lead compound for developing new drugs to treat sepsis and systemic inflammatory disorders.
Arginine methylation modulation of T cell responses (IRM11P.636)
Authors: Guerau-De-Arellano Mireia , Savardekar Himanshu , Jablonski Kyle , Amici Stephanie , Panfil Amanda , Webb Lindsay , Thornton Kelsey , Li Linsen , Green Patrick , Baiocchi Robert , Li Chenlong
Published: January 2023

Inhibition of methylation reactions suppresses T cell proliferation and inflammatory responses but the specific methylation enzyme target responsible for these effects is unknown. Protein Arginine Methyltranferases (PRMT), enzymes that catalyze dimethylation of arginine in histones and other proteins, have been proposed to mediate these effects. Among PRMTs, PRMT5 symmetrically dimethylates protein arginine and is associated with embryonic development and cancer but has no known role in T cells. Here, we used myelin-specific memory Th cells that model autoimmune responses in Multiple Sclerosis to investigate the role of PRMT5 in memory Th1 cell responses. PRMT5 was up-regulated as Th1 cells undergoing activation, with kinetics that closely matched the cycle of T cell proliferation. PRMT5 protein up-regulation correlated with loss of miR-96, an inhibitor of PRMT5 translation. Further, a highly selective, small molecule PRMT5 inhibitor blunted memory T cell proliferation, an effect that could be partially rescued by IL-2. These data implicate PRMT5 in regulation of adaptive memory T helper cell responses and open novel therapeutic opportunities for T cell-mediated autoimmune disease.
DNA damage repair enzyme APE1 is a non-classical secretory protein and acts as a mediator of IL6 dependent inflammatory responses (IRM11P.626)
Authors: Nath Somsubhra , Shukla Ashima , Joshi Shantaram , Bhakat Kishor
Published: January 2023

Apurinic/apyrimidinic endonuclease 1 (APE1) is a pleiotropic nuclear protein with roles in DNA base excision repair (BER) pathway as well as in transcriptional regulation. APE1 has been found to be systematically released in blood plasma during endotoxemia in rats. However, the physiological importance of this secretion remains elusive. Here, we show that both normal human monocytes and monocytic cell line THP-1 secrete APE1 upon inflammatory challenges. This secretion follows a non-classical route of transportation and requires ATP-binding cassette transporters. Challenging the THP-1 cells with purified recombinant APE1 (recAPE1) in culture media shows increased expression and secretion of pro-inflammatory cytokine IL6. Phosphorylation of transcription factor NFҡB-p65 upon recAPE1 treatment precedes increased occupancy to IL6 promoter resulting in induced expression of the cytokine. Extracellular IL6 further promotes the secretion of APE1 thus working in a positive feedback loop. Moreover, release of APE1 by necrotic cells indicates stress responsive behavior of this secretion. Together, our study demonstrates a novel role of extracellular APE1 and identifies secretory APE1 as a mediator of IL6 dependent inflammatory responses.
Adenovirus Serotype 5 Vectors Trigger IL-27-Dependent Inhibitory CD4+ T Cell Responses
Authors: Larocca Rafael Assumpção , Provine Nicholas M , Iampietro Mark Justin , Borducchi Erica N , Penaloza-MacMaster Pablo A , Badamchi-Zadeh Alexander , Bricault Christine A , Blass Eryn , Parenteau Lily R , Blackmore Stephen , Stephenson Kathryn E , Barouch Dan H
Published: January 2023

Viral vectors are attractive vaccine platforms that elicit robust innate and adaptive immune responses, but they vary greatly in their ability to induce protective immunity. Adenovirus serotype 5 (Ad5) vectors elicit robust CD8+ T cell responses but are typically characterized by an exhausted phenotype. The mechanisms by which Ad5 vectors induce dysfunctional CD8+ T cells have not been fully elucidated. Here we demonstrate that Ad5 vectors, but not Ad26 vectors, elicit exhausted antigen-specific IL-10+PD1+ CD4+ T cells with a dysfunctional transcriptional profile, and these cells effectively suppress CD8+ T cell responses in vivo. The blockade of IL-10 increased the frequency of antigen-specific CD8+ T cells with enhanced cytokine polyfunctionality and protective capacity against a Listeria monocytogenes challenge. Induction of inhibitory CD4+ T cells by Ad5 vectors is associated with increased IL-27 expression, and IL-27 blockade improves CD4+ T cell cytokine polyfunctionality. Together our data highlight a novel role for IL-27 induced inhibitory CD4+ T cells in regulating responses to viral vector vaccines.
Exhaustion of Human Dendritic Cells Results in a Switch from the IL-15R/IL-15- to IL-2R/IL-2-driven Expansion of Antigen-specific CD8+ T Cells
Authors: Kalinski Pawel , Obermajer Natasa , Edwards Robert , Svane Inge-Marie , Hansen Morten
Published: January 2023

Maturation of dendritic cell (DC) is considered to be crucial for their ability to promote clonal expansion of antigen-specific T cells. Here, we show that the ability to expand human antigen-specific CD8+ T cells is a feature of specialized “effector” DCs at early stages of maturation, and it is suppressed in “exhausted” DCs at late stages of maturation and in DCs matured in the conditions of chronic inflammation.

Effector DCs, at early stages of maturation induced by TLR ligands and interferons, express membrane IL-15Rα/IL-15 complex, which promotes highly efficient and IL-2/IL-2Rα-independent expansion of MART-1-specific IL-15Rαhigh CD8+ T cells and cooperates with DC-produced IL-12p70 in inducing poly-functional CTL phenotype. In sharp contrast, exhausted DCs which lack membrane IL-15Rα/IL-15 complex, utilize a significantly less efficient IL-2Rα/IL-2 trans-presentation pathway to co-stimulate CD8+ T cells.

The currently-reported switch from the IL-15Rα/IL-15- to IL-2Rα/IL-2 costimulatory pathway in exhausted DCs helps to understand the role of inflammation in clonal expansion and contraction of CD8+ T cells at different stages of immune responses and helps to design improved modes of immunotherapy of cancer and chronic infections.
Myeloid cytokines distinctly and reversibly control LAIR-1 (CD305) expression on monocytes-mΦs/monocyte-DCs.
Authors: Santiago-Schwarz Frances , Son Myoungsun , Diamond Betty
Published: January 2023

Cross-linking of the inhibitory immunoreceptor LAIR-1 (CD305) on the cell surface phosphorylates LAIR-1 inhibitory motifs (ITIM) and restricts the growth of monocyte-derived dendritic cells (mono-DCs). Despite the suggestion that LAIR-1’s ability to prevent unwarranted immunity is compromised in autoimmunity, much remains to be learned about the control of LAIR-1 expression.

Here, we compared the ability of various myeloid cytokines to control LAIR-1 expression on the surface of monocytes/macrophages (mϕs) and mono-DCs. Treatment of freshly isolated blood monocytes from healthy donors with mono-DC growth factors (GM-CSF+IL-4/GM-CSF+IL-13) led to reduced levels of LAIR-1 at the critical monocyte-to-mono-DC juncture. In contrast, M-CSF (a steady state/M2 cytokine) sustained LAIR-1+CD14+CD33m/M+CD16+ mono-mϕs. M-CSF effects on LAIR-1 occurred under serum-supplemented and serum-free conditions.

Addition of M-CSF to mono-DCs previously cultured in GM-CSF+IL-4 promptly reversed the down-regulatory effects of GM-CSF+IL-4 producing increases in LAIR-1+CD14+ cells. Conversely, addition of GM-CSF+IL-4 to M-CSF exposed cultures lowered levels of LAIR-1 and CD14. M-CSF treatment of SLE blood myeloid cells (featuring activated monocytes/mono-DCs lacking LAIR-1, CD14) produced high levels of LAIR-1 and CD14 vs GM-CSF/IL-4. Thus: 1. The expression of LAIR-1 on monocytes/mϕs and mono-DCs is distinctly and reversibly controlled by myeloid cytokines. 2. LAIR-1 expression on circulating myeloid cells in SLE may be regulated by M-CSF.
Ly6C expression on T cells is modulated by IL-27, interferon-gamma and TCR stimulation
Authors: DeLong Jonathan Howard , Hall Aisling O’Hara , Muallem Gaia , Hunter Christopher A
Published: January 2023

Ly6C is a GPI-anchored glycoprotein expressed on a range of hematopoietic cell lineages. On T cells, its expression distinguishes short-lived effector CD4+ T cells from memory precursor effector cells. The function of Ly6C is unclear but this surface molecule is associated with homing to lymph nodes and crosslinking Ly6C with antibodies reduces T cell production of IL-2. We are investigating the factors that regulate the expression of Ly6C and their possible roles in infection. Using sorted Ly6C-negative naive T cells we found that TCR stimulation is not sufficient to induce Ly6C expression, whereas the cytokines IL-27 and interferon-gamma (IFNg) alone can induce Ly6C expression. However, this cytokine-mediated induction is enhanced by TCR stimulation. The in vivo significance of this pathway is seen during toxoplasmosis, when both naïve and activated parasite specific effector CD4+ and CD8+ T cells upregulated Ly6C expression. This upregulation was reduced in mice that lack IL-27. While these results suggest that cytokines have a major role in the regulation of Ly6C expression, it remains unclear how this affects T cell function and what role cytokine-mediated expression plays in the specific expression of Ly6C on effector but not memory populations.
CD103 expression on DCs regulates the onset and severity of TH17 airway inflammation
Authors: Bernatchez Emilie , Langlois Anick , Brassard Julyanne , Marsolais David , Blanchet Marie-Renée
Published: January 2023

Pulmonary dendritic cells (DCs) drive TH17 immunopathological responses to foreign antigens, including Saccharopolyspora rectivirgula (SR), a causative agent of hypersensitivity pneumonitis (HP). More specifically, CD103+ DCs, a DC cell subset found in the lung, are associated with the induction of TH17 responses; however, this remains controversial, as they are also associated with regulatory activity. Furthermore, the importance and modulation of CD103 expression on DCs and T cells in homeostasis maintenance and the regulation of TH17 hypersensitivity responses is elusive. We aimed to verify whether modulation of CD103 expression is important in the onset and regulation of the TH17 hypersensitivity lung response in HP. Exposure to SR in Cd103−/− mice led to an exacerbation of the inflammatory response as early as 18h after the first exposure to SR. Using DC and T cell transfers experiments, we demonstrate that CD103 expression on DCs specifically (and not T cells) is crucial in regulating this TH17 response. Interestingly, we observed that SR exposure (in vivo and in vitro) reduces rapidly CD103 protein and mRNA expression by DCs. In conclusion, we demonstrate that CD103 expression by DCs at the onset of the TH17 inflammatory response is crucial for homeostasis maintenance and regulation of the TH17 hypersensitivity response, and that loss of CD103 expression following antigen exposure may be a new mechanism leading to homeostasis breakdown in the lung.
Identifying exosome binding and internalization in blood cell subsets by imaging flow cytometry.
Authors: Pugsley Haley Renee , Friend Sherree , Probst Christine , Morrissey Phil
Published: January 2023

Only recently has the importance of extracellular vesicles as key mediators of intercellular communication been appreciated. Extracellular vesicles are membrane derived structures that include exosomes, microvesicles and apoptotic bodies. Exosomes have been shown to transfer molecules between cells and have the potential to transfer signals between cells. Exosomes are released under normal physiological conditions; however, they are also believed to serve as mediators in the pathogenesis of neurological, vascular, hematological and autoimmune diseases as well as cancer. Quantifying and characterizing exosomes in a reproducible and reliable manner has been difficult due to their small size (50 – 100 nm in diameter). Exosomes analysis can be done using high-magnification microscopy however this technique has a very low throughput. Attempts to analyze exosomes using traditional flow cytometers has been hampered by the limit of detection of such small particles and low refractive index. To overcome these limitations we have employed multispectral imaging flow cytometry that has the advantage of combining high throughput flow cytometry with higher sensitivity to small particles and the added benefit of imaging that can provide visual confirmation of particle integrity and characterization. In this study we use multispectral imaging flow cytometry to investigate the interaction of exosomes with white blood cells. Exosomes derived from different cell types will be investigated for their preferential interactions with blood cell subsets by combining immunophenotyping with morphological parameters to measure their binding and internalization.
Hepatocyte-dependent cross-presentation of soluble antigens induces antigen-specific CD8+ T cell tolerance
Authors: Damo Martina , Wilson D. Scott , Hubbell Jeffrey A.
Published: January 2023

The ability of hepatocytes to cross-present soluble extracellular antigens and the related immunological outcomes remain poorly investigated.

Our work shows that murine primary hepatocytes actively uptake and process extracellular antigens in EEA1+ and TAP1+ phagosomes, which are functionally related to cross-presentation. In fact EEA1+TAP1+ organelles, which are one of the signatures of professional cross-presenting cells, are also found in the cytoplasm of hepatocytes. Moreover, in vitro hepatocyte-dependent cross-presentation of OVA to OT-I cells is sensitive to specific drug inhibitors of endosomal and proteasomal activity, providing direct evidence that endocytosed antigens enter the cross-presentation pathway.

In vivo, cross-presenting hepatocytes induce tolerance of adoptively transferred OT-I cells by clonal deletion, as pro-apoptotic markers are upregulated on hepatocyte-educated OT-I cells. Non-deleted OT-I cells show instead significantly reduced response to vaccination, indicative of anergy. Active engulfment or emperipolesis of antigen cross-presenting hepatocytes by OT-I cells is also detected in vitro, as an additional means of T cell deletion.

PD-1/PD-L1 interactions participate in the induction of hepatocyte-dependent cross-tolerance, as specific blockage of PD-L1 on hepatocytes significantly reduces the development of hepatocyte-dependent cross-tolerance.

Due to their physiologic metabolic functions and their capacity to cross-present extracellular antigens here described, we propose hepatocytes as one of the major players in the establishment of antigen-specific CD8+ T cell peripheral tolerance.
Transcriptional control of RORγt across innate and adaptive immunity.
Authors: Pokrovskii Maria , Kim Sang Yong , Ciofani Maria , Littman Dan R
Published: January 2023

RORγt is the transcription factor responsible for the development and function of T helper 17 (Th17), γδT17, and innate lymphoid cells (ILC3s), which are key drivers of both immunity in tissues proximal to barrier surfaces and of autoimmune pathogenesis. An understanding of how regulation of the RORγt locus contributes to the programming of these separate lineages is crucial to dissecting the confounding ability of RORγt to drive such divergent responses. Examination of mice deficient in individual transcription factors that we found to constitute a key regulatory module driving Th17 cell development revealed divergent control of RORγt in innate and adaptive immune cells. This evidence suggests that distinct genomic cis-acting regulatory elements drive RORγt expression in a context dependent manner. Indeed, we found that cell-specific combinatorial usage of enhancers regulates the development and function of RORγt+ lineages in-vivo. Specifically, Th17 and ILC3 have largely unique enhancer usage while innate-like gdT17 cells share both innate and adaptive elements. We show that in vivo deletion of individual enhancer elements can modulate RORγt expression in a cell-type specific manner, the tuning of which affects cell frequency and function. Our findings provide new opportunities to study the contributions of individual RORγt+ lineages to immunity and inflammation. Moreover, these enhancers, which are highly conserved in humans, can be used to locate novel polymorphisms involved in the stratification of RORγt-dependent disease.
Structure-functional dissection of VTCN1 protein: Impact of individual extracellular domains and N-linked glycosylation modifications on its co-inhibitory function.
Authors: Amatya Christina , Radichev Ilian , Savinov Alexei
Published: January 2023

V-set Domain-Containing T Cell Activation Inhibitor 1 (VTCN1), also known as B7-H4, is a negative co-stimulatory ligand that inhibits T cell activation/proliferation. We have previously shown that VTCN1 signaling is impaired in context of type I diabetes in both mouse models and human patients. Moreover, aberrant VTCN1 expression has been associated with certain ovarian and renal carcinomas. These findings highlight the importance of VTCN1 as a potential therapeutic target.

At present, neither the receptor, nor the mechanism of VTCN1 action is known. Structurally, VCTN1 is a 283 a. a.-long heavily glycosylated protein with a very short intracellular tail followed by a type 1 hydrophobic transmembrane domain and a long extracellular part consisting of Ig-like V-set (IgV) and Ig-like C-set (IgC) domains. Here, we used reductionist approach to study discrete contributions of VTCN1 Ig-like domains, as well as impact of separate/combined N-linked glycosylation modifications in VTCN1 co-inhibitory function. Accordingly, we generated a set of mouse VTCN1 protein mutants, engineered to contain each of its two domains alone and/or in combination, and carrying either deletion(s) or point mutation(s) of the potential proteins’ glycosylation sites. We then evaluated the ability of these mutant proteins to bind pre-activated T cells in culture and influence T cell proliferation and cytokine production.

We show that both IgV and IgC VTCN1 domains retain inhibitory activities, even though IgV domain mostly constrains T cell proliferation, while IgC domain predominantly inhibits cytokine production. Additionally, we reveal that glycosylation of VTCN1 is crucial for its membrane trafficking, folding and negative co-stimulatory functions.
N-glycan branching regulates B cell peripheral functions
Authors: Mortales Christie-Lynn , Demetriou Michael
Published: January 2023

N-glycosylation is important for regulating many immune cell functions. To date, the role of N-glycan branching on influencing B cell antigen presenting cell (APC) function is not known. Proper CD4+ TH cell activation requires APC peptide presentation together with CD80/CD86 co-stimulation. CD80 and CD86 respectively promote TH1 cell-mediated and TH2 humoral immune responses. APCs then release cytokines that further dictate TH cell polarization into TH1, TH2, TH17 or TREG cells. In vitro stimulated N-glycan branching deficient B cells have higher CD80 and lower CD86 surface levels, and produce more pro-inflammatory TNFα and less anti-inflammatory IL-10. Furthermore, N-glycan branching deficient B cells have increased toll-like receptor 4 (TLR4) surface levels resulting from lack of endocytosis. Sustained surface TLR4 enhanced intracellular signaling towards NFκB activation as measured by increased phosphorylation of TAK1 and p38. Endosomal TLR4 signaling, which can lead to IRF3 activation, was diminished as seen by decreased expression of TRAF3 and phosphorylation of TBK1. Our results show N-glycan branching on peripheral B cells restricts NFκB activation to limit pro-inflammatory cytokine expression and CD80 surface levels, ultimately dampening TH1 cell-mediated responses. This study demonstrates N-glycosylation is important for regulating B cell APC function.
Inflammatory monocytes enhance disease progression during post-influenza MRSA pneumonia
Authors: Fischer Karl Jacob , Sun Keer , Bauer Christopher , Yajjala Vijaya Kumar
Published: January 2023

Morbidity associated with influenza pandemics of the last century has been significantly associated with secondary bacterial infections. It is generally believed that the enhanced susceptibility to secondary bacterial infection is due to influenza-suppressed innate immunity. Interestingly, preceding influenza infection does not inhibit inflammatory cell recruitment during secondary bacterial infection. In fact, due to the recruitment of inflammatory monocytes, there are increased numbers of phagocytes in influenza and bacterial coinfected airways. The protective role of monocytes during influenza infection alone is well recognized; however, it is not clear how this phagocyte population contributes to post-influenza bacterial pneumonia. Using CC-Chemokine Receptor 2 deficient (CCR2−/−) mice defective in airway monocyte recruitment, we are currently investigating the role of inflammatory monocytes during influenza and methicillin-resistant Staphylococcus aureus (MRSA) coinfection. Specifically, C57BL/6 mice were infected intranasally with a sublethal dose of PR8 and subsequently MRSA on day seven post-influenza infection. Despite their delay in viral clearance, coinfected CCR2−/− mice exhibited significantly reduced bacterial burdens and importantly, improved survival rates compared with WT mice. Moreover, pro-inflammatory cytokine response in coinfected CCR2−/− mice was significantly decreased, suggesting an attenuation in lung inflammation. In summary, we found that by inhibiting bacterial clearance and perpetuating lung inflammation, inflammatory monocytes exacerbate adverse disease outcome during post-influenza MRSA pneumonia.
Platelets mediate host-defense against S. aureus through direct bactericidal activity and by enhancing macrophage activities
Authors: Ali Ramadan , Weuscher Leah , Dona Keith , Worth Randall
Published: January 2023

Platelets are chief effector cells in hemostasis. However, recent evidence suggests that platelets have multiple roles in host defense against infection. Recent reports by us and others showed that platelets functionally contribute to protection against Staphylococcus aureus infection. In the current study, the capacity of mouse and human platelets to participate in host defense against S.aureus infection was determined by assessing two possibilities: First, the ability of platelets to directly kill S. aureus; and second, the possibility that platelets enhance macrophage phagocytosis and intracellular killing of S. aureus. Both human and mouse platelets effectively killed S. aureus (>35% and >40% killing in 60 min., respectively) in an activation-dependent (0.1U.ml thrombin) manner. Interestingly, thrombin-stimulated mouse platelets also significantly enhanced peritoneal macrophage phagocytosis of S. aureus by 70% and intracellular killing by 43%. To determine if platelet-enhanced uptake and killing was due to direct interaction or through releasable products, peritoneal macrophages were exposed to releasates collected from resting or thrombin-activated platelets. The data show that releasates collected from thrombin-activated platelets induce similar increases in both uptake and killing of S. aureus by macrophages. These data confirm our hypothesis that platelets participate both directly and indirectly in protection against S. aureus infection.
The Characterization of Nonclassical T cells and their Responses during Mtb Infection
Authors: Simpson Allison , Foreman Taylor W , Rout Namita , Kaushal Deepak
Published: January 2023

Tuberculosis (TB) is a catastrophic infectious disease, affecting roughly one third of the world’s population. Typically 5–10% of infections will develop into active TB, however co-morbidities such as HIV present an increased risk of TB reactivation, leading to life-threatening active infection. While it is well-known that T cells, and in particular, Class II restricted, IFN-γ expressing classical CD4+ T cells are essential for immunity to TB, these are by themselves not sufficient correlates of protection, especially during reactivation of LTBI by HIV co-infection. Recently lymphocytic subsets that recognize nonpeptidic antigens have been classified as nonclassically activated T cells. These cells are, however, poorly characterized as regulators of the immune system, particularly in patients infected with M. tuberculosis (Mtb). We hypothesize that non-classically activated T cells recognize bacterial antigens during Mtb infection, as well as Mtb/HIV co-infection, and may play a role in protective immunity. As part of this project, we will enumerate and phenotype the populations of nonclassical T cells based on expression of their surface antigens at different time points of Mtb and Mtb/SIV infection in rhesus macaques. Furthermore, we will characterize the function of the non-classical T cells by analyzing their secreted factors. Better understanding the role of nonclassical T cells in TB pathogenesis may enable us to develop better vaccines and treatments against TB.
Impact of Silver and Carbon Nanoparticle Exposures on Macrophage Responses to Mycobacterium tuberculosis (M.tb)
Authors: Sarkar Srijata , Carranza Claudia , Theodorou Ioannis , Fen Leo Bey , Ellis Timothy , Porter Alexandra , Ryan Mary , Zhang Junfeng , Tetley Teresa , Schwander Stephan
Published: January 2023

To assess biological risks of nanoparticle (NP) exposures, we examined the effects of silver (Ag) core Ag20-citrate (Ag20) and carbon black (CB) on human host innate immune responses to M.tb, a respiratory pathogen that causes tuberculosis. Human peripheral blood monocyte-derived macrophages (MDMs) were exposed to 1, 10, 25 and 50 μg/mL of Ag20 and CB for 4 and 24 h. At 4 h no significant cytotoxicity was observed with neither of the NPs at none of the concentrations examined relative to unexposed MDMs. At 24 h, viability of the MDMs was reduced by 60–70% and 20% following Ag20 and CB exposure, respectively, at doses ≥25 μg/mL. M.tb-induced host immune responses are mediated by the activation of TLR signalling pathways that culminate in NF-κB activation. Effects of Ag20 and CB (10μg/mL for 4 h, a nontoxic dose) on TLR signalling in M.tb-infected and uninfected MDMs were compared using a human TLR signaling pathway-specific profiler array. Ag20 exposure suppressed the M.tb-induced expression of a subset of NF-κB-mediated target genes (CSF2, CSF3, IFNG, IL1A, IL1B, IL6, IL10, TNFA, NFKB1A). Ag20-mediated suppression of M.tb-induced activation of the TLR-signalling pathway was not due to decreased uptake of M.tb by MDMs or mycobacteriocidal effects of Ag20. Ag20 exposure increased the expression of HSPA1A mRNA that encodes for the stress-induced Hsp72. In contrast, exposure to CB resulted in little effects on M.tb-induced host gene expression indicating that immunosuppressive effects of Ag20 are NP-specific. Since Hsp72 has been shown to suppress NF-κB activation, we propose that Ag20-mediated Hsp72 upregulation contributes to the suppression of M.tb-induced NF-κB activation and host immune responses.

Funding NIEHS 5U19 ES019536
Ankyrin Repeat Domain 22 Mediates Host Defense Against Viral Infection Through STING Signaling Pathway
Authors: Bin Lianghua , Li Xiaozhao , Feng Juntao , Richers Brittany , Leung Donald Y.M.
Published: January 2023

The innate immune system is critical for early detection of invading viruses and for initiating cellular host defense countermeasures, which include the production of interferons. Ankyrin repeat domain 22 (ANKRD22) encodes a protein of 191 amino acids containing 2 ankyrin repeating domain, and its biological function was unknown. In this study, we find that ANKRD22 expression can be significantly induced in human peripheral blood mononuclear cells (PBMCs) by DNA virus herpes simplex virus -1(HSV-1) and RNA virus respiratory syncytial virus (RSV) as well as IFN-α and IFN-γ. Its induction in PBMCs by viruses is largely a secondary effect of virus-induced interferons. We further demonstrate that ANKRD22 was majorly induced in monocytes and dendritic cells from PBMCs. Over-expression of ANKRD22 confers protection against HSV-1 and RSV infection in multiple tested cell lines. Vice versa, inhibition of ANKRD22 leads to enhanced HSV-1 and RSV replication in host cells. Furthermore, we identifies that ANKRD22 specifically interacts with STING, a critical protein function in multiple antiviral innate immune pathways. ANKRD22 by itself can only slightly induce type I IFNs IFN-α and IFN-β, and can’t induce type III IFN IL-29. However, it significantly synergizes STING-induced both type I and type III interferon production. Collectively, our studies indicate that ANKRD22 plays an important role for host defense against both DNA and RNA viruses by optimizing STING’s function on inducing type I and type III IFNs.