bluuebunny/crossref_metadata_2025 huggingface dataset:
Flagellin encoded in gene-based vector vaccines is a route-dependent immune adjuvant (P4302)
Authors: Rady Hamada , Huang Weitao , Ramsay Alistair
Published: January 2023

TLRs play an important role in the initiation of immune responses. Their ligands may, therefore, have adjuvant properties in vaccine settings. Flagellin (TLR5L) promotes strong antigen-specific humoral responses, however its capacity to enhance specific T cell responses is controversial. Here, we evaluate adjuvant properties of flagellin in gene-based vaccination strategies. Intramuscular (IM) immunization with DNA or Ad vaccine vectors encoding Ag85B of M. tuberculosis (Mtb) and flagellin enhanced Ag85B-specific CD4+ and CD8+ T cell responses over vectors encoding Ag85B alone. In contrast, intranasal (IN) co-immunization with Ad encoding flagellin led to decreased numbers of mucosal Ag85B-specific CD4+ and CD8+ T cells. Following DNA/Ad prime-boosting, mice co-primed with DNA-Ag85B-flagellin IM and then boosted with Ad-Ag85B IM or IN, had significantly enhanced Ag85B-specific CD4+ (IM and IN boosting) and CD8+ (IM boosting) T cell responses without obvious morbidity. Moreover, co-priming with flagellin enhanced protection against pathogenic aerosolized Mtb. Our data indicate that the capacity of flagellin to enhance Ag85B-specific cellular immune responses is route dependent, either when encoded in DNA or Ad vaccines, or following DNA/Ad prime-boosting. Inclusion of flagellin at the priming phase of DNA/Ad prime-boost vaccination has strong adjuvant activity enhancing protective immune responses to co-expressed vaccine antigen.
Stability of vaccine induced Th2 cells following intranasal infection with group A streptococcus (P4258)
Authors: Dileepan Thamotharampillai , Scully Ingrid , Anderson Annaliesa , Jansen Kathrin , Cleary Paul
Published: January 2023

Group A streptococcus (GAS) naturally colonizes the human pharynx and can cause pharyngitis. We have found that intranasal administration (i.n.) of GAS elicits a Th17 response in mice; in contrast, intramuscular (i.m.) injection of recombinant GAS protein vaccines elicits a Th2 response. The influence of GAS infection on the vaccination-induced Th2 population was assessed using IL-4 reporter mice (4get and KN2) immunized with a GAS protein vaccine plus alum. Vaccination caused expansion of IL-4+ T helper cells which did not express IL-17A. Purified GFP+IL-4+ Thy1.1+ CD4+ T cells from vaccinated 4get mice were adoptively transferred to Thy1.2+ congenic recipients to evaluate their response to subsequent GAS infections. Recipient mice were infected i.n. with GAS and cytokine profiles of transferred cells were determined. In 5 of 6 recipient mice, GFP+Thy1.1+ cells retained their original Th2 phenotype and did not express IL-17A. In the sixth mouse, 8.6% of Thy1.1+ cells expressed IL-17A after exposure to GAS. As expected, endogenous Thy1.2+ cells from infected recipients expressed IL-17A. Thus, im vaccination primarily induced Th2 cells, and most cells were terminally differentiated and unable to develop the capacity to express IL-17A upon subsequent mucosal exposure to GAS. Thus vaccination with recombinant GAS proteins is unlikely to create a reservoir of GAS-specific T cells that can become polarized toward a Th17 response upon GAS infection.
Systemic immunization with a recombinant Pneumocystis protein vaccine candidate induces systemic and mucosal immune responses in non-human primates (P4260)
Authors: Kling Heather , Burton Andrew , Mcmanus Alexandria , Guyach Siobhan , Tarantelli Rebecca , Norris Karen
Published: January 2023

Despite widespread antiretroviral therapy, pulmonary disease remains a leading cause of morbidity and mortality in HIV+ patients, likely resulting from increased colonization rates by Pneumocystis (Pc) jirovecii, which causes pneumonia and is associated with chronic obstructive pulmonary disease (COPD). We have previously shown that early detection of antibodies to Pc kexin-like protease (KEX1) in the bronchoalveolar lavage fluid (BALF) correlates with protection from Pc colonization and subsequent COPD development in a macaque model of HIV. Here, we test the capacity of a systemic recombinant KEX1 vaccine to induce detectable KEX1-antibody responses in the lungs. Rhesus macaques (n=6) were intramuscularly (i.m.) immunized with 100ug of truncated, recombinant KEX1 protein + aluminum hydroxide (Alum), rested, then boosted with 50ug of KEX1 + Alum. Mock-immunized macaques (n=6) were injected (i.m.) with proteins from empty E.coli expression vector (pET28b) + Alum. Following boost inoculation, all macaques had plasma KEX1-IgG RET >250,000 (range=256,000-2,000,000) and BALF KEX1-IgG RET >1800 (range=1900-31,800). Five of 6 macaques had detectable BALF KEX1-IgA (range=100-550). Thus, systemic immunization with recombinant protein and Alum results in significant boosting of both circulating and mucosal KEX1 antibody titers. These data support KEX1 as a potent vaccine candidate against Pneumocystis infection.
Are antibodies necessary for a Chlamydia T cell vaccine? (P4278)
Authors: Karunakaran Karuna , Yu Hong , Jiang Xiaozhou , Li Yuling , Shen Caixia , Brunham Robert
Published: January 2023

The murine Chlamydia muridarum model demonstrates that CD4 T cells are necessary and sufficient to resolve primary infection and that either CD4 T cells or antibody is necessary and sufficient to resolve reinfection. It remains uncertain whether a Chlamydia vaccine should accelerate clearence or prevent reinfection. Using immunoproteomics we have identified five Chlamydia outer membrane proteins (PmpG, PmpE, PmpF, PmpH and MOMP) that when individually used as protein vaccine accelerated clearance of Chlamydia infection. All 5 proteins are located in the chlamydial outer membrane complex (COMC). We tested PmpG and MOMP (major outer membrane protein that constitute more than 60% of COMC) as recombinant proteins or in native conformation as part of COMC preparations for their efficacy in accelerating clearance, providing significant protection and reducing tubal pathology. Our results show that COMC, PmpG and MOMP formulated with a Th1 polarizing adjuvant accelerated clearance but that only COMC exhibited near complete protection to challenge infection and reduced genital tract pathology. High titres of Chlamydia neutralizing antibodies were only detected following COMC immunization and not following PmpG or MOMP immunization. The superior efficacy of COMC as Chlamydia vaccine may be due to native conformation of outer membrane proteins that induce functional antibodies as well as CD4 T cell responses. These results suggest COMC may be a promising Chlamydia vaccine vehicle.
High plasma TNF-α levels and TNF-α/IL-10 ratios are associated with TNF2 allele in severe P. falciparum malaria patients in Sri Lanka (P3023)
Authors: Handunnetti Shiroma , Perera Maureen , Herath Nimali , Pathirana Sisira , Mendis Kamini , Premawansa Sunil
Published: January 2023

Plasma levels of pro- and anti-inflammatory cytokines of P. falciparum-infected patients with severe malaria (SM; n=62) and uncomplicated malaria (UM; n=69) from Sri Lanka were assessed. SM patients had significantly higher levels of TNF-α (P<0.01), IL-6 (P<0.01) and IL-10 (P<0.05) compared to the UM patients. Plasma IL-2 levels of these patients were undetectable. TNF-α levels of a third group of patients with uncomplicated P. falciparum malaria, who were recruited during their fever episodes (UMF; n=14) were significantly higher than those of the UM patients (P<0.001) and comparable to SM patients. Plasma IFN-γ levels of SM patients were higher compared to UM patients, but was not statistically significant. Body temperature in both SM and UMF groups were significantly higher compared to UM group whereas percent parasitaemia in all three groups were comparable. Analysis of plasma TNF-α levels and the ratio of TNF-α/IL-10 in UM (n=34) and SM (n=34) patients carrying TNF1 and TNF2 allelic types showed that SM patients carrying TNF2 had significantly higher TNF-α levels as well as TNF-α/IL-10 ratio compared to UM patients carrying TNF1, UM patients carrying TNF2 and SM patients carrying TNF1 (P<0.05). These results suggest that the high circulating TNF-α levels and the inadequate IL-10 response in the SM patients carrying TNF2 allele could have contributed to the development of severe falciparum malarial disease.
Elevated proinflammatory cytokine production by monocytes in HIV(+) individuals at risk for cardiovascular disease (P3033)
Authors: Jalbert Emilie , Parikh Nisha , Seto Todd , Chow Dominic , Shikuma Cecilia , Ndhlovu Lishomwa , Barbour Jason
Published: January 2023

Despite virologic suppression by HIV antiretroviral therapy, residual inflammation associated with chronic HIV infection increases the risk of developing cardiovascular disease. Monocytes have been shown to be major players in the development of atherosclerosis due to their proinflammatory responses to oxidized Low Density Lipoproteins. Our study sought to assess the functional properties of monocytes from peripheral blood of HIV-infected individuals. The cohort consisted of 33 HIV(+) subjects on HAART and 14 HIV(-) risk- and age- matched subjects. Our flow cytometry-based functional assay measured monocyte production of IL-1β, IL-8 and IL-6 in the absence of stimulation and in response to LPS or oxLDL. Without stimulation, HIV(+) subjects had a greater frequency of cells producing IL-1β and IL-8. In the presence of either oxLDL or LPS, both groups increased the frequency of responding cells compared to no stimulation, but HIV(+) subjects maintained a higher frequency of IL-1β(+) and IL-8(+) cells compared to HIV(-). There was no IL-6 production in either group in the absence of stimulation, but upon stimulation with either oxLDL or LPS, there was a higher frequency of IL-6 producing cells in the HIV(+) group. The higher level of inflammatory cytokine production in HIV(+) adults compared to HIV(-), both at rest and in the presence of stimulation, may in part account for increased risk of cardiovascular disease seen in the HIV(+) population.
IL-17 has a critical role to promote protection against Leishmania infantum infection (P3011)
Authors: Nascimento Manuela , Carregaro Vanessa , Lima Djalma , Costa Diego , Dias Fabrício , Ryffel Bernhard , Rossi Marcos , Jesus Amélia , Almeida Roque , Santana João
Published: January 2023

During visceral leishmaniasis the Th1 profile is associated with protection, but the role of IL-17-producing cells remains poorly defined. Our aim was to evaluate whether IL-17 participates in the host immune response against L. infantum and such mechanisms involved. We found that infected humans strongly produce IL-17 during the symptoms peak. Similarly, in murine model IL-17 was induced at the same time of parasitism peak. The IL-17 participates on the parasite control since IL-17R-/- infected mice showed higher parasite burden then WT. In accordance, a lower inflammatory infiltrate was observed in IL-17R-/- mice as a consequence of significant lesser expression of cxcl10, cxcr3, ccl5 and ccr5 in the liver and spleen. Interestingly, an up regulation of ccl17, ccr4 and foxp3 and IL-10 expression was observed in KO mice. Corroborating these data, IL-17 production was potentiated in IL-10-/- infected mice, which were more resistant to infection. Besides, in the absence of IL-17R a smaller expression of IFN-γ, Tbet and iNOS was detected. Indeed, IL-17R-/- infected-macrophages showed a deficiency to kill the parasite and the stimulus with IL-17 and/or IFN-γ in WT infected-macrophage showed that IL-17 acts synergistically with IFN-γ potentiating the NO production and the macrophage leishmanicidal capacity. Together, our results showed that L. infantum triggers IL-17-producing cells that promote the control of parasitism by inducing NO expression and IL-10 modulation.
IL-9-producing cells promote Th1 responses against Mycobacterium tuberculosis. (P3015)
Authors: Alvarez Ivana , Rovetta Ana , Hernández Del Pino Rodrigo , Pasquinelli Virginia , Peña Delfina , Musella Rosa , Palmero Domingo , Chuluyán Héctor , Samten Buka , Barnes Peter , García Verónica
Published: January 2023

IFNγ-secreting cells are critical in the defense against M. tuberculosis (Mtb) infection, but other cells might be required to eradicate the pathogen. IL-9-producing cells were reported to be involved in inflammation and immune diseases, but their role during tuberculosis remains to be clarified. Here we analyzed the involvement of IL-9-secreting cells in the immunity of patients with active disease. We demonstrated that Mtb induced a marked expansion of CD4 IL-9+ cells in peripheral blood from tuberculosis patients. After antigen stimulation, patients with robust Th1 responses to Mtb (high responders) produced higher amounts of IL-9 compared to those with weak Th1 responses (low responders) and a direct significant correlation between IL-9 and IFNγ production against Mtb was detected. To investigate the role of the IL-9 secreted by those lymphocytes, we inhibited IL-9 expression with siRNA, which significantly diminished the production of IFNγ against Mtb. Addition of IL-9 markedly increased the secretion of IFNγ against Mtb, even in low responders. Furthermore, treatment with IL-9 inhibited Mtb-induced apoptosis of IFNγ+ cells, enhancing the survival of the cells. Finally, by studying the effect of neutralizing IL-9 antibodies on polarized Th9 cells, we confirmed that IL-9 participates in the up-regulation of IFNγ production against Mtb. Thus, our results indicate that IL-9 enhances Th1 responses during tuberculosis promoting the cell-mediated immunity to the pathogen.
Deletion of OLFM4 rescues defective host defense against Staphylococcus aureus, but not Aspergillus fumigatus in murine X-linked chronic granulomatous disease (P1259)
Authors: Liu Wenli , Sugui Jancy , Li Hongzhen , Kwon-Chung Kyung , Rodgers Griffin
Published: January 2023

Chronic granulomatous disease (CGD) patients have recurrent life-threating bacterial and fungal infections due to the mutation of one of four subunits of the respiratory burst oxidase (NADPH-oxidase). Olfactomedin 4 (OLFM4) is a neutrophil granule protein, which has been recently identified as a negative regulator of host innate immunity against bacteria infection in mice through modulation of neutrophil protease activity. The goal of this study was to evaluate the impact of OLFM4 deletion on host innate immunity against Staphylococcus aureus and Aspergillus fumigatus, in a murine X-linked CGD model. We found that neutrophil intracellular killing and in vivo clearance of S. aureus have been significantly increased in gp91phox- and OLFM4-double deficiency mice compared with CGD mice. The mice survival to S. aureus sepsis in gp91phox- and OLFM4-double deficiency mice has also been significantly prolonged compared with CGD mice. Cathepsin C activities in OLFM4 deficient as well as double deficient mice neutrophils were significantly higher than those in WT mouse neutrophils. Accordingly, the elastase and cathepsin G activities in the neutrophils of OLFM4 deficient and double deficient mice were also substantially higher than those in WT mice as well as CGD mice. However, we have not observed enhanced innate immunity against A. fumigatus in OLFM4 deficiency mice. OLFM4 might prove to be an important target in CGD patients to augment host defense against bacterial infection.
Systems biology analysis of the ceca and bursa revealed unique innate immune responses to Campylobacter jejuni in two genetically distinct lines of chickens (P1269)
Authors: Zhou Huaijun , Abernathy Jason , Nazmi Ali , Li Xianyao , Swaggerty Christina , Pevzner Igal , Drake Kenneth , Kogut Michael
Published: January 2023

Campylobacter jejuni (C. jejuni) is one of the most commonly reported causes of foodborne illness worldwide, although C. jejuni is a commensal in chickens. Two genetically distinct broiler lines, either resistant (line A) or susceptible (line B) to C. jejuni colonization, , were used to understand innate immune response to C. jejuni in a time-course (1, 4, 24, and 36 h post-inoculation) study. A total of 128 1-d-old chicks (64 per line), were either orally challenged with C. jejuni or inoculated with PBS. Total RNA were isolated from ceca and bursa, and a chicken 44K Agilent microarray was performed on all samples. Microarray data was then analyzed using a dynamic Bayesian network machine learning approach. Bursa samples from infected line A birds showed significantly more perturbations in carbohydrate and lipid metabolism compared to non-infected controls while susceptible line B birds responded with perturbations in signal transduction and amino acid biosynthesis. Differences were also observed in the ceca where apoptosis, MAPK and Toll-like receptor signaling pathways were significantly perturbed in line A birds while cell adhesion molecules, Jak-STAT signaling and fatty acid metabolism were significantly perturbed in line B birds. The distinct perturbed signaling pathways between these two lines and between two tissues provide new insights into the early immune response of chickens to C.jejuni and will allow for in silico hypothesis testing for future functional studies.
The role of miRNA in regulating bacterial clearance (P1273)
Authors: Tay Hock , Kaiko Gerard , Hansbro Philip , Foster Paul
Published: January 2023

RATIONALE MicroRNAs (miRNAs) are small non-coding RNA that can bind to multiple target mRNA to repress protein production. While the expressions of specific miRNAs are known to be dysregulated in multiple human diseases, the role of miRNAs in regulating bacterial infection is unclear. Our aim is to investigate the roles of miRNA in regulating these processes. METHODS BALB/c mice were challenged with low doses of NTHi. Lung miRNAs that were differentially expressed were identified by microarray. miRNA inhibitors (antagomirs) were used to knockdown miRNA expression to investigate the roles of miRNA in bacterial clearance in vitro and in vivo. RESULTS Upon NTHi infection, 20 miRNAs were up-regulated while 52 were down-regulated by > 2.5 fold. Interestingly, knockdown of a specific miRNA in vitro decreased bacterial load and increased phagocytosis by macrophages and neutrophils. Similarly, transfer of macrophages or neutrophils deficient in miRNA into naïve recipient mice leads to enhance clearance of the bacteria in vivo. This enhance clearance is independent of cellular infiltration. Inhibiting miRNA may play a protective role by enhancing the function of innate immune cells during bacterial infection. CONCLUSION Our study suggests that miRNA may play important roles in regulating the innate immune response to bacterial infection.
Gene expression of SOAT1, a key enzyme for cholesteryl ester biosynthesis, is regulated by LPS and correlates with the antibacterial activity of human bronchial epithelial cell secretions against Pseudomonas aeruginosa (P1249)
Authors: Bahrami Bita , Anouseyan Rabin , Glavatska Maryna , Escobar Oswaldo , Porter Edith
Published: January 2023

Airway epithelial cells contribute to the first line of mucosal defense by secretion of antimicrobial peptides and antimicrobial lipids, including the cholesteryl ester (CE) cholesteryl arachidonate. Sterol O-acyltransferase 1 (SOAT1) is a key enzyme for CE biosynthesis. However, little is known about the regulation of the lipid-mediated arm of innate host defense. Polarized human bronchial epithelial cells (HBE) were incubated for 6 h with 100 ng/mL LPS, 10 μg/mL peptidoglycan (PG), 25 μg/mL PamCys3, and solvent control. SOAT1 mRNA was quantified by qRT-PCR with duplexing and RPLP0α as housekeeping gene. Lipid content of apical HBE secretions was analyzed by rpHPLC. SOAT1 expression was blocked with siRNA/lipofection. Antibacterial activity of HBE secretions was assessed by a 3 h colony forming unit assay with Pseudomonas aeruginosa (PA). SOAT1 gene expression was significantly increased by LPS (14.6 ±11.4, means ± SD, n=4, p=0.037). Stimulation with PG and PamCys3 increased SOAT1 expression by ~ 4- and 2-fold, respectively. Secretions from LPS-stimulated HBE showed an increase of total lipids including CA by ~30-50%. Selective inhibition of SOAT1 mRNA expression by 80% reduced the antibacterial activity of HBE whereby PA showed enhanced bacterial growth reaching >170% of the controls. These data suggest that SOAT1 is regulated by conserved innate immunity pathways and further support the role of antimicrobial lipids as effector molecules in mucosal airway defense.
Induction of human Beta-Defensin-1 gene expression is dependent on IRF-7 and PU.1. (P1379)
Authors: Ryan Lisa , Dai Jihong , Fitzgerald-Bocarsly Patricia , Schwartz Kyell , Diamond Gill
Published: January 2023

Beta-defensins have been gaining recognition as immunomodulators and regulators of the inflammatory response. Human beta-defensin (hBD) peptides 1, 2 and 3 are differentially modulated in primary gingival and tracheal epithelial cells (EC) by viruses. We showed that both HSV-1, Sendai and PR8 influenza viruses increase the gene expression of hBD-1 in monocytes and plasmacytoid dendritic cells (PDC) and suppress gene expression of hBD-1 in target EC. Deleting the hBD-1 homologue in mice also increased influenza-induced morbidity, mortality and lung inflammation compared with wild type C57BL/6 mice, highlighting its importance in viral innate immunity. In this study, we investigated how hBD-1 gene expression is regulated. In PDC, no increased hBD-1 gene expression occurred when cells were incubated with the TLR9 agonist, CpG-A, indicating that hBD-1 is not induced via TLR9. When PDC were incubated with anti-interferon (IFN)-alpha-receptor-1 antibody, no change in viral induction by HSV-1 occurred. In addition, in a luciferase reporter system in A549 cells co-transfected with expression vectors containing IRF3, IRF5, IRF7 and PU.1, IRF7 and PU.1 were the only transcription factors that increased activation of the hBD-1 promoter. These results suggest that HSV-1 induction of hBD-1 occurs independently of IFN-alpha at the receptor level but that transcription factors common to IFN-alpha are involved in the regulation of hBD-1 gene expression.
EBI2 regulates type I IFN responses to TLR stimulation (P1409)
Authors: Chiang Eugene , Johnston Robert , Grogan Jane
Published: January 2023

Epstein-Barr virus induced receptor 2 (EBI2), a G protein-coupled receptor that has been shown to have important roles in segregating B cells within follicles, has recently been implicated as a regulator of an interferon regulatory factor 7 (IRF7)-driven inflammatory network (IDIN) that is associated with type I diabetes. Using plasmacytoid dendritic cells (pDCs) and CD11b+ myeloid cells from knockout mice deficient in EBI2, elevated production of type I IFN and IFN-α-dependent proinflammatory cytokines, respectively, were observed in response to in vitro stimulation with various TLR agonists. EBI2 appears to act as a negative regulator of IRF7-mediated type I IFN and IDIN in pDCs, as constitutive expression of IRF7 and IDIN genes were higher in EBI2 KO pDCs, likely accounting for the heightened primed state of responsiveness to endosomal TLR stimulation. Similar to its role on B cells, EBI2 plays a role in pDC localization. EBI2 KO pDCs failed to migrate in response to the EBI2 ligand 7α,25-dihydroxycholesterol and EBI2 KO mice had significantly lower frequencies of pDCs in the spleen and peritoneal cavity. Together, dysregulated migration of pDCs and elevated type I IFN responsiveness may be responsible for increased levels of IFN-α and IFN-β detected in the peritoneum of EBI2 KO mice following in vivo challenge with endosomal TLR agonists. These findings support a role for EBI2 as an important regulator of pDC and myeloid cell responses.
HSV-1 serine/threonine kinase US3 hyperphosphorylates IRF3 and inhibits the IFN-β production (P1413)
Authors: Zheng Chunfu , Wang Shuai
Published: January 2023

Viruses have evolved a variety of strategies to modulate host IFN-mediated immune responses. HSV-1 US3, a Ser/Thr kinase conserved in α-herpesviruses, has been demonstrated to be involved in multiple functions during HSV-1 infection. In this study, we reported the novel observation that US3 significantly inhibited Sendai virus (SeV) mediated activation of the IFN-β and IFN stimulated response elements (ISRE) promoters and the transcription of IFN-β, ISG54 and ISG56. US3 was also shown to block SeV-induced dimerization and nuclear translocation of IRF3. This inhibitory function required the kinase activity of US3, as kinase dead (KD) US3 mutants K220M and D305A could not inhibit the IFN-β production. US3 also inhibited IRF-3-responsive PRD(III-I) promoter activity induced by ectopic expression of the N-terminus of RIG-I (RIG-IN), MAVS, TBK-1, IKKϵ and IRF3/5D, indicating that US3 may block the IFN-β pathway at the IRF3 level. US3 was demonstrated to interact with and hyperphosphorylate IRF3 to prevent IRF3 activation. Finally, we constructed the US3 kinase dead mutant viruses, denoted as K220M or D305A HSV-1 respectively, and infection by both mutant viruses resulted in remarkable increase of IFN-β production compared with wild-type virus. To our knowledge, this is the first report that IRF3 serves as a substrate of HSV-1 Us3. These findings provide convincing evidence that US3 hyperphosphorylates IRF3, blocks the production of IFN-β, and subverts of the host innate immunity.
Inflammatory disease and impaired antiviral immunity due to unbalanced NF-kB and IRF3 activation result from a de novo human NEMO mutation (P1415)
Authors: Wessel Alex , Hsu Amy , Zilberman-Rudenko Jevgenia , Goldbach-Mansky Raphaela , Siegel Richard , Hanson Eric
Published: January 2023

The RIG-I-like receptors mediate antiviral signaling through NEMO-dependent activation of transcription factors NF-kB and IRF3, which are recognized for their roles in inflammation and the induction of Type I Interferon, respectively. We identified an individual with increased susceptibility to viral infection and persistent inflammatory disease. Sanger sequencing revealed a de novo synonymous mutation in the gene encoding NEMO that results in an in-frame splicing event and exclusive production of a shortened protein. To understand the role of NEMO in regulating the antiviral response, we stimulated patient and healthy control fibroblasts with RIG-I-like receptor ligand poly(I:C), which mimics viral RNA. Induction of IRF3 response genes IFNB1 and CXCL10 was reduced 35-fold and 500-fold (n=2), respectively, compared to healthy control, and patient cells were unable to control viral infection. In contrast, TNF induced the expression of proinflammatory genes TNFSF10 and IL17 7-fold and 4-fold higher in patient cells than in healthy control cells. Increased TNF induced gene expression was accompanied by increased p65 nuclear translocation indicative of increased canonical IKK activation. These results suggest that aberrant expression of an alternately spliced NEMO isoform constitutes a novel autoinflammatory disease characterized by unbalanced NF-kB and IRF3 mediated responses to viral infection.
Novel function of Trim44 promotes an antiviral response by stabilizing VISA (P1417)
Authors: Sun Bing , Yang Bo
Published: January 2023

VISA functions as a critical adaptor in the regulation of both the production of type I interferons (IFNs) and the subsequent control of the innate antiviral response. In this report, we demonstrate that Trim44 interacts with VISA and promotes VISA-mediated antiviral responses. The overexpression of Trim44 enhances the cellular response to viral infection, whereas Trim44 knockdown yields the opposite effect. Trim44 stabilizes VISA by preventing VISA ubiquitination and degradation. These findings suggest that Trim44 functions as a positive regulator of the virus-triggered immune response by enhancing the stability of VISA.
Intrinsic upregulation of critical innate immune effectors in a novel transgenic mouse model confers viral resistance (P1389)
Authors: Painter Meghan , Rodriguez Moses
Published: January 2023

Our laboratory generated a transgenic mouse that ubiquitously expresses 3D, a picorno viral RNA-dependent RNA polymerase. 3D transgenic mice display an extreme antiviral phenotype when challenged with viruses from distinct families. To probe this protective molecular mechanism, we assayed differential gene expression in tissues of uninfected 3D transgenic mice. Microarray analysis revealed increased levels of 78 genes >4-fold, including a distinct upregulation of critical innate antiviral effectors across tissues. RT-PCR analysis of liver, lung, kidney, CNS and heart tissue from uninfected 3D mice showed a significant upregulation of antiviral genes (up to 300-fold). Following infection with EMCV, 3D transgenic mice exhibited significantly decreased viral tissue titers and survived lethal viral infection. This effect is specific to the 3D transgene since other mice expressing the picorno viral transgenes VP1 or VP2 did not display an antiviral phenotype. Studies of 3D-Ifnar1-/- or 3D-Ifngr1-/- mice, supported the conclusion that type I IFN signaling is necessary for the antiviral state in 3D mice, whereas IFNg signaling is dispensable. Maintenance of a viral-resistant state in uninfected 3D mice is independent of adaptive immunity since uninfected 3D-Rag1-/- mice are able to highly upregulate antiviral factors. Collectively, our data indicate that intrinsic upregulation of critical innate immune effectors confers broad-spectrum viral resistance in 3D mice.
STING recognition of cytoplasmic DNA instigates cellular defense (P1393)
Authors: Xia Tianli , Abe Takayuki , Harashima Ai , Konno Hiroyasu , Konno Keiko , Morales Alejo , Ahn Jeonghyun , Gutman Delia , Barber Glen
Published: January 2023

How the cell recognizes cytosolic DNA including DNA based microbes to trigger host defense related gene activation remains to be fully resolved. Here, we demonstrate that STING (for Stimulator of Interferon Genes), an endoplasmic reticulum (ER) translocon associated transmembrane protein, acts to detect cytoplasmic DNA species. STING homodimers were able to complex with self (apoptotic, necrotic) or pathogen related ssDNA and dsDNA and were indispensible for HSV-1-mediated transcriptional activation of a wide array of innate immune and pro-inflammatory genes in addition to type I IFN. Our data indicates that STING instigates cytoplasmic DNA-mediated cellular defense gene transcription and facilitates adoptive responses that are required for protection of the host. In contrast, chronic STING activation may manifest inflammatory responses and possibly autoimmune disease triggered by self-DNA.
α4β7 integrin-mediated lymphocyte adhesion to VCAM-1 under physiologic shear requires L-selectin function (P5116)
Authors: Loppnow Jessica , Kadono Takafumi , Sato Sinichi , Tedder Thomas , Steeber Douglas
Published: January 2023

Lymphocyte migration is facilitated by a cascade of adhesion molecule interactions. Specifically, lymphocyte rolling is mediated by selectins, while slow rolling and adhesion are attributed to integrin and Ig family members. α4β7 integrin interactions with its primary ligand, MAdCAM-1, synergize with L-selectin function to regulate the recirculation of naïve lymphocytes to the gut. However, subsets of regulatory T cells also express high levels of L-selectin and α4β7 integrin, and may instead bind to VCAM-1, a secondary ligand for α4β7 integrin. Therefore, potential synergism between L-selectin function and α4β7 integrin/VCAM-1 interactions were examined. To determine the ability of α4β7 integrin to interact with VCAM-1 under conditions of shear, transfected TK-1 T cell interactions with EA.hy926 endothelial cell lines were assessed using an in vitro flow chamber assay. While TK-1 cells, expressing α4β7 but not α4β1 integrin, showed little interaction with VCAM-1-transfected endothelial cells, the presence of L-selectin function enabled α4β7 integrin/VCAM-1 interactions to support slow rolling and adhesion. Importantly, α4β7 integrin interactions with VCAM-1 supported slow rolling and adhesion of murine regulatory T cells under shear stress in the presence of L-selectin function. Thus, functional synergy between L-selectin and α4β7 integrin/VCAM-1 interactions may promote regulatory T cell recruitment to sites of inflammation.
Isoforms of vitamin E differentially regulate inflammation and lung function (P5136)
Authors: Cook-Mills Joan , Abdala-Valencia Hiam , Berdnikovs Sergejs
Published: January 2023

Vitamin E regulation of disease has been extensively studied but most studies focus on the α-tocopherol isoform of vitamin E. These reports indicate contradictory outcomes for anti-inflammatory functions of the α-tocopherol isoform of vitamin E with regards to animal and clinical studies. The seemingly disparate results are consistent with our studies demonstrating that purified natural forms of vitamin E have opposing regulatory functions during inflammation. We have determined that α-tocopherol inhibits whereas γ-tocopherol elevates allergic inflammation, airway hyperresponsiveness, leukocyte transendothelial migration, and endothelial cell adhesion molecule signaling through protein kinase Cα. Moreover, we determined that α-tocopherol is an antagonist and γ-tocopherol is an agonist of PKCα through direct binding to a regulatory domain of PKCα. Furthermore, in a recent clinical study, we determined that increasing serum concentrations of γ-tocopherol associated with worse lung function, while increasing serum concentrations of α-tocopherol associated with better lung function. In summary, we have determined mechanisms for opposing regulatory functions of α-tocopherol and γ-tocopherol on inflammation. Information from our studies will have significant impact on the design of clinical studies on inflammation and on vitamin E consumption.
The divergent roles of Leptin and Wnt5a on mast cell function (P6020)
Authors: Kolawole Elizabeth , Ryan John
Published: January 2023

The prevalence of obesity has reached epidemic proportions in western countries. Increased levels of adipose tissue are strongly associated with disease states such as chronic heart disease, atherosclerosis, insulin resistance and type II diabetes. Obesity is also associated with increased risk of inflammatory conditions such as allergic asthma and allergic rhinitis. Furthermore, the obese state has been described as chronic low-grade inflammation. Thus, obesity and inflammation are inextricably linked. The role of mast cells in inflammatory disease and allergic reaction is unequivocal. Moreover, mast cells are instrumental effector cells in IgE mediated allergic diseases such as asthma and rhinitis. The receptor for IgE is the high affinity multimeric Fc receptor FcϵRI, which when activated through an antigen on mast cells results in signal transduction. This includes degranulation and the production of inflammatory and chemotactic mediators. We hypothesize that the adipokine Wnt5a enhances IgE mediated mast cell function and migration. We further hypothesize that the adipokine leptin suppresses IgE mediated mast cell cytokine production, a phenomenon seen in the Th1 prone genetic background (C57BL/6) but absent in the Th2 prone genetic background (BALB/c and 129/sv).
Mast cell-MDSC interactions promote chemokine secretion and parasite expulsion. (P6022)
Authors: Ryan John , Morales Johanna , Urban Joseph , Conrad Daniel
Published: January 2023

Mast cells and myeloid-derived suppressor cells (MDSC) are both increased at sites of growing tumors and parasitic infection. We have tested the effects of mast cell-MDSC interactions in vitro and in vivo. Surprisingly, co-culture of these cells resulted in a significant increase in pro-inflammatory chemokine production, which was attributed to Ly6C+ MDSC. MDSC depletion via gemcitabine treatment in mice infected with Nippostrongylus brasiliensis or Trichenlla spiralis drastically increased parasite burden, reminiscent of mast cell-deficient mice. Gemcitabine also decreased mast cell-dependent antigen-induced airway hyperresponsiveness (AHR) and inflammation, while MDSC injection worsened AHR. These data suggest that the evolutionary purpose of MDSC may be to potentiate Th2-induced, mast cell-dependent inflammatory responses.
Structural analysis of epitopes recognized by two anti-CϵmX mAbs reveals intrinsic disorder of the CϵmX domain (P6002)
Authors: Chu Hsing-Mao , Wright Jon , Chan Yueh-Hsuan , Wu Pheidias C. , Lim Carmay , Chang Tse Wen
Published: January 2023

In human membrane-bound IgE (mIgE), a CϵmX domain containing 52 aa residues is located between the CH4 domain and the C-terminal membrane-anchoring segment. Antibodies specific for CϵmX, which can cause lysis of mIgE+ B cells, are being developed to treat allergic diseases and an anti-CϵmX mAb h47H4 is in phase IIb clinical trials. Hence, structural characterization of CϵmX free and complexed with anti-CϵmX mAb is of wide interest and importance. We have solved the crystal structures of (i) free CϵmX fused to IgG1-Fc and (ii) part of CϵmX complexed with an anti-CϵmX mAb, h4B12-Fab, at 1.94 Å resolution. The crystal structure of the IgG1-Fc•CϵmX fusion protein of homodimer exhibits a lack of well-defined electron density for residues 10 to 52 of A subunit and in absence of B subunit in dimeric CϵmX domain. The first 33 residues are also predicted to be intrinsically disordered. The 3D complex structure and epitope mapping by ELISA showed that the anti-CϵmX mAb, h4B12, binds to GLAGGSAQSQRAPDR, which has been reported to bind another anti-CϵmX mAb, h47H4, except for the first 6 residues (in italics). Comparison of the 3-D complex structure of h4B12 and h47H4 revealed different conformations of AQSQRAPDR, a feature characteristic of the intrinsically disordered regions in the CϵmX domain. This is the first report showing that the drug target, CϵmX, contains an intrinsically disordered region that can switch to different conformations upon binding to different anti-CϵmX mAbs.
T cell dependent induction of novel mast cell-lineage cells in spleen (P6006)
Authors: Toyoshima Shota , Abe Ryo
Published: January 2023

Mast cells and basophil are known to exert a variety of functions in normal as well as pathological immune responses. Although mutual dependencies between mast cell lineage cells (MCs) and T cells have been recently well documented, how antigen-specific T cell responses leads to differentiate MCs and to MCs-dependent immune responses is not fully understood. When we attempted to generate antigen-specific T cell lines from OVA and KLH immunized mice by repeatedly stimulating their spleen cells or lymph node (LN) cells in vitro, we observed dominant expansion of FcϵR1 and c-kit positive cells in spleen cell culture, not in LN cell culture. Although this surface phenotype has been classified as that of MC, toluidine blue staining and electron microscopic analysis demonstrated that granules found in these cells appeared more like those seen in classic basophils rather than MCs. The functions of these cells were equivalent to those of classical MCs or basophils such as β-hexomidase release in vitro and PCA reaction in vivo. Consistently, expression pattern of MCPs revealed mixed phenotypes of MCs and basophils. These cells produced IFN-γ when they were co-cultured with antigen specific T cells in the presence of relevant antigen, which occurred in mast cell-T cell contact manner. These results suggested the existence of unique mast cell lineage in spleen contributing T cell dependent immune responses. Roles of these cells in vivo are currently under investigation.
Cardif regulates mouse natural killer cell development and maturation. (P1454)
Authors: Haynes LaTeira , Hedrick Catherine
Published: January 2023

Cardif, also known as IPS-1, VISA and, MAVS, is an intracellular adaptor protein that functions downstream of the RIG-I family of pattern recognition receptors. Cardif is required for the production of type I-IFNs and other inflammatory cytokines after RIG-I like receptors recognize intracellular antigenic RNA. Studies have recently shown that Cardif may have other roles in the immune system in addition to its role in viral immunity. In our study, we find that the absence of Cardif hinders normal natural killer cell development and maturation. Cardif-/- mice have a 35% loss of stage 5 mature NK cells in the periphery. There are also fewer pre-NK cells in the bone marrow of Cardif-/- mice. However, Cardif-/- NK cells produce more IFNg upon stimulation. Mixed chimeric mice revealed that the retarded progression from early NK cell progenitors to pre-NK cells is a cell-intrinsic mechanism. In addition, Cardif-/- NK cells are at a competitive disadvantage in the periphery of chimeric mice. Our results reveal a novel role for Cardif signaling in the development and maturation of NK cells.
The impact of short CYLD in medullary thymic epithelial cell development (P1425)
Authors: Reissig Sonja , Hövelmeyer Nadine , Riemann Marc , Weih Debra , Weih Falk , Waisman Ari
Published: January 2023

CYLD is a deubiquitinating enzyme and acts as a negative regulator of NF-κB signaling. To analyse the function of CYLD in vivo we used CYLDex7/8 mice, which are characterized by loss of the full-length transcript and overexpression of a short splice variant of cyld (sCYLD). In these mice thymic and peripheral CD4 and CD8 T cells were reduced compared to controls. Hence, we were interested to investigate the contribution of CYLD in positive and negative selection in the thymus in vivo. For this purpose we crossed the HY-TCR transgene (HYtg) to CYLDex7/8 mice. The analysis of CYLDex7/8 HYtg males revealed an increase in CD4+CD8+ DP as well as in CD8 SP thymocytes, suggesting a less pronounced negative selection in CYLD mutant mice compared to HYtg control mice. Since medullary thymic epithelial cells (mTECs) play an essential role in the negative selection process of thymocytes, the levels of mTECs in the medullary compartment were examined by histology and FACS analysis. We observed reduced numbers of mTECs, combined with decreased expression levels of the mTEC markers UEA-1, keratin-5, claudin-3 and claudin-4. Interestingly, specifically immature mTECs were significantly reduced compared to controls, suggesting a developmental block in the transition of immature to mature mTECs. Taken together, these results display a crucial role of sCYLD in the development and maturation of mTECs.
The role of commensal microbiota in the formation and maintenance of antigen-specific memory CD4 T cells (P3174)
Authors: Spasova Darina , Surh Charles
Published: January 2023

The mammalian intestinal mucosa harbors over one trillion microbes, predominantly comprised of bacterial species, which easily surpass the number of host cells. Given this large number and the possible threat of opportunistic infections, the host immune system constantly monitors the epithelial mucosal barriers and regulates microbial interactions at these surfaces. This crosstalk and mutualism established between host and microbe has been heavily researched in recent years. The main efforts have focused on identifying the gut’s role in the formation of a functional immune system, whereas little light has been shed on the specific role of gut bacteria on T cell responses and memory to systemic and local pathogens. Here, we interrogate the role of gut flora on CD4 T cell responses to pathogens by comparing germ-free and specific-pathogen-free mice during systemic (acute LCMV) or localized (oral Listeria monocytogenes) infection as models pathogens. Our results indicate a marked difference between T cell responses in germ-free and specific pathogen free mice, suggesting a role for commensal bacteria not just in shaping the immune system but also in preparing it to respond with effector cells and later sustaining memory cells.
High fat diet-induced obesity affects CD4+ T cell differentiation in the small intestine (P3176)
Authors: Hong Chun Pyo , Yang Bo Gie , Kim Jung-Hwan , Jang Min Seong , Lee Eun-Jung , Jeun Eun Ji , Kim Chan , Seoh Ju-Young , Jang Myoung Ho
Published: January 2023

Obesity-induced metabolic diseases are caused by the excess infiltration of pro-inflammatory cells to metabolic tissues including adipose and liver. However, systematic understanding of correlation between obesity and alterations of gut immunity is unclear. We hypothesized that immune cells in small intestine may be affected substantially upon high-fat feeding since dietary lipids are absorbed at luminal surface of small intestine. Here we found that small intestinal CD4+ and CD8+ T cells but not B cells were decreased in obese state. In CD4+ T cell subsets, the proportion of TH1 cells was increased in obese state, whereas the proportion of TH17 cells was decreased. The regulation of Intestinal T cells is dependent on antigen presenting cells such as macrophages and dendritic cells. We found intestinal macrophage subsets but not dendritic cells were changed in obese state. We next investigated that which antigen presenting cells from small intestine control CD4+ T cell differentiation during obese state ex vivo. We found that two macrophage subsets control balance of TH1 and TH17 cells inversely. These results show that small intestinal macrophages can have a key role in modulating small intestinal CD4+ T cell differentiation during obesity.
Initiation of food allergy by a CD4+ T cell-intrinsic IL-4 program, controlled by OX40L (P6204)
Authors: Chu Derek , Mohammed-Ali Zahraa , Walker Tina , Goncharova Susanna , Llop-Guevara Alba , Barra Nicole , Gillgrass Amy , Ashkar Ali , Bramson Jonathan , Humbles Alison , Kolbeck Roland , Waserman Susan , Jordana Manel
Published: January 2023

Intestinal Th2 immunity in food allergy results in the production of IgG1 and IgE, and upon antigen challenge, anaphylaxis and eosinophilic inflammation. Although allergic sensitization critically requires IL-4 to develop, the source and control of IL-4 during the initiation of Th2 immunity remains unclear. Non-intestinal and non-food allergy systems have suggested a role for innate lymphocytes such as NKT or γδ T cells as a rapid source of IL-4 required to induce Th2 polarization. In contrast, we show here that NKT-deficient IL-15 KO, β2m KO and anti-NK1.1 treated mice have completely intact Th2 food allergic responses comparable to NKT-sufficient mice, including antigen-specific IgG1 and IgE, anaphylaxis, eosinophilic inflammation and cytokine production. Likewise, γδ T cell-deficient TCRδ KO mice mount comparable Th2 immune responses to oral antigen as their WT counterparts. By restricting IL-4 expression to only CD4+ Th cells, we find that IL-4 from CD4+ Th cells themselves induce food allergy. Further, IL-4 reporter mice show that CD4+ Th cell production of IL-4 in vivo is dependent on OX40L, a costimulatory molecule that we have shown to be highly expressed on allergen-exposed dendritic cells (DCs). Although NKT and γδ T cells express IL-4, this is not upregulated after allergen priming. Together, these data show that intestinal Th2 immunity in food allergy is initiated by a CD4+ Th cell-intrinsic IL-4 program that is controlled by DC-OX40L and not NKT or γδ T cells.