bluuebunny/crossref_metadata_2025 huggingface dataset:
Defining the targets of Dengue virus infection in human skin (VIR8P.1069)
Authors: Duangkhae Parichat , Smith Amanda , Marques Ernesto , Ryman Kate , Barratt-Boyes Simon
Published: January 2023

Dengue is the most important arboviral infection of humans. Dengue virus (DENV) is introduced into human skin by the bite of infected mosquitoes. While Langerhans cells (LCs) have been implicated as the main targets of DENV infection the dynamics of infection of human skin remain ill-defined. Here, we exposed skin explants from healthy human donors to DENV to determine the targets of infection. Skin explants were inoculated with DENV and analyzed by immunofluorescence after intervals of incubation using antibodies to cell-specific markers and viral protein NS3. Time course experiments showed that the first targets of DENV infection were basal keratinocytes, with infection detected within 8 h. From 12-48 h of infection abundant virus replication was detected in epidermal LCs and dermal macrophages (MØs) as well as tubular structures consistent with lymphatic endothelium. Quantitative analysis indicated that exposure to DENV resulted in significant infiltration of MØs into dermis. These preliminary data suggest that DENV initially infects basal keratinocytes which may release factors promoting the mobilization of infected LCs out of the epidermis, and the influx of MØs into the dermis, which subsequently replicate DENV to high levels. Ongoing experiments are designed to determine the mechanism for MØ recruitment. These studies are revealing that DENV infection of human skin is a dynamic process involving sequential infection and recruitment of distinct cellular targets.
Hepatocytes intrinsic STAT4 regulates auto-immune liver inflammation (BA3P.209)
Authors: Kumar Pawan , Chen Kong , Pociask Derek , Kolls Jay
Published: January 2023

Autoimmune hepatitis is a condition where immune cells target and destroy liver cells. The role of STAT4 in autoimmune hepatitis is ill-defined. Therefore, we investigated the role of STAT4 signaling in Concanavalin A (ConA)-induced auto-immune hepatitis model. Our data indicate Stat4-/- mice but not WT, Il12p35-/- or Il12p40-/- mice were resistant to ConA-induced hepatitis as measured by liver pathology and the serum alanine aminotransferase (ALT) level. Furthermore, no significant difference in serum ALT and necrotic lesions were observed when WT or Stat4-/- spleenocytes were adoptive transfer into lymphocyte deficient Rag2gc-/- mice, suggesting that protection in Stat4-/- is independent of lymphocyte STAT4. Interestingly, serum IFNg level were significantly reduced in both Il12p35-/- and Stat4-/- mice, and injection of recombinant IFNg into Stat4-/- mice increase the serum ALT, but it does not reach the level observed in WT mice, suggesting a non-IFNg mediated protection in Stat4-/- mice. Surprisingly, there were no significant difference in FAS and FasL transcript in ConA injected liver tissue in WT and Stat4-/- mice. In addition, anti-FAS antibody injections lead to significant necrotic changes in WT, but not in Stat4-/- mice. Our data strongly indicate absence of STAT4 signaling potentially in hepatocytes regulates cell survival, which is further confirmed by significantly more pSTAT3 staining in liver tissue of Stat4-/-.
The nuclear receptor Nur77 suppresses CNS autoimmunity by limiting myeloid catecholamine production (BA3P.134)
Authors: Shaked Iftach , Hanna Richard , Ley Klaus
Published: January 2023

In multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalitis (EAE), recruitment of peripheral autoreactive leukocytes to the central nervous system (CNS) is central for pathogenesis. Although sympathetic norepinephrine (NE) has been described as a key driving force of leukocyte recruitment, molecular mechanisms that control this process have not been fully elucidated. Macrophages can also produce NE, but the importance of this activity has not been addressed in the MS context. Nur77, an orphan nuclear receptor involved in differentiation of monocytes, can be activated by both inflammatory and sympathetic stimuli. Here we show that in macrophages, Nur77 is essential to regulate expression of tyrosine hydroxylase (TH), a rate-limiting enzyme in catecholamine biosynthesis. Mice with global or myeloid-specific Nur77 deletion exhibited enhanced TH expression and elevated blood NE levels, which triggered excessive production of IL-6, CXCL1, CCL2 and CCL5, ultimately leading to accelerated and exacerbated EAE. Live cell imaging of the spinal cord revealed enhanced infiltration of encephalitogenic 2D2 T cells and monocytes, and hastened activation of microglia in Nur77-deficient mice. Consistently, exacerbated EAE in Nur77-deficient mice was ameliorated by TH inhibition or by α1 adrenergic receptor blockade. Our data identify Nur77 as a key nuclear receptor regulating myeloid catecholamine production and CNS autoimmunity.
X chromosome dose and sex bias in autoimmune diseases: increased 47,XXX in systemic lupus erythematosus and Sjögren’s syndrome (BA4P.211)
Authors: Liu Ke , Kurien Biji , Zimmerman Sarah , Kaufman Kenneth , Mariette Xavier , Kottyan Leah , Jonsson Roland , Ng Wan-Fai , Omdal Roald , Rischmueller Maureen , Wahren-herlenius Marie , James Judith , Lessard Christopher , Thompson Susan , Gaffney Patrick , Montgomery Courtney , Siminovitch Katherine , Edberg Jeffrey , Kimberly Robert , Tsao Betty , McCune W. , Salmon Jane , Sivils Kathy , Harley John , Scofield R.
Published: January 2023

Mechanism for female predominance in autoimmunity is unknown. We suspected an X chromosome dose effect and predicted if so, triple X (47,XXX, 1 in ~1,000 live female births) would be increased in female predominant diseases (systemic lupus erythematosus [SLE], primary Sjögren’s syndrome [SS], primary biliary cirrhosis [PBC] and rheumatoid arthritis [RA])compared to diseases without female predominance (sarcoidosis, granulomatosis with polyangiitis [GPA]) and healthy controls. We used single nucleotide polymorphism (SNP) arrays to identify 47,XXX and fluorescent in situ hybridization, or q-PCR to confirm when possible. 47,XXX was found in 7 of 2,948 SLE and 3 of 1,053 SS female patients, but in none of the 4,822 female controls (OR≥21.31, 95% CI: 2.36-∞, p=0.001 and OR≥22.95, 95% CI: 1.89-∞, p=0.006, respectively). One 47,XXX was present for every ~421 SLE women and ~351 SS women. In addition, we identified one 47,XXX from each of 1,159 women with PBC and 943 with sarcoidosis. No 47,XXX was identified among 453 women with RA or 247 with GPA. In conclusion, 47,XXX was present in excess among SLE and SS subjects as predicted by X chromosome dose effect. These estimated prevalence of SLE and SS with 47,XXX being respectively ~2.4 and ~2.8 times higher than in women with 46,XX and ~24 and ~39 times higher than in men with 46,XY. There was no increase of 47,XXX is other female-biased diseases, suggesting multiple pathways to such a bias in autoimmunity.
A new mTOR-dependent, Akt-independent pathway differentiates the migratory behavior of regulatory and conventional T cells (CAM1P.235)
Authors: Raimondi Giorgio , Chen Leo , Rosborough Brian , Thomson Angus
Published: January 2023

The vitamin A metabolite all-trans retinoic acid (ATRA) induces a gut-homing phenotype in activated CD4+ conventional T cells (Tconv) by upregulating the integrin α4β7 and the chemokine receptor CCR9. We report that in contrast to mouse Tconv, only half of regulatory T cells (Treg) upregulate CCR9 when stimulated in presence of ATRA, even though Tconv and Treg express identical levels of receptor (RAR). The bimodal distribution of CCR9+ versus CCR9- Treg subsets is not associated to differences in proliferation, level of FoxP3 expression, or segregation between thymic-derived and induced Treg. Furthermore, exposure to the mTOR inhibitor rapamycin suppresses upregulation of both CCR9+ and α4β7 on Treg, an inhibitory effect not evident in Tconv. This suggests that ATRA-induced upregulation of CCR9 and α4β7 on Treg is dependent on activation of an mTOR signaling pathway. This mTOR involvement is independent of Akt activity, since specific inhibition of Akt or its downstream target GSK-3B did not prevent CCR9 expression. Additionally, Rictor-/- Treg (that lack mTORC2) showed unaltered expression of CCR9 compared to WT control, suggesting participation of mTORC1 or another mTOR complex. These findings reveal a novel difference between ATRA signaling and chemokine receptor induction in Treg versus Tconv, and provide a framework via which the migratory behavior of Treg versus Tconv can be differentially influenced for therapeutic purposes.
Absence of L-selectin accelerates atherosclerosis via the modulation of B cell subsets and local immune response within the aortas of L-selectin-deficient Apoe-/- mice. (CAM1P.236)
Authors: Galkina Elena , Taghavie-Moghadam Paresa , Ley Klaus , Gjurich Breanne
Published: January 2023

To date, a significant body of evidence implicates the immune system in atherosclerosis. Studies report that follicular B cells are pro-atherogenic whereas B1a cells play a protective role; yet the role of other B cell subsets is unclear. The mechanisms behind the regulation of cell subset specific homing into aortas are not well understood. To determine the role of L-selectin in atherosclerosis, we bred L-selectin deficient (Sell-/-) and Apoe-/- mice and generated Sell-/-Apoe-/- mice. L-selectin deficiency resulted in accelerated atherosclerosis that was accompanied by a 1.3-fold increase in the number of leukocytes within aortas of Sell-/-Apoe-/- mice. However, Sell-/-Apoe-/- aortas displayed a 2-fold reduction in aortic B cells. Importantly, L-selectin deficiency significantly reduced the percentage and number of B1a and regulatory B (Breg) cells within Sell-/-Apoe-/- compared to Apoe-/- aortas and spleens. We found more B1a expressing L-selectin compared with splenic FO B cells, suggesting a potentially high dependence of B1a cells from L-selectin. With the reduction of aortic B1a and Breg, there were reduced IL-10 levels in Sell-/-Apoe-/- aortas. In line with the increased aortic leukocyte cellularity, there was a 1.5-fold increase in aortic CD68hi macrophages. These data provide evidence for a functional role of L-selectin in atherosclerosis via the regulation of the content of aortic B1a and Breg cells and the modulation of the local immune response within the aorta.
Dynamic regulation of LFA-1 and Mac-1 during neutrophil extravasation in live mice (CAM1P.232)
Authors: Hyun Young-Min , Capece Tara , Lim Kihong , Wong Jennifer , Kim Minsoo
Published: January 2023

LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) are key integrins that regulate leukocyte adhesion and migration in inflamed tissues. Despite considerable advances in our understanding of leukocyte migration, real-time regulation of leukocyte integrins in vivo is not well characterized. To this end, we generated knock-in mice in which CD11a, CD11b, and CD18 were fused with mYFP and mCFP, respectively, to generate the CD11a-mYFP, CD11b-mYFP, and CD18-mCFP mice. We then crossed these mice to generate LFA-1 FRET (CD11a-mYFP/CD18-mCFP) and Mac-1 FRET (CD11b-mYFP/CD18-mCFP) mice. Using FRET assay with multiphoton intravital microscopy (MP-IVM), we now can directly measure LFA-1 and Mac-1 activation during neutrophil extravasation in inflamed tissues. Neutrophils showed distinct patterns of distribution of active integrins and their clustering at different steps in the multistep extravasation cascade. Experiments with blocking antibodies and knock-out mice confirmed that LFA-1 and Mac-1 are regulated by distinct activation kinetics in vivo. Our data suggest that these knock-in mice will be useful to visualize the subcellular distribution and activation of LFA-1 and Mac-1 in real-time during leukocyte migration in intact tissues.
Mouse iNKT cell development and functions are differentially regulated by SHIP-1 (HEM3P.288)
Authors: Anderson Courtney , Salter Alexander , Reilly Emma , Toussaint Leon , Kerr William , Brossay Laurent
Published: January 2023

The SH2-containing inositol phosphatase-1 (SHIP-1) is a 5’ inositol phosphatase known to negatively regulate the signaling product of phosphoinositide-3 kinase (PI3K), phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3). SHIP-1 can be recruited to a large number of inhibitory receptors expressed on iNKT cells. We hypothesized SHIP-1 deletion would have major effects on iNKT cell development by altering the thresholds for positive and negative selection. In SHIP-1 deficient animals, iNKT cell frequency and number are impaired when compared to littermate controls. However, using both mixed bone marrow chimera and mice conditionally deficient for SHIP-1 in the T cell lineage, we show iNKT cell development is globally intact, demonstrating SHIP-1 is not required intrinsically for iNKT cell development. In addition, although SHIP-1 is necessary extrinsically for iNKT positive selection, it is dispensable on CD1d positive selecting cells. Interestingly, SHIP-1 deficient iNKT cells display functional defects including reduced cytokine production. These results further our understanding of how iNKT cell activation is regulated and provide insights into the biology of this unique cell lineage.
Id3 and Id2 collaboratively regulate innate-like γδ T cell development (HEM3P.275)
Authors: Zhang Baojun , Lin Yenyu , Dai Meifang , Zhuang Yuan
Published: January 2023

γδ T cells expressing the Vγ1.1 and Vδ6.3 TCR chains are one of growing numbers of recently characterized innate-like lymphocytes. Id3 has been shown to be a major transcription regulator to prevent the development and expansion of this population. We showed that deletion of Id3 resulted in a dramatic expansion of Vγ1.1Vδ6.3 T cells on the C57BL/6 background. This phenotype is in contrast to the much milder phenotype when the Id3 mutation was initially characterized on the 129/sv background. We conducted a quantitative trait analysis in search of the presumed Id3 modifier(s). Id2 was mapped as a major modifier of Id3 in suppressing Vγ1.1Vδ6.3 cell expansion. This mapping result was further confirmed by introduction of the targeted allele of Id2 to the Id3 mutant background. Our study led to four major observations: first, a decrease in Id2 expression on Id3 deficiency background promotes expansion of Vγ1.1Vδ6.3 cells. Second, complete elimination of both Id2 and Id3 genes triggers apoptosis instead of further expansion of Vγ1.1Vδ6.3 cells. Third, Id3 and Id2 collaboratively control survival and expansion of the γδ lineage through regulating a proper threshold of E proteins. Finally, Id proteins seem to control cell survival and expansion post Vγ1.1Vδ6.3 lineage specification. Based on these findings, we propose a dual safety mechanism to explain the role of Id3 and Id2 in regulating the population size of Vγ1.1Vδ6.3 T cells.
Injury-induced vascular intimal hyperplasia is mediated through a HMGB1- and TLR4- driven mechanism (HUM1P.304)
Authors: Cai Jingjing , Yuan Hong , Wang Qingde , Yang Huan , Billiar Timothy , Chen Alex F.
Published: January 2023

Aim: To determine whether high-mobility group box 1 (HMGB1) and Toll like receptor 4 (TLR4) drive the inflammatory cascade that promotes intimal hyperplasia (IH) following acute vascular injury. Methods and Results: Carotid artery wire injury in C57BL/6 mice induced a significant increase in intima to media (I/M) ratio at four weeks. Global deletion of HMGB1 using an inducible knockout mouse strain caused prevention of IH. IH was decreased by over 50% in WT mice treated with HMGB1 neutralizing antibody. Of knockout mouse strains deficient in putative receptors for HMGB1, TLR4-/- mice showed the greatest inhibition of IH. Both anti-HMGB1 treated mice and TLR4-/- mice exhibited a marked decrease in monocytic recruitment. Mice with selective depletion of TLR4 from macrophage exhibited a similar level of inhibition of IH to that seen in the global TLR4-/-. In vitro, dithiol HMGB1 dose-dependently promoted SMC migration and MCP-1/CCR2 expression, which was abolished by TLR4 inhibitory peptide. Conditioned media from HMGB1 treated macrophage induced SMC proliferation and SMC mitogen production, which was also diminished by blocking TLR4 on macrophages. Conclusion: Innate immune mechanism known to be involved in the sterile inflammatory response are involved in the acute vascular injury responses leading to IH. HMGB1 promotes IH by inducing vascular SMC migration and chemokine production via TLR4 pathway while simultaneously triggering SMC mitogen release by infiltrating monocytes.
Host genotype interaction with cigarette smoke determines modulation of immune response (HUM2P.342)
Authors: Taneja Veena
Published: January 2023

While the etiology of rheumatoid arthritis (RA) is unknown, both genetic and environmental factors are involved in pathogenesis. Among genetic factors, HLA-DRB1*0401/DQ8 provide the strongest association. Among environmental factors, Cigarette smoking (CS) has been associated with a 2-fold increase in risk for developing seropositive RA; specially individuals carrying DRB1*0401 who smoke cigarettes. It has been hypothesized that the response to Vimentin precedes the onset of RA. To determine the interaction between genetic factors and CS, we generated transgenic mice expressing RA-susceptible DRB1*0401 and DQ8 genes in the absence endogenous class II molecules. To determine how cigarette smoke (CS) interacts with host genetic factors in the induction of RA-associated autoimmunity, we used transgenic mice carrying the RA-susceptible HLA genes DR4 and DQ8, but lacking all endogenous murine class II molecules. Cigarette smoke exposure augmented immune responses to citrullinated synovial antigens, collagen and vimentin. Interestingly, CS exposure suppressed collagen-induced arthritis (CIA) in DRB1*0401 mice even though innate immune response was enhanced. On the other hand, CS exposure exacerbated CIA in DQ8 mice, which was accompanied by an increased expression of Th17 gene transcripts in lungs. These observations suggest that cigarette smoke promotes antigen-specific autoimmunity that is profoundly influenced by host genetic factor.
Inflammatory disease due to selectively impaired NF-kB activation and type I interferon response resulting from a de novo human NEMO hypomorphic mutation (HUM2P.333)
Authors: Wessel Alex , Hsu Amy , Goldbach-Mansky Rapheala , Zilberman-Rudenko Jevgenia , Siegel Richard , Hanson Eric
Published: January 2023

Hypomorphic mutation of the NF-kB essential modulator (NEMO) causes a spectrum of inflammatory disease and immunodeficiency. NEMO is critical in immunity, regulating the activation of transcription factors NF-kB and IRF3 to control inflammation and antiviral type I interferon. In an individual with persistent inflammatory disease, we identified a synonymous mutation in the gene encoding NEMO that enhances alternative splicing and production of a mutant protein (Δ-NEMO). Co-immunoprecipitation studies in patient T cells revealed impaired association of Δ-NEMO with TANK (TRAF family member-associated NF-kB activator) and, consequently, with the IRF3 kinase TANK-binding kinase 1 (TBK1). Concomitantly, patient fibroblasts displayed impaired production of IRF3-dependent type I and III interferon in response to poly(I:C) and permitted increased virus propagation. However, cells containing Δ-NEMO exhibited enhanced IRF3 nuclear translocation and binding to the IFNB1 promoter. Our results suggest that defective interferon production in this patient lies instead in impaired NF-kB activation, as poly(I:C)- and 5’pppRNA induced translocation of NF-kB in patient fibroblasts was reduced. This impairment in NF-kB was specific, as TNF-induced NF-kB activation and gene induction was normal. These data describe a novel mechanism of autoinflammatory disease due to a type I interferon deficiency. Thus, this represents a model of inflammatory diseases responsive to type I IFN therapy.
Structure-function relationships that govern FcεRI signaling by allergens (HYP3P.344)
Authors: Wilson Bridget , Mahajan Avanika , Lidke Diane , Tung Chang-Shung , Manavi Kasra , Tapia Lydia , Bradbury Andrew , Hlavacek William
Published: January 2023

Multivalent antigens trigger robust FcεRI-dependent signaling responses in mast cells and basophils. Most prior studies have relied on chemically heterogenous artificial ligands, rendering it difficult to predict receptor cluster size and orientation and to translate the observations to clinical relevance. This work focuses on the use of structurally defined polyvalent antigens to crosslink IgE-FcεRI, including a symmetrical trivalent ligand (DNP3-fibritin) and natural allergen (shrimp tropomyosin). In silico docking and computer simulation methods were used to build structural models of ligand-IgE-FcεRI ectodomain complexes and to estimate aggregation properties, including distances between individual receptors after crosslinking. Signaling responses were studied in “humanized” RBL-2H3 cells and in human basophils from allergic subjects. High resolution imaging approaches were employed to provide insight into the kinetics of crosslinking and the nanoscale organization of FcεRI signaling. The bell-shaped secretory responses are shown to be linked to the balance of positive (Syk-mediated) and negative (SHIP-mediated) signaling, as well as to differences in aggregate size, complexity and time course. Structurally defined antigens provide an improved basis for understanding FcεRI signal initiation and termination. These studies lay the foundation for understanding the essential structural features of natural allergens, such as valency, epitope distance and affinity.
The ribonucleotide reductase inhibitors didox and trimidox suppress IgE-stimulated mast cell cytokine secretion (HYP3P.350)
Authors: McLeod Jamie , Elford Howard , Ryan John
Published: January 2023

Ribonucleotide reductase is the enzyme responsible for catalyzing the production of deoxynucleotide triphosphates (dNTPs), essential for proper DNA synthesis and cell cycle progression. Inhibitors of ribonucleotide reductase have become attractive therapeutic agents for the treatment of conditions characterized by excessive cell proliferation or inappropriate immune cell activation, including myeloproliferative disorders, psoriasis and cancer. Late phase IgE-stimulated mast cell (MC) activation elicits sustained inflammation and is marked by synthesis and secretion of cytokines that have been implicated in the inflammatory responses associated with rheumatoid arthritis, multiple sclerosis and heart disease. Given the potential utility of ribonucleotide reductase inhibitors as cytostatic agents that may influence immune cell activation, we investigated the anti-inflammatory abilities of Didox and Trimidox as a therapeutic approach to attenuate the allergic response. Our results have found that Didox and Trimidox pre-treatment yields a dose-dependent reduction in IgE-stimulated bone marrow-derived mast cells (BMMC) cytokine secretion. These effects are not directly linked to an increase in cell death. Together this preliminary data could indicate a role for Didox and Trimidox in the treatment of MC-associated inflammatory and autoimmune diseases.
LjC1qDC-1, a novel C1q-domain containing protein from lampetra japonica involving in the immune response (INM2P.428)
Authors: Li Qingwei , Pang Yue , Liu Ge , Wang Shiyue , Pan Xiong
Published: January 2023

The C1q domain containing (C1qDC) proteins are defined as a family of all proteins containing the globular C1q (gC1q) domain in the C-terminus. They participate in a series of immune responses depending on versatile gC1q domain which can bind a variety of self and non-self ligands. In this study, a novel C1qDC protein was identified from Lampetra japonica (designated as LjC1qDC-1). Its full-length cDNA was of 1662 bp with an open reading frame (ORF) of 711 bp encoding a polypeptide of 236 amino acids. The deduced protein contained a signal peptide, a collagen-like sequence with Gly-Xaa-Yaa repeats, and a typical gC1q domain. Real-time PCR analysis showed that the transcript of LjC1qDC-1 gene was distributed in various immune associated tissues of lamprey. This observation was further confirmed by FACS and confocal laser scanning microscope. In addition, LjC1qDC-1 interaction with specific VLR was analysised by co-immunoprecipitation and western blot. All these results indicated that LjC1qDC-1 gene identified may play an important role in the immune responses of lamprey, which provided a clue to understand the universal function of C1qDC proteins in different species.
Differential expression profile of long non-coding RNAs from Gr-1+ CD11b+ myeloid cells in response to sepsis in mice (INM2P.424)
Authors: Shu Qiang
Published: January 2023

Background: Emerging evidence shows that small noncoding RNAs are involved in the immune response to sepsis. While, whether long noncoding RNAs (lncRNAs) are associated with the immune response to sepsis is currently unexplored. So we performed a global expression profile of lncRNAs from myeloid-derived suppressor cells (MDSCs), which are an important population that mediate immune suppression in response to sepsis, defined by the cell-surface phenotype Gr-1+ and CD11b+. Methods: Sepsis was induced by cecal ligation puncture (CLP) in mice. MDSCs were sorted from spleens by magnetic selection and FACS 72 h after CLP or sham surgery. The Arraystar lncRNA microarray was used to profile the genome-wide lncRNA expression. The expression pattern and classification of lncRNAs were evaluated by bioinformatics. Results: The microarray provided 16,551 lncRNAs in the septic group and 17,381 in the sham group. After sepsis, among the lncRNAs expressed in both groups, 139 were upregulated and 268 downregulated by ≥ 1.5-fold compared with sham. Nearly half of the differentially-expressed lncRNAs were intergenic RNAs. qRT -PCR confirmed the lncRNA expression pattern of microarray hybridization. Conclusions: The present study provided the first global profile of lncRNA expression in response to sepsis. The expression pattern of lncRNAs in MDSCs was affected by sepsis. Future researches are needed to determine candidate lncRNAs as biomarkers or therapeutic targets for sepsis.
PD-1 inhibits Th1 responses during cryptococcus neoformans infection (IRC2P.452)
Authors: McBerry Cortez , Sakai Shunsuke , Kauffman Keith , Barber Daniel
Published: January 2023

PD-1 is an inhibitory receptor expressed on the surface of activated T cells. PD-1 previously has been shown to play a significant role in regulating effector T cell responses against viral infections and tumors. However, its role in the control of CD4 T cell responses during infection with fungal pathogens remains poorly understood. Here we examine the role of PD-1 in regulating host responses and resistance to the opportunistic fungal pathogen Cryptococcus neoformans. We find that in wild-type mice C. neoformans elicits Th1, Th2, and Th17 responses, and PD-1 is expressed on each of these effector T cell subsets. In PD-1 deficient mice, the frequency of IFN-γ-producing and t-bet expressing CD4+ T cells were increased, while CD4+ T cells making IL-13, IL-5, and IL-4 were decreased and Th17 responses were unchanged. Also, PD-1 deficient mice show a decrease in eosinophils in the lung, and in fungal burden. These data suggest that PD-1 impairs host resistance to C. neoformans by selectively inhibiting Th1 responses. The work was supported by the NIH Intramural Research Program.
Actin-binding protein 1 links B-cell receptor to negative signaling pathways (IRC3P.462)
Authors: Song Wenxia , Seeley-Fallen Margaret , Onabajo Olusegun , Tan Tse-Hua , Upadhyaya Arpita
Published: January 2023

Prolonged or uncontrolled B-cell receptor (BCR) signaling is associated with autoimmunity. We previously demonstrated a role for actin in BCR signal attenuation. This study reveals that Actin-binding protein 1 (Abp1/HIP-55/SH3P7) is a critical negative regulator of BCR signaling, and is responsible for linking actin remodeling to negative regulatory pathways of the BCR. In Abp1 knockout mice, the numbers of spontaneous germinal center B cells and marginal zone B cells are significantly increased, while early B-cell development is unaltered. Serum levels of autoantibody and total antibody are elevated, while T-dependent antibody responses are markedly reduced. Upon activation, surface BCR clustering is enhanced and B-cell contraction is delayed in Abp1-/- B cells; concurrent with slow but persistent increases in F-actin at BCR signalosomes. Furthermore, BCR signaling is enhanced in Abp1-/- B cells compared to wild-type B cells, including increased levels of Ca2+ flux and phosphorylated BLNK, MEK1/2 and ERK, coinciding with reductions in the recruitment of the inhibitory signaling molecules HPK1 and SHIP-1 to BCR signalosomes. Our results indicate that Abp1 contributes to the maintenance of B-cell tolerance via negatively regulating BCR signaling, by promoting actin-dependent B-cell contraction and activation of inhibitory signaling molecules. This reveals a new mechanism for negative regulation of BCR signaling.
A unique population of induced age associated B cells (iABC) are generated in aged mice in response to IAV (IRC3P.465)
Authors: Zhang Wenliang , Kuang Yi , Swain Susan
Published: January 2023

With age, mice generate lower levels of isotype-switched IgG antibody (Ab) following immunization with influenza A (IAV). The total IgG and all the IgG isotypes in serum are 10-100-fold lower in aged mice. In contrast the levels of IgM are roughly equivalent to those of young ones. We find defects in germinal center Tfh in the aged that likely contribute to poor IgG response. We analyzed whether there were also intrinsic changes in the aged B cells that favor IgM production. We found a unique population of Fashi/GL7neg induced age-associated B cells (iABC) after IAV infection containing many IgM-secreting plasma cells (PC). The iABC were detected as early as 3 days post infection in secondary lymhoid sites and their generation was CD4 T cell independent. In the bone marrow 60% of the IgM+ PC were from iABC, while traditional follicular B cells gave rise only to IgM- plasma cells. Thus an alternate source of IAV-specific Ab is generated in infected aged mice that may contribute promote viral clearance. This finding may provide a new target for immunotherapies and strategies for improving antibody response in the elderly.
Regulatory T cells use LAG-3 as the “brake” in autoimmune diabetes but the “accelerator” in tumor environment (IRC4P.487)
Authors: Zhang Qianxia , Beres Amy , Vignali Dario
Published: January 2023

Lymphocyte Activation Gene-3 (LAG-3, CD223) is an inhibitory receptor that is expressed upon TCR activation, is highly up-regulated on exhausted conventional T cells, and regulates T cell expansion, homeostasis and function via both cell intrinsic and extrinsic mechanisms. LAG-3 is also required for maximal regulatory T cell (Treg) function. Our lab has previously shown that LAG-3 modulates tolerance mechanisms, but the specific function of LAG-3 expressed by Tregs remains unknown. To elucidate the role of LAG-3 in Treg-mediated immune suppression, we generated mice in which active, membrane-expressed LAG-3 is specifically deleted in Foxp3+ Tregs. We used both the non-obese diabetes (NOD) and B6 mouse backgrounds to study the role of LAG-3-deficient Tregs in autoimmune diabetes and B16 melanoma, respectively. Surprisingly, the deletion of LAG-3 in Tregs both protected NOD mice from autoimmune diabetes and resulted in decreased B16 melanoma growth. In an autoimmune environment, the number of Tregs infiltrating the islets was limited by LAG-3, which facilitated the breakdown of self-tolerance. However, in the tumor microenvironment, LAG-3 activity was required for Treg-mediated immune suppression. These data suggest opposing roles for LAG-3 on Tregs in an inflammatory versus an immunosuppressive microenvironment. Therefore, specifically targeting LAG-3 on Tregs may provide a strategy for limiting tumor growth without subsequent development of autoimmunity.
Reduced T-bet induction in proliferation- interferon-γ decoupled CD4+ T cells as a mechanism for propagated T cell tolerance (IRC4P.491)
Authors: Dutta Avijit , Drake Charles , Powell Jonathan , Pardoll Drew , Huang Ching-Tai
Published: January 2023

Infectious tolerance of immunity has been proposed as a mechanism of tolerance propagation. Limited effort of tolerance induction may be exploited for maintaining stable long-term tolerance. In our hemagglutinin (HA) antigen-specific transgenic mouse model system, adoptively transferred naïve HA-specific CD4+ T cells became tolerized in HA bearing mice and developed into regulatory T cells (Treg) with LAG-3 expression. These Treg suppressed the activation of secondly transferred naïve HA-specific CD4+ T cells. The suppressed HA-specific CD4+ T cells of second transfer were impaired for their IFN-γ capacity with reduced T-bet induction and STAT1 phosphorylation. However, they still substantially proliferated and acquired LAG-3 expression. They themselves became regulatory T cells as well. They further suppressed HA specific naïve CD4+ T cells both in vitro and in vivo. Compromised T-bet induction and IFN-γ capacity with T cell activation under Treg suppression ensure the transition into Treg without significant effector activity and ultimate propagated tolerance.
Structure-functional analysis of Foxp3 (IRC4P.478)
Authors: Zeng Wei-ping
Published: January 2023

Foxp3 is responsible for the major immunological features of Treg cells, including hypoproliferation in vitro, immune suppression of conventional T cells and resistance to Th2 cell differentiation. In addition to the Forkhead domain, the Foxp3 protein contains the N-terminal, zinc finger and leucine zipper domains. To understand how these domains contribute to Foxp3 functions, we systematically compared the roles of these domains in determining the 3 major immunological features of Treg cells. We designed a bridge-mediated mutagenesis method to generate Foxp3 mutants with complete deletion of each of the domains. CD4 T cells expressing the Foxp3 mutant with deletion of the N-terminal, leucine zipper or the forkhead domain showed robust TCR dependent proliferation in vitro, differentiated into Th2 cells, and lost immune suppressive activities in vitro and in vivo, demonstrating a complete loss of all 3 functions of Foxp3. In contrast, deletion of the zinc finger domain only partially impaired these functions of Foxp3. This result suggests that mutations in the zinc finger domain could lead to nonlethal autoimmune and allergic diseases, in which reduction rather than complete loss of Foxp3 functions is expected. In any case, deletion of a particular domain showed similar effects on all 3 functions of Foxp3. Therefore defining each of the immunological features of Treg cells requires intact Foxp3 proteins.
Differential requirement for IRF4 in CD8+ T cell responses to acute versus chronic LCMV infection (IRM4P.498)
Authors: Nayar Ribhu , Schutten Elizabeth , Brehm Michael , Welsh Raymond , Berg Leslie
Published: January 2023

The magnitude of the CD8+ T cell response to acute infection is regulated by the strength of TCR signaling. A key component to this is the level of expression of the transcription factor IRF4, which controls CD8+ T cell expansion in a dose-dependent manner. During an acute response to LCMV-Armstrong, a modest reduction in IRF4 levels, such as that achieved by a haplo-deficiency at the irf4 locus, leads to a 2-4 fold reduction in the number of LCMV specific CD8+ T cells without impacting viral clearance (RN manuscript submitted). These data indicate that acute LCMV infections are associated with an expansion of cognate CD8+ T cells that is in excess of that needed to clear the virus. In contrast, during LCMV-clone 13 infection, the modestly-reduced CD8+ T cell response of irf4+/- T cells fails to control the infection, resulting in long-term viral persistence. In this system, loss of one allele of irf4 results in reduced CD8+ T cell function, increased exhaustion due to loss of PD1 low Tbet high precursors, and less weight loss than in LCMV-clone 13-infected WT mice. These data indicate differential requirements for high levels of IRF4 in generating functional CD8+ T cell responses capable of clearing acute versus chronic virus infections.
A new genetic model for separating and analyzing Treg cells in unchallenged mice based on their proliferative history (LYM3P.738)
Authors: Zhang Baojun , Dai Meifang , Zhuang Yuan , Li Qijing
Published: January 2023

Apart from other cell types, antigen-primed conventional CD4 T cells integrate their functional differentiation into the process of proliferation. However, it is unknown whether the functional maturation of Tregs is also associated with their proliferative history, especially in unchallenged mice. To aid dissection of Treg populations, we developed a reporter system for marking and isolating Tregs undergone proliferative expansion during development or tolerance response towards endogenous antigens. We designed a tandem loxP cassette, named Tlox, to prevent intramolecular recombination. Tlox also acts as a genetic switch placed in front of a fluorescent marker. Cell division provides an opportunity for inter-molecular pairing between the duplicated Tlox on sister chromatids. Upon pairing, Cre recombinase can convert Tlox to a single loxP and consequently express the fluorescent marker permanently. We showed that, in vitro and in vivo, reporter activation is indeed dependent on both active cell cycles and Cre expression. By combining Ox40-Cre with the Tlox reporter, we separated Tregs based on their proliferative history in naïve animals. Functional and transcriptomic analysis indicted that, within the Treg pool, proliferated Tregs form a distinct population and are highly enriched with effector phenotypes. Thus, the newly established lineage tracking system provides a novel tool for assessing dynamitic changes among Treg cells during their tolerance response in live animals.
The RNA-binding protein HuR is a posttranscriptional on switch for CD4+ Th2 and Th17 differentiation (LYM3P.730)
Authors: Atasoy Ulus , Chen Jing , Techasintana Patsharaporn , Gubin Matthew , Magee Joseph , Cascio Jason , Calaluce Robert , Yu Shiguang
Published: January 2023

Posttranscriptional control by RNA binding proteins (RBPs) in CD4+ T cells is poorly understood. The RBP, HuR, regulates IL-4, IL-13 and GATA-3, by binding to AU-rich (ARE) regions in their 3’ UTRs. We hypothesized HuR is regulating CD4+ Th2 differentiation by controlling mRNA stability. We previously showed that HuR over-expression results in increased Th2 differentiation and Th2 cytokines. We generated a new model to study Th2 differentiation, distal Lck-cre ROSA HuRfl/fl, in which HuR is ablated prior to T cell activation. This resulted in profound suppression of Th2 but not Th1 cytokines, under in vitro conditions. We used the ova challenge model of airway inflammation to study antigen presentation and in vivo cytokine regulation. HuR KO mice had completely abolished lung cellular inflammation to levels similar to non-immunized mice. This further resulted in significant reductions in IL-4, IL-13 secretion, and serum IgE levels. We also investigated effects of HuR ablation in Th17 differentiation. We show HuR regulates IL-17 expression by controlling transcript stability. HuR KO T cells have greatly reduced Th17 differentiation (66% reduction) and diminished onset and severity of inflammation in an EAE model. These data indicate that HuR is required for efficient CD4+ Th2 and Th17 differentiation both in vitro and in vivo but not Th1. These findings reveal nuanced control by HuR upon different CD4+ T cell subsets.
Resistance to invasive pneumococcal infection in mice with acute allergic lung inflammation (MPF1P.766)
Authors: Sanfilippo Alan , Furuya Yoichi , Metzger Dennis
Published: January 2023

A recent publication by Talbot et al (N Engl J Med 2005) first identified asthma as a risk factor for invasive pneumococcal disease (IPD). However, more recent reports suggest that mortality is unaffected following IPD in patients with asthma and that COPD, an obstructive condition similar to asthma, protects against the development of complicated pneumonia. To clarify the effects of asthma on the immune response to a pneumococcal infection in the lung, we used an acute murine model of ovalbumin (OVA)-induced allergic lung inflammation (ALI) and hypothesized that mice with acute ALI would show increased susceptibility to IPD. Surprisingly, mice with acute ALI demonstrated enhanced resistance to infection. Resistance in mice with ALI correlated with a greater capacity for bacterial clearance from the lungs within eight hours post infection. This early clearance resulted in decreased bacterial invasiveness into the lung tissue as well as a blunted cytokine response to infection. Despite an increase in alveolar macrophage levels following OVA challenge, the increased bacterial clearance appeared to be mediated by MHCII- SiglecF+ eosinophils. These findings suggest that while eosinophils are pathogenic in asthma they could be protective against IPD, highlighting the need for a clearer understanding of the risk of IPD in patients with different asthma phenotypes.
Leukotriene B4 controls methicillin-resistant Staphylococcus aureus skin infection (MPF2P.815)
Authors: Brandt Stephanie , Wang Sue , Delafield Nathan , Serezani Henrique
Published: January 2023

Methicillin resistant Staphylococcus aureus (MRSA) was traditionally a nosocomial pathogen but community-associated MRSA clones have emerged as a cause of severe necrotizing cutaneous infection. Leukotriene B4 (LTB4) is a potent lipid mediator produced by 5-lipoxygenase (5LO) that is important in host defense against many pathogens including Streptococcus pneumoniae lung infections and Leishmania amazonensis skin infections. We hypothesize that 5LO-/- and mice lacking LTB4 receptor (BLT1-/-) are more susceptible to MRSA skin infection than wild type (WT) mice. MRSA was inoculated subcutaneously in shaved backs of 5LO-/- or BLT1-/- and WT mice. We observed delayed skin lesion healing in 5LO-/- or BLT1-/- compared to WT. Both genotypes had higher bacterial burden in the site of infection than WT. H&E staining showed more leukocyte recruitment and epidermis thickening in both genotypes compared to the WT. We did not observe any change in phagocytosis of MRSA in macrophages from WT and BLT1-/-, which suggests a defect in killing. However, macrophages from 5LO-/- mice produced less nitric oxide, a potent microbicide, than WT macrophages. Topical treatment of 5LO-/- mice with an ointment containing LTB4 at the site of infection reduced lesion size when compared to untreated mice. These findings suggest that LTB4 is an important agent in skin host defense mechanisms. LTB4 is a likely candidate to be used as an immunotherapeutic agent to control MRSA infections.
Antibody and complement-mediated enhancement of red cell invasion by Plasmodium falciparum. (MPF2P.806)
Authors: Biryukov Sergei , Stoute José
Published: January 2023

Malaria is an infectious disease caused by an intracellular protist of the genus Plasmodium that invades red blood cells (RBCs). Experimental vaccines that target the blood stage of the parasite (merozoites) induce a potent humoral immune response but there is no protection despite inhibition of invasion in vitro in the presence of heat-inactivated (HI) serum. The reason for this discrepancy is unknown. We hypothesize that complement activation and opsonization of merozoites enhances complement receptor 1 (CR1)-mediated invasion of RBCs. We tested the effect of anti-merozoite surface protein 1 (MSP-1) antibody 5.2 (mAb5.2) in the presence of fresh or HI serum on the in vitro invasion of RBCs by P. falciparum. We observed inhibition of RBCs invasion with inactivation of serum for 3 min, 5 min, or 30 min at 56°C. The inhibitory effect of 3 min HI serum was rescued upon the addition of C2 and Factor B, but only in the presence of mAb5.2. Compstatin, a C3 inhibitor, negated the enhancing effect of mAb5.2 in fresh serum. The invasion enhancing effect of mAb5.2 was absent in 3 min HI serum. Similar results were seen in fresh serum when sCR1 was used as a competitor. Purified antibodies from MSP-1 vaccinees showed inhibitory activity in 3 min HI serum, but inhibition was reduced in fresh serum. Our results suggest that anti-merozoite antibodies in the presence of complement can enhance RBCs invasion, constituting a novel mechanism of immune evasion by P. falciparum.
CCL21 defect impairs dendritic cell trafficking in SAMP1/YitFc mice (MUC2P.817)
Authors: Mikulski Zbigniew , Johnson Rebecca , Shaked Iftach , Kim Gisen , Nowyhed Heba , Chodaczek Grzegorz , Cominelli Fabio , Ley Klaus
Published: January 2023

SAMP1/YitFc (SAMP) mice develop chronic ileitis similar to Crohn’s disease. The disease mechanism is unknown but thought to involve adaptive and innate immune responses. Here, we discovered that the chemokine CCL21, a ligand for CCR7, is almost completely absent in mesenteric lymphatics and other tissues of SAMP mice. Lymphatic CCL21 is known to be required for dendritic cell (DC) trafficking. Absence of CCL21 results in a severe defect of CD11b+CD103+ DC migration from the ileal lamina propria to the mesenteric lymph nodes (MLN), similar to the defect seen in CCR7-deficient mice. The ability of DCs to produce retinoic acid (RA) supporting regulatory T cells (Tregs) is also drastically reduced in SAMP mice. In young mice, the defects in CCL21 expression and DC trafficking preceded the clinical manifestation of ileitis. As a therapeutic intervention, we mobilized DCs by oral treatment with the TLR7 ligand R848, which increased Tregs in MLN and dramatically improved disease scores. Thus, absence of CCL21 expression represents a major pathogenic defect contributing to ileitis in SAMP mice. Our data suggest that therapies aimed at improved DC trafficking might be useful in patients with Crohn’s disease.
Poor expression and activation of the NLRP3 inflammasome in resident but not inflammatory colonic monocytes/macrophages (MUC2P.831)
Authors: Filardy Alessandra , He JianPing , Silveira Danielle , Kelsall Brian
Published: January 2023

Colonic macrophages (cMPs) play an important role in maintaining intestinal homeostasis since they are able to tolerate commensals and kill pathogenic microorganisms. One critical inflammatory pathway tightly controlled in cMPs is the assembly of the NLRP3 inflammasome that drives the maturation and secretion of IL-1β and IL-18 in response to multiple stimuli, including ATP and bacterial toxins. Here we found poor secretion of IL-1β by F4/80+CD64+ cMP after NLRP3 inflammasome activation. Steady-state cMPs expressed low constitutive levels of pro-IL-1β and NLRP3 protein. Following LPS/P3C4 stimulation, both cMPs and bone marrow-derived MP (BMDMs) increased expression of pro-IL-1β and NLRP3 mRNA. Surprisingly, however, pro-IL-1β and NLRP3 protein expression was significantly impaired in cMPs compared with BMDMs. Using the adoptive transfer model of colitis, recently recruited F480+CD64+ inflammatory monocytes/cMPs expressed similar levels of pro-IL-1β mRNA to cMPs under resting conditions; however in contrast, inflammatory monocytes/cMPs secreted high levels of mature IL-1β after NLRP3 inflammasome activation. Collectively, these results show that cMPs, but neither BMDMs nor newly recruited inflammatory monocytes/cMPs, have a functional impairment of NLRP3 inflammasome activation; and suggest that the poor NLRP3 activity observed in resident cMPs is largely due to the selective inhibition of the translational of NLRP3 and pro-IL-1β mRNA to protein.