bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptomic signatures convergent across multiple studies of upper and lower airway allergic disease support the unified airway hypothesis (HUM7P.321)
Authors: Berdnikovs Sergejs , Schleimer Robert , Bryce Paul , Abdala-Valencia Hiam
Published: January 2023

Asthma is frequently associated with co-morbid diseases of the upper airways, including chronic rhinosinusitis (CRS). This has led to a hypothesis that the upper and lower airway may be immunologically linked, functioning as a continuous mucosal epithelial barrier interacting with the environment. Despite a growing number of transcriptomics studies, the differences and similarities between upper and lower airways in allergic disease are still poorly understood. We analyzed raw microarray data from three independent studies of CRS and four studies of asthma, and conducted a secondary multivariate and pathway analysis to identify genes commonly altered in both diseases. Despite regional differences, close to 20% of genes found to be differentially expressed in CRS were also found to be differentially expressed in the lower airways of asthmatics. Notable examples are IL6R, IL13RA2, MSMB, MUC5B, POSTN, SERPINB2, TFF2, WIF1 and WNT5A. Pathway analysis revealed that key processes represented by these shared genes are developmental pathway signaling, epithelial-mesenchymal transition and innate immune response, suggesting co-existence of epithelial remodeling and persistence of innate immune signals. Interestingly, many of the genes altered in CRS that we identified in the upper airways are also asthma susceptible genes previously identified through GWAS studies (e.g., IL1RL1, ORMDL3), indicating a common role for these genes in the pathogenesis of respiratory allergic diseases.
Regulatory function of interferon regulatory factor 3 in obesity, inflammation and insulin resistance (HUM7P.287)
Authors: Tang Peng , Zhang Yongliang
Published: January 2023

Obesity is a major risk factor for diseases ranging from insulin resistance, type 2 diabetes mellitus to nonalcoholic fatty liver disease. Obesity is associated with a state of chronic, low-grade inflammation. In this study, we found the important regulatory role of interferon regulatory factor 3 (IRF3), a master regulator of type I interferon-dependent immune responses, in obesity. We found that the expression of IRF3 was increased in white adipose tissue (WAT) from mice fed with high fat diet compared with that from mice fed with normal chow. IRF3 knockout (KO) mice developed obesity spontaneously with increased adiposity compared with wild-type (WT) controls. Deficiency of IRF3 in mice resulted in impaired insulin sensitivity and lipid homeostasis. The development of obesity in IRF3 KO mice was associated with increased infiltration of macrophages which was more inflammatory in the WAT compared with macrophages in WT mice. IRF3 deficient macrophages exhibited increased M1 polarization than WT cells. Additionally, IRF3 KO mice developed hepatic steatosis with increased expression of lipogenesis genes in the liver. Our study therefore demonstrated that IRF3 played an important role in the regulation of insulin sensitivity, inflammation and lipid homeostasis, which may provide a new strategy for treatment of obesity, type 2 diabetes mellitus and hepatic steatosis.
Cyclin-dependent kinase 5 contributes to adipose tissue inflammation in obesity through suppression of Treg differentiation (HUM7P.288)
Authors: Pareek Tej , Lam Eric , Letterio John
Published: January 2023

Homeostatic imbalance of regulatory and conventional T cells is linked with obesity-induced inflammation and insulin resistance. A decline in the number of both circulating and locally residing T regulatory (Treg) cells in adipose tissues is hallmark of adiposity, inflammation, and glucose intolerance. Recently, the signal transducer and activator of transcription 3 (Stat3) has been implicated as a critical mediator in this process; however, the factors that initiate this inflammatory cascade remain largely unclear. We recently reported a previously unknown function for Cyclin-dependent kinase 5 (Cdk5) in T cells, a kinase renowned otherwise for its role in regulating the development and function of cells in the neuronal lineage. Here we report for the first time that the mice lacking Cdk5 in T cells (Cdk5TKO-mice) are lean compared to their wild type littermates as confirmed by their body mass index, histopathological analysis of adipose tissue as well as complete body MRI scans measuring adiposity index. Moreover we observed a significant increase in circulating CD4+ CD25+ Foxp3+ Treg cells along with a marked decrease in Ser-727 phosphorylation of Stat3. Finally, both pharmacologic and genetic deletion of Cdk5 abrogates the IL6-mediated suppression of TGF-β-induced Treg differentiation. These results further solidify the role of Cdk5 as an essential regulator of T cell function and implicate Cdk5 as a potential therapeutic target for prevention and treatment of obesity.
Silencing of IL-17 modulates inflammatory and antimicrobial defense responses mediated by Propionibacterium acnes (HUM7P.291)
Authors: Agak George , Yu Yang , Qin Min , Park Andrew , Krutzik Stephan , Garban Hermes , Kim Jenny
Published: January 2023

Inflammation resulting from immune responses targeting Propionibacterium acnes plays a significant role in acne pathogenesis. We demonstrate that IL-17-expressing cells are present in skin biopsies from acne patients, and that P. acnes is a potent inducer of Th17-related cytokines, including IL-17 (inflammatory) and IL-22 (antimicrobial defense and tissue repair). Here we investigated whether silencing of IL-17 and IL-22 modulates Th17-related inflammatory and antimicrobial responses mediated by P. acnes. P. acnes stimulated peripheral blood mononuclear cells (PBMCs) demonstrated elevated expression of antimicrobial peptides (AMPs); psoriasin, cathelicidins, defensins, and antimicrobial chemokines; CXCL-1/-9/-10/-11 and CCL15 expression. siRNA-mediated knockdown of IL-17 led to loss of capacity to express and secrete IL-17, IL-22 and AMPs upon P. acnes stimulation. However, knocking-down IL-22 alone led to decreased AMP expression, but had no effect on the expression of genes known to directly signal Th17 responses including IL-17A, RORα and RORc. Both IL-17 and IL-22 were required for antimicrobial chemokines CXCL1 and CXCL10 expression, but not CXCL9, CXCL11 and CCL15. Importantly, IL-22 may play a role in modulating the IL-17-driven chronic inflammatory responses initiated by P. acnes and that treatments targeting the IL-17 pathway may be effective tools for acne therapy.
Myocyte enhancer factor-2 plays critical roles in HTLV-1 infection and transformation of CD4+ T cells associated with adult T cell leukemia (ATL) by stabilizing complex between Tax and CREB (HUM8P.335)
Authors: Jain Pooja
Published: January 2023

The exact molecular mechanisms regarding HTLV-1 Tax-mediated viral gene expression and CD4+ T cell transformation have yet to be fully delineated. Herein, utilizing virus infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the role of myocyte enhancer factor-2 (MEF-2) in Tax-mediated transactivation of the LTR. Inhibition of MEF-2 expression by shRNA and its activity by HDAC9 led to reduced viral replication and HTLV-mediated T cell transformation in correlation with a heightened expression of MEF-2 in ATL patients. Mechanistically, MEF-2 was shown to be recruited to the HTLV-1 LTR followed by an assessment of MEF-2 binding site(s). A novel promoter-binding assay confirmed that MEF-2 constitutes the transcriptional complex that assembles on the LTR. Furthermore, an increase in MEF-2 expression was observed upon infection in an extent similar to CREB (the known Tax-interacting transcription factor) and HATs - p300, CBP, and p/CAF. Confocal imaging confirmed MEF-2 co-localization with Tax and these proteins were also shown to interact by co-immunoprecipitation. A number of MEF-2-integrated signaling pathways were activated during HTLV-1 infection of primary CD4+ T cells, including PI3K/Akt, NF-κB, MAPK, JAK/STAT and TGF-β possibly regulating MEF-2 activity. Overall, these studies are the first to describe the involvement and regulation of a novel transcription factor during the course of a retroviral infection and associated disease syndrome.
IL-7/Lymphopenia enhanced Type-I IFN response by modulating STAT-1 levels: its impact in CD4 T cell homeostasis (HUM8P.338)
Authors: Le Saout Cecile , Hasley Rebecca , Imamichi Hiromi , Tcheung Lueng , Hu Zonghui , Luckey Megan , Park Jung-Hyun , Durum Scott , Smith Mindy , Rupert Adam , Sneller Michael , Lane H. Clifford , Catalfamo Marta
Published: January 2023

HIV infection is associated with chronic immune activation and alteration of T cell homeostasis (CD4 T cell depletion and CD8 T cell expansion). While the acute CD4 depletion observed in the initial phase of HIV infection is likely due to direct cytopatic effects of the virus, the mechanism/s underlying the steady decline of the CD4 T cell pool during the chronic phase of infection are unclear and are felt to be associated with “immune activation”. In the current study, we addressed the impact of the combination of two distinct forces: homeostatic (CD4 T cell depletion) and inflammatory (HIV-driven IFN-α) on T cell homeostasis. We present evidences that IL-7 and lymphopenia enhanced CD4 T cell responsiveness to IFN-α by modulating expression of the Signal Transducers and Activators of Transcription (STAT) 1, 2 and 3. In a murine model, CD4 T cell depletion and CD8 T cell expansion were observed in a lymphopenic host chronically treated with IFN-α. These findings suggest that a synergistic interaction between lymphopenia and IFN-α may play a role in the pathogenesis of HIV infection. The analysis of this pathway may contribute to the development of new strategies to reverse the dysregulation of the T cell pools seen in patients with HIV infection.
Mucosal immune responses predict clinical outcomes during influenza infection independently of age and viral load (HUM8P.346)
Authors: Thomas Paul , Gartland Andrew , Wong Sook-San , Jeevan Trushar , Wang David , Roddam Philippa , Caniza Miguela , DeVincenzo John , Hertz Tomer , Webby Richard , Oshansky Christine
Published: January 2023

Children are an at-risk population for developing complications following influenza infection, but immunological correlates of disease severity are not understood. We hypothesized that innate cellular immune responses at the site of infection would correlate with disease outcome. To test this hypothesis, we established an observational cohort study with longitudinal sampling of peripheral and mucosal sites in 84 naturally influenza-infected individuals, including infants. Cellular responses, viral loads, and cytokines were quantified from nasal lavages and blood, and correlated to clinical severity. We found that while viral loads in children and adults were similar, innate responses in the airways were stronger in children and varied considerably between plasma and site of infection. Adjusting for age and viral load, an innate immune profile characterized by increased nasal lavage MCP-3, IFNα2 and plasma IL-10 levels at enrollment predicted progression to severe disease. Increased plasma IL-10, MCP-3, and IL-6 levels predicted hospitalization. This inflammatory cytokine production correlated significantly with monocyte localization from the blood to the site of infection, with conventional monocytes positively correlating with inflammation. Increased frequencies of CD14lo monocytes were in the airways of participants with lower inflammatory cytokine levels. An innate profile was identified that correlated with disease progression independent of viral dynamics and age.
Age-related gut dysbiosis contributes to age-related colitis and colon cancer risk and appears B7-H1-dependent. (HUM8P.352)
Authors: Cardenas Kim , Dao Vinh , Hurez Vincent , Curiel Tyler
Published: January 2023

Gut microbial imbalance (dysbiosis) can contribute to intestinal inflammation. Inflammation-driven colon cancer is a leading cause of death in the aged. We hypothesize that intestinal dysbiosis occurs with age and contributes to colon cancer susceptibility. We found that aged mice (~20 mos) had increased basal colon inflammation. Young mice (~3 mos) treated with AOM/DSS, an established model of inflammation-driven colon cancer, got transient colitis followed by colon cancer as expected. By striking contrast, aged mice on AOM/DSS developed severe, fatal, acute colitis preventing carcinogenesis studies. We also found that the colon microbiome is age-dependent. Gut flora-ablating antibiotics (abx) in young mice on AOM/DSS did not alter acute colitis or cancer. Strikingly, old mice on abx were highly protected from AOM/DSS-induced colitis and colon cancer, consistent with age-related dysbiosis causing increased risk for colitis and inflammation driven-colon cancer. PD-1 regulates gut flora in young mice and B7-H1 is its ligand. Aged B7-H1-/- mice given AOM/DSS, but no abx, did not develop acute colitis or colon cancer, suggesting that B7-H1 mediates age-related gut dysbiosis, inflammation and colon cancer risk. Taken together, our data suggest that age-related, B7-H1-dependent gut dysbiosis increases colon inflammation and susceptibility to colon carcinogenesis. Ongoing co-housing (WT/B7-H1 and young/aged) and other studies are defining specific immune mechanisms.
The NAD glycohydrolase CD38 regulates macrophage effector function and defense against Listeria monocytogenes. (INC7P.409)
Authors: Botta Davide , Rivero-Nava Laura , Lund Frances
Published: January 2023

CD38, an ecto-enzyme with ADP-ribosyl cyclase activity, is expressed on myeloid cells and has been shown to be a regulator of innate immunity and inflammation. Our study aimed to determine whether CD38 plays a role in the immune response to the Gram-positive bacterial pathogen Listeria monocytogenes (Lm). Following an intravenous Lm infection, Cd38-/- mice exhibited increased mortality and morbidity compared to wild-type mice. Analysis of tissue homogenates showed greater bacterial burden in the spleen and liver of Cd38-/- mice 3-4 days post infection, suggesting that early Lm resistance is CD38-dependent. Bone marrow chimera experiments revealed that the increased susceptibility of Cd38-/- mice was due to defects on myeloid lineage cells. While recruitment of PMNs, CD11b+ cells and NK cells to the spleen was not affected in Lm-infected CD38-/- mice, the efflux of monocytes from the bone marrow to the blood and spleen was significantly reduced. Expression levels of IL-10, an inhibitor of macrophage effector function, were elevated in serum and spleens of Lm-infected Cd38-/- mice, and splenocytes isolated from Lm-infected Cd38-/- mice had decreased nitric oxide production and TNFα secretion. Furthermore, in vitro stimulation experiments showed decreased expression of key inflammatory cytokines in activated CD38-deficient macrophages. Our data suggest that CD38 deficiency impairs macrophage effector function that is crucial for an effective early immune response to Lm.
Hematopoietic stem and progenitor cells expand into functional PMN at sites of S. aureus-infected wounds (INC7P.426)
Authors: Falahee Patrick , Granick Jennifer , Dahmubed Delsheen , Simon Scott
Published: January 2023

Staphylococcus aureus is a frequent cause of chronic nosocomial skin and soft tissue infections that can spread to muscle, bone and connective tissue. In response to infection, a heterogeneous population of circulating and bone marrow (BM)-derived cells are recruited to the site of injury where they collectively contribute to host defense and tissue repair. Polymorphonuclear leukocytes (PMN) serve a critical role as the first-line effectors responsible for innate immune protection against bacterial challenge. Using a transgenic mouse model of S. aureus-infected skin wounds, we recently reported that a subset of hematopoietic stem and progenitor cells (HSPC) that are lineage negative, Sca-1+ and c-kit+ (LSK cells) undergo local granulopoiesis and account for as much as 30% of total PMN within the abscess (Granick et al., 2013). Follow up studies show that LSK cells are stimulated directly by the agonist PAM3CSK4 in a manner dependent on TLR2/MyD88 and PGE2 signaling. We show that local granulopoiesis differs from myeloid proliferation within the BM revealing functional divergence of PMN produced at the site of wounding. Current efforts are focused on signaling effects of PGE2 that can manipulate the expansion capability of LSK during infection in wounds in order to gain an advantage over chronic and persistent Staphylococcus aureus infections.
Hepatic stellate cell-derived retinoic acid regulates group 3 innate lymphoid cells and modulates viral hepatitis (INC8P.432)
Authors: Jie Zuliang , Liang Yuejin , Hou Lifei , Soong Lynn , Cong Yingzi , Sun Jiaren
Published: January 2023

IL-17 and IL-22 are important for T cell priming and hepatoprotection in viral hepatitis, respectively; however, the source and regulation of these cytokines in the liver microenvironment are not well defined. Here, we present evidence for a significant expansion of intrahepatic cells producing IL-17 or IL-22 in mice during adenovirus infection. A subset of IL-17+ or IL-22+ cells expressed no myeloid/lymphoid lineage markers. Instead, they expressed high levels of stem-like markers, IL-7Rα (CD127) and RORγt, consistent with the newly described group 3 innate lymphoid cells (ILC3s). These cells contributed significantly to the establishment of the early cytokine milieu in virus-infected liver. Interestingly, retinoic acid increased in the liver following viral infection. Moreover, hepatic stellate cell (HSC)-derived retinoic acid promoted IL-22 production by ILC3s and protected liver from inflammatory response-related injuries. Notably, hepatic IL-7 was important for ILC3-derived IL-22 production. Blockade of IL-7Rα in vivo significantly decreased IL-22 levels and exacerbated hepatic inflammation. Collectively, this study has demonstrated the importance of a unique intrahepatic subpopulation ILC3 and its cytokine production during the initial stages of viral infection in the liver. Moreover, the cross talk among the hepatic stellate cell and innate immune cells plays a critical role in modulating immune responses in viral hepatitis.
Apocynin inhibits reactive oxygen species (ROS) and downregulates chemokine and cytokine production induced by avian influenza H5N1and H7N9 virus infection. (INC8P.433)
Authors: Stambas John , Lowther Sue , Ye Siying
Published: January 2023

Influenza A virus infection causes acute inflammation resulting in increased reactive oxygen species (ROS) production and lung injury. ROS are produced following activation of the NADPH oxidase enzyme complex. Apocynin, an inhibitor of NADPH oxidase complex formation has been shown to reduce the accumulation of inflammatory cells in the lungs of influenza-infected mice. As hypercytokinemia is thought to contribute to severe disease pathogenesis of both H5N1 and H7N9 infection in humans, we hypothesized that apocynin could reduce virus-induced cytokine/chemokine production. Human lung epithelial and chicken cell lines were infected with low pathogenic avian influenza H7N9 (A/Anhui/1/2013) and HPAI H5N1 (A/chicken/Vietnam/0008/2004) viruses in the absence or presence of apocynin. Quantitative real-time PCR demonstrated that apocynin inhibited influenza-induced cytokines/chemokines and ROS production following infection. Interestingly, addition of apocynin to in vitro cultures significantly increased SOCS1 and SOCS3 mRNA and protein expression, contributing to the negative regulation of overall cytokine expression. We suggest that intervention strategies targeting both the virus (current therapeutics) and the host e.g. apocynin (future therapeutics) could be used to ameliorate disease in infected individuals, reducing hypercytokinemia and pathology and allowing full development of adaptive immune responses.
Dendritic cell CD209a expression is critical for the development of pathogenic Th17 cell responses in murine schistosomiasis (INC9P.443)
Authors: Ponichtera Holly , Shainheit Mara , Liu Beiyun , Raychowdhury Raktima , Larkin Bridget , Russo Joanne , Salantes Danielle , Lai Chao-Qiang , Parnell Laurence , Yun Tae , Cheong Cheolho , Bunnell Stephen , Hacohen Nir , Stadecker Miguel
Published: January 2023

The magnitude of immunopathology and pro-inflammatory cytokine production in murine Schistosoma mansoni infection is strain-dependent. Severe hepatic egg-induced granulomatous inflammation in CBA mice is associated with Th1 and Th17 cytokine responses, whereas BL/6 mice develop milder lesions in a Th2-polarized cytokine environment. Pathogenic Th17 cell responses in CBA mice are dependent on the production of IL-1β and IL-23 by egg-stimulated dendritic cells (DC); by comparison, such Th17 cells fail to develop in BL/6 mice. The reasons for strain-dependent differences in DC reactivity to eggs remain unclear. Genome-wide gene profiling revealed significant differences between CBA vs. BL/6 DCs in C-type lectin receptors (CLRs), a family of pattern recognition receptors that binds glycans such as those produced by schistosome eggs. Expression of the CLR CD209a, a murine homologue of human DC-specific ICAM-3-grabbing non-integrin (DC-SIGN), was strikingly higher in several APC populations from CBA mice; however, only CBA DC, but not macrophages, B cells, or granulocytes elicited Th17 cell differentiation in response to schistosome eggs. Gene silencing in CBA DC, and over-expression in BL/6 DC, demonstrated CD209a to be necessary for egg-induced DC production of ERK1/2 map kinase-dependent IL-1β and IL-23 as well as subsequent Th17 cell development. These findings reveal a novel mechanism controlling the development of Th17 cell-mediated immunopathology in helminthic disease.
Inhibition of TLR2 signaling by small molecule inhibitors targeting a novel putative pocket within the TLR2 TIR domain (INM9P.448)
Authors: Mistry Pragnesh , Laird Michelle , Schwarz Ryan , Greene Shannon , Dyson Tristan , Jabre Sandra , Snyder Greg , Deredge Daniel , Chauhan Jamal , Wintrode Patrick , Xiao Tsan , Sundberg Eric , Fletcher Steven , Toshchakov Vladimir , MacKerell Alexander , Vogel Stefanie
Published: January 2023

Overexuberant TLR2 signaling has been implicated in numerous diseases. Visual analysis of the TLR2 TIR domain crystal structure suggested the presence of a “pocket” adjacent to the highly conserved and functionally important proline and glycine residues of the BB loop. Given the importance of the BB loop in mediating TLR signal transduction, we hypothesized that Computer-Aided Drug Design (CADD) could be used to identify a small molecule inhibitor(s) that would fit within this “pocket” and blunt TLR2 signaling. Using CADD, ~150 small compounds were identified based on their predicted ability to bind in this pocket. Based on inhibition of IL-8 mRNA induced by TLR2 agonists, compound “C29” was found to inhibit TLR2/1 and TLR2/6 signaling in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 blocks heat-killed and live bacterial TLR2 agonist-induced proinflammatory cytokine mRNA. C29 prevents the early activation of TLR2-mediated signaling, including NF-κB and MAPKs, as well as MyD88 recruitment to TLR2 in THP-1 cells. C29 is cleavable and the byproduct, o-vanillin, reproduces comparable TLR2 inhibitory activity. O-vanillin covalently interacts with recombinant human TLR2 TIR domain dose-dependently. Mutagenesis of “pocket” residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Collectively, these results provide proof-of-principle for using CADD to identify inhibitors of TLR2 signaling.
CD40 ligation by T cells downregulates macrophage TNF production by IRAK1-mediated IL-10 expression and controls innate hyperinflammation (INM9P.462)
Authors: Inoue Makoto , Shinohara Mari
Published: January 2023

Endotoxemia is caused by excessive inflammation, but immune system has various mechanisms to avoid collateral organ damages in endotoxemia. A handful of reports have shown that innate immune responses are suppressed by the adaptive immune system. However, the molecular mechanism by which adaptive immune cells suppress innate inflammatory responses is not clear. Here, we found that T cell-macrophage interaction prolonged survival of mice in lipopolysaccharide (LPS)-induced enodotoxemia through controlling TNF levels, demonstrated by T cell reconstitution in Rag2-deficient recipients. Downregulation of macrophage TNF expression was achieved by crosstalk between CD40 and TLR4 signaling pathways, which mediated IRAK1 nuclear translocation and its binding to the Il10 promoter in macrophages, without interfering the NFκB pathway. IL-10 is then detected by macrophages in an autocrine fashion to destabilize Tnfa mRNA, resulting in attenuated TNF levels. Simultaneous stimulation of CD40 and TLR4 signaling pathways was essential to induce IRAK1-mediated IL-10 expression; because CD40 signaling licenses IRAK1 nuclear translocation by IRAK1 sumoylation in the presence of TRAF2 and intracellular isoform of osteopontin (iOPN), while TLR4 signaling provides IRF5 as a chaperone of sumoylated IRAK1 for its nuclear translocation. Our study demonstrates a mechanistic basis of the immunosuppressive role of macrophage CD40 in LPS endotoxemia.
Molecular mechanisms responsible for the dynamic modulation of macrophage responses to varying dosages of LPS (INM9P.453)
Authors: Morris Matt , Li Liwu
Published: January 2023

Monocytes and macrophages can be dynamically programmed into distinct states depending upon the strength of external stimulants. Innate programming may bear significant relevance to the pathogenesis and resolution of human inflammatory diseases. However, systems analyses of the molecular mechanisms responsible for the dynamic programming of innate leukocytes are lacking. In this study, we focused on the macrophage responses to lipopolysaccharide (LPS), a ubiquitous bacterial endotoxin recognized by Toll-like receptor 4 (TLR4). We employed both immortalized human monocytic THP-1 cells and primary murine bone-marrow derived macrophages (BMDM). We observed that varying doses of LPS induce distinct expression profiles of selected pro- and anti- inflammatory cytokines. Lower doses of LPS preferentially suppressed anti-inflammatory mediators, and selectively induced pro-inflammatory mediators. Rising LPS dosages caused a shift in expression profiles, giving rise to double-positive cells. Higher LPS dosages shifted the landscape to anti-inflammatory mediators and drove down the expression of pro-inflammatory mediators. By utilizing BMDM from IRAK-M knockout mice, we identified IRAK-M as a key player in this process, as IRAK-M deletion skews the dynamic responses to varying dosages of LPS. Our study provides a systems view of macrophage programming, and may shed light on our understanding of the dynamic balance between pathogenesis and resolution of inflammatory diseases.
Mesenchymal stem cells differentially modulate effector CD8+ T cells and exacerbate EAE. (IRC8P.484)
Authors: Glenn Justin , Smith Matthew , Whartenby Katharine , Calabresi Peter
Published: January 2023

Auto-reactive T cells are a significant cause of myelin destruction and eventual axon loss in EAE and probably multiple sclerosis (MS). Both CD4+ and CD8+ T cells acquire pathogenic IFNγ-producing- (Type I) and IL-17A-producing- (Type 17) effector phenotypes in MS and EAE. On the frontier of MS therapy, mesenchymal stem cells (MSC) have emerged as a promising candidate for inflammatory suppression and disease amelioration. Although MSC have been extensively demonstrated to suppress pathogenic effector CD4+ T cells and CD4+ T cell-mediated EAE, surprisingly few studies have addressed their modulation of effector CD8+ T cells in MS or EAE. We find that MSC differentially modulate CD8+ T cell development depending on effector T cell subtype. MSC drive activated low-IFNγ producers toward an enhanced high-IFNγ Tc1-like phenotype but strongly inhibit the production of IL-17A and Tc17 polarization in vitro. These observations are underscored by differential MSC modulation of T cell activation, proliferation, and signature transcription factor up-regulation. In addition, MSC constitutively produced a panel of Type I inflammatory cytokines associated with the IFNγ/IL-17A axis.Based on these in vitro effects on CD8+ T cells, we next evaluated their impact on the severity of EAE. To better evaluate CD8+ T cells, we immunized mice with MOG 37-50, which is a CD8-targeted epitope. Our results revealed a disease worsening, consistent with the in vitro stimulation of Tc1 cells.
Local intravaginal microencapsulated interleukin 12 treatment enhances adaptive immunity to Neisseria gonorrhoeae and induces cross-protection against heterologous strains of gonococci (IRC8P.492)
Authors: Liu Yingru , Phillips Tara , Egilmez Nejat , Russell Michael
Published: January 2023

Uncomplicated genital infection with N. gonorrhoeae does not induce specific protective immunity or immune memory. Our previous studies in a mouse genital tract infection model have demonstrated that N. gonorrhoeae selectively elicits Th17-mediated innate host defenses that it can survive and concomitantly suppresses Th1/Th2-driven adaptive immune responses that would eliminate it. Here we show that intravaginally administered IL-12 encapsulated in sustained-release polymer microspheres led to significantly faster clearance of infection. Enhanced resistance to secondary infection with the same strain of N. gonorrhoeae induced by intravaginal treatment with IL-12 microspheres during primary infection persisted for 6 months. Furthermore, treatment of mice with IL-12 microspheres during gonococcal challenge induced resistance to reinfection with unrelated strains of N. gonorrhoeae. ELISA, flow cytometry and PCR studies demonstrated that IL-12 microsphere treatment in infected mice effectively enhanced Th1 immune responses in the vaginal tract and the draining lymph nodes, without hindering Th17 responses. Although IL-12 microspheres did not change Th2 responses, the treatment significantly increased the production of gonococcus-specific circulating IgG and vaginal IgA and IgG antibodies. These results suggest that local administration of microencapsulated IL-12 may serve as a novel, effective immunomodulatory and therapeutic strategy against gonorrhea.
Pneumococcal choline-binding proteins contain an activity that mediates Streptococcus pneumoniae-induced immunosuppression (IRC8P.478)
Authors: Saumyaa Saumyaa , Colino Jesus , Snapper Clifford
Published: January 2023

We previously demonstrated that intact, inactivated Streptococcus pneumoniae (strain R36A) inhibits murine IgG, T follicular helper, germinal center and plasma cell responses to co-immunized chicken ovalbumin (cOVA), whereas intact, inactivated Streptococcus agalactiae and Neisseria meningitides did not. Since phosphorylcholine (PC), reported to mediate immunosuppression by filarial organisms, was selectively expressed by R36A, we determined whether R36A lacking PC (R36APC-) lost this inhibitory activity. R36APC- indeed, exhibited a significant loss of inhibition of the anti-cOVA response. However, R36APC- is also devoid of a family of choline binding proteins (CBPs) that are attached to the bacterial cell surface via PC binding. We thus prepared CBP-depleted R36A (R36ACBP-) by stripping CBPs from the cell surface using choline chloride. R36ACBP-, which continues to express PC, also exhibited a significant reversal in inhibition of the anti-cOVA response. Inhibition of the anti-cOVA response was also mediated by isolated R36A cell walls, supernatant from choline chloride-treated R36A, and purified CBPs. In contrast, trypsinized intact R36A and trypsinized CBP-containing supernatant lost their inhibitory activity. These results suggest a novel immunosuppressive property expressed by CBPs and CBP-expressing S. pneumoniae. Studies are continuing to specifically identify this activity.
Bias in synapse stability leads to suppression of naive CD8 T cell activation by memory CD8 T cells during secondary responses (IRC9P.709)
Authors: Mayya Viveka , Judokusumo Edward , Medina Ricardo , Zairis Sakellarios , Wiggins Chris , Kam Lance , Dustin Michael
Published: January 2023

T cells can form either a stable synapse or a mobile junction termed kinapse when interacting with antigen presenting cells during an immune response. Stable synapses can lead to long dwell times with antigen presenting cells, whereas mobile kinapses likely favor transient interactions with antigen presenting cells. The underlying cell-intrinsic mechanisms determining the mode of interaction as well as their specific immunological consequences are not known. We have implemented methods borrowed from computer-vision and machine-learning disciplines to accurately quantify the positional stability of T cell junctions formed on model antigen presenting surfaces coated with ICAM-1 and anti-CD3. We have found that human memory CD8 T cells form stable synapses whereas naive CD8 T cells form kinapses or transient contacts. This bias in synapse stability is neither observed in human CD4 T cell subsets nor in murine CD8 T cell subsets under the conditions we have tested. Increased surface expression of LFA1 in human memory CD8 T cells contributes to their increased synapse stability. Using patterned surfaces to simulate competition for antigen presenting cells, we show that synapse stability exhibited by memory CD8 T cells causes suppression of naive cell activation by preventing access to antigen. Increased synapse stability of memory CD8 T cells is expected to promote the development of CTL escape variants during hyper-mutable viral infections and thus prove detrimental to the host.
Helper T cell differentiation and regulation of inflammatory lymphangiogenesis (IRC10P.468)
Authors: Elhadad Sonia , Mehrara Babak
Published: January 2023

Lymph node lymphangiogenesis (LNL) is a critical regulator of inflammation. Although it is clear that pro lymphangiogenic factors VEGF-A and C play a central role in the regulation of this process, recent studies showed that these effects are balanced with anti-lymphangiogenic forces like IFN-g. However, although it is clear that T cells play a negative regulatory role in LNL, the effect of T cell differentiation remains unknown. We analyzed the effects of T cell differentiation on LNL in a mouse model of inflammation and analyzed draining lymph nodes at various time point by flow cytometry, histology, and RT-PCR. We found that induced inflammation, resulted in a robust inflammatory response in the draining lymph nodes beginning 7 days after injection and lasting until day 21. This process was associated with generation of a mixed Th1,Th2 responses, decreased expression of IFN-g, and increased expression of VEGF-A/C. Loss of CD4 or CD8 T cells resulted in augmentation of lymphangiogenesis, increased expression of IFN-g, and VEGF-C. Treatment of mice with IFN-g, IL13, or IL17A neutralizing antibodies resulted in augmented LNL and increased expression of VEGF-C and IFN-g. Loss of either CD4 or CD8 T cells resulted in augmentation of LNL as a result of increased VEGF-A/C expression. Similarly, inhibition of Th1, Th2, or Th17 cytokines increase LNL and increased VEGF-A/C expression. In conclusion, we showed that T cell differentiation modulates inflammatory LNL.
Evaluation of IL-2 therapy on human immune system homeostasis in humanized mice (LYM8P.731)
Authors: Durost Philip , Shultz Leonard , Brehm Michael
Published: January 2023

Regulatory T cells (Treg) are an IL-2 dependent subset of CD4 T cells with the capacity to suppress immune responses. In mice, IL-2 treatment can prevent autoimmune disease and rejection of allogeneic tissues by selective expansion of Treg populations. Moreover clinical trials have shown that low dose IL-2 therapy increases Treg in humans and is effective in ameliorating immune disorders. However, low dose IL-2/rapamycin therapy in type 1 diabetic individuals was less effective and had potentially detrimental effects on beta cell function. The unpredictability of IL-2 therapy in humans emphasizes the need for novel models to fully evaluate therapies targeting human Treg before being used in the clinic. Here we have evaluated the impact of IL-2 treatment on human immune systems using a humanized mouse model. For these studies we used NOD-scid IL2rγnull (NSG) mice engrafted with fetal thymus and liver tissues and human HSC [NSG-BLT (bone marrow, liver, thymus) mice]. Human T cell populations develop efficiently in NSG-BLT mice making this model ideal to study T cell specific therapies. NSG-BLT mice treated with an AAV vector expressing human IL-2 showed a significant increase in the number of both functional human Tregs and human NK cells. IL-2 expression did not alter T effector/memory T cells subsets. Collectively, these data show that humanized mouse models are an ideal platform for testing human-specific immune therapies.
The role of CD4+ T cell subsets in the clearance of Pneumocystis murina pneumonia in mice (MPF6P.734)
Authors: Zheng Mingquan , Ripple Michael , Yu Amy , Cleveland Hillary , Stefanovski Matthew , Chen Kong , Kolls Jay
Published: January 2023

Pneumocystis (PC) Pneumonia remains a serious complication in the immuno-compromised host. The host defense against PC is critically dependent upon CD4+ T cells. The role of specific T-cell subsets that mediate host resistance against PC infection remains unclear. We use STAT transcription factor knockout (KO) mice to determine the role of Th1, Th2, and Th17 cells in PC infection. After 28 days infection with PC, serum, lung tissue, and mediastinal lymph node (LN) were collected. LN and lung tissue cells were measured for cytokines by ELISpot and intracellular staining. Total lung RNA was measured for PC burden by RT-qPCR. Cytokines were assayed from cell culture supernatant. Anti-PC antibody was measured by ELISA.Stat4, Stat6 single KO, and Stat4/6 double KO mice all were resistant to PC compared to the Rag2/gc KO mice. Stat4 was required for a Th1 response and Stat6 was required for a Th2 response. STAT3fl/fl/CD4Cre+ mice had partial susceptibility of PC. Stat3 expression was required in CD4+ T cells for PC specific Th17 responses. STAT4/6 double KO crossed to STAT3fl/fl/CD4Cre+ (Triple KO) mice were highly susceptible to PC infection compare to single and double KO mice. Individual Stat4 and 6 and double mutants clear PC. STAT3fl/fl/CD4Cre+ conferred partial susceptibility to PC. Triple KO were as susceptible for PC as Rag2/gc KO mice. These data support the hypothesis that PC is a true opportunist and that various T-cell subsets can confer protection against infection.
Cell stress molecules expression in periodontal disease (MUC7P.762)
Authors: Aguilera Galaviz Luis , Aceves Medina Maria , Hernandez Montoya Martha , Diaz Rosas Cristal , Frausto Esparza Silverio , Estrada Garcia Iris
Published: January 2023

Periodontal Disease is bacterial infection of tooth-supporting tissues leading to chronic inflammation and loss of teeth, the immunopathologic mechanisms include inflammatory cells and proinflammatory cytokines; persistent local inflammation develops the expression of stress molecules as Hsp90, Hsp70 and P53 also autoantibodies. The rol of cellular stress and autoimmunity in periodontal disease is not understood. Objetives: Determine the expression of cell stress molecules and auto-antibodies in periodontal disease patients. Methods: 34 patient with severe chronic periodontitis and 14 healthy people were clinical evaluated. Periodontal tissue and sera were obtained. Electroforesis, Western blot and Immunodetection to identify Hsp70, Hsp90 and P53 was set up, Hsp 90 were evaluated by RT-PCR and IFI for ANCA, antiepithelial antibodies in mouse skin and ANA using Hep2 cells. Results: 57% were positives for ANCA, total of sera were positives for nuclear and/or cytoplasmic antibodies and 57% presented anti-epithelial antibodies, Hsp90, Hsp70 and P53 is expressed according with the severity of disease. Conclusions: The expression profile of Hsps in periodontal tissue is according with the severity, and could be associated with cellular stress because of the damage mechanism induced by periodontal pathogens and trigger necrotic or apoptotic phenomena. Destruction of extracellular matrix in periodontal tissue could be mediated by auto-antibodies that recognize self structures.
Human chorionic gonadotropin supports human pregnancy by induction of regulatory T cells (MUC7P.766)
Authors: Schumacher Anne , Poloski Eileen , Zenclussen Ana
Published: January 2023

Regulatory T cells (Treg) are proposed to be involved in fetal tolerance induction. However, the precise mechanisms underlying their generation and function are still not defined. Recently, we proved that the pregnancy hormone human Chorionic Gonadotropin (hCG) efficiently attracts human Treg to trophoblasts favoring a local Treg accumulation. Here we analyzed whether hCG is also involved in Treg generation and activity. Naïve T cells were isolated from blood of pregnant women and co-cultured with either hCG-producing trophoblasts (JEG-3, primary cells) or non-hCG-producing trophoblasts (HTR8, Swan71) and keratinocytes (HaCat). To confirm hCG specificity, we either blocked hCG in JEG-3 co-cultures or used urine-purified or recombinant hCG as culture supplements. Treg induction was then assessed by the expression of specific Treg markers. Furthermore suppressive function of hCG-treated naïve T cells was evaluated in a mixed leukocyte reaction. Co-culture of naïve T cells with JEG-3, primary trophoblasts or supplemented hCG resulted in a statistically significant increase in the expression of specific Treg markers as compared to co-cultures with HTR8, Swan71 or HaCat cells. Moreover, blockage of hCG in JEG-3 co-cultures impaired the up-regulation of Treg markers. Interestingly, we further confirmed an increased suppressive activity of naïve T cells after hCG treatment. Our results strongly suggest a key role for hCG in Treg generation and induction of suppressive activity.
Uterine natural killer cells: their modulation by carbon monoxide and their impact on the remodeling of spiral arteries (MUC7P.768)
Authors: Zenclussen Ana , Schumacher Anne , Linzke Nadja
Published: January 2023

Pre-eclampsia (PE) and intrauterine growth restriction (IUGR) are linked to impaired remodeling of maternal spiral arteries (SA). We previously introduced Heme Oxygenase-1 (HO-1) as a positive modulator of pregnancy. Its main mediator, carbon monoxide, could mimic HO-1 protective effects in a mouse model of Hmox1 genetic deficiency. Here, we investigated whether HO-1 deficiency is related to gestational hypertension and if CO is able to prevent PE and IUGR through the modulation of uterine NK cells (uNK) that are necessary for initiation of SA remodeling. Hmox1+/- or Hmox1-/- implantations presented fewer uNK cell numbers and lower expression of uNK-related angiogeneic factors than Hmox1+/+ sites. Quantitative histology revealed that Hmox1+/- and Hmox1-/- implantations had shallow SA development. This was accompanied by IUGR and gestational hypertension. Application of CO at low dose during early to mid-gestation prevented IUGR in Hmox1+/- mothers, being this associated with enhanced in situ proliferation of uNKs and normalization of angiogenic parameters. Most importantly, CO improved SA remodeling and normalized blood pressure, ensuring a proper fetal growth. Thus, CO emerges as a key molecular player in pregnancy success by modulating uNKs, which results in promotion of SA remodeling, adequate fetal support/growth and prevention of gestational hypertension.
Characterization of IL-22 and IL-17 expressing cells in cervix (MUC7P.769)
Authors: Makinde Hadijat , Lurain Nell , Martinson Jeffrey , Mirmonsef Paria , Landay Alan , Spear Greg
Published: January 2023

IL22 and IL17 have important antimicrobial and epithelial repair functions. However their role in the female lower genital tract has not been well defined. We determined the frequency and phenotype of cells producing these cytokines at this mucosal site, because we postulated that they contribute to resistance to sexually transmitted infections. Cervical tissue was obtained from benign hysterectomies. Cells were obtained by collagenase digestion, stimulated with PMA/ionomycin/Monensin for 4 hrs and stained for flow cytometry. On average in endocervical tissue, the CD3+CD45+ cells were 71% (CD4+ ~50%) and CD3- CD45+ cells were 24%. 20% of CD45+ cells were Lin- (negative for CD3, CD14, CD16, CD19, CD20 and CD56). Unstimulated CD4+ T-cells were < 0.05% IL22+, IL17+ and IL22+IL17+, while after stimulation the percentages were 1.9%, 5.5% and 1.6% respectively. Unstimulated CD3- cells in cervical tissue were 2.3% IL22+, 3% IL17+ and 4.6% of IL22+IL17+, and the percentages after stimulation were 1.6%, 6% and 1.9% positive respectively. Our data show that in cervical tissue, there are CD3+ and CD3- cells capable of making IL22 and IL17. This is important because this is the first study to show what types of cells in the genital tract are making these cytokines, and suggests that these cells could induce antimicrobial peptide production by cervical epithelial cells in response to sexually transmitted pathogens.
Uterine integrity is required to maintain human fetal immunologic naiveté (MUC7P.770)
Authors: Loewendorf-Snead Andrea , Nguyen Tina , Yesayan Maria , Kahn Daniel
Published: January 2023

Introduction: In healthy pregnancy, maternal and fetal compartments are physically separated by multiple cell layers and the fetal immune system displays an ‘in-experienced’ phenotype in utero. The uterus as an immunologic barrier contributing to the naïve state of the fetal immune system has not been explored thus far. Methods: A prospective cohort study of fetuses born to mothers with prior uterine scar was undertaken (UCLA IRB # 11-002962). Cord blood lymphocytes were analyzed for memory status of the T regulatory cells (CD4+FoxP3+RO/RA) and blinded from placental location prior to final analysis. Results: In this prospective cohort study, we identified placental implantation in apposition to a uterine scar as a sufficient factor for fetal in utero immune activation (CD45RO+), specifically in the regulatory T cell compartment. Our results (N=20) identify a risk difference of 90% with a relative risk of 10 (p<0.05) of activation (RO+) of the FoxP3+ regulatory T cells when the placenta was implanted over the prior uterine scar. Discussion: Our study demonstrates that intrauterine formation of tolerogenic fetal responses is caused by fetal exposure to a scarred uterus. Our results highlight the role that uterine integrity plays as an immunologic barrier.