bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-4- or IL-13-mediated modification of structural molecules in murine and human epidermal keratinocytes (P6015)
Authors: Omori-Miyake Miyuki , Yamashita Masakatsu , Yagi Junji
Published: January 2023

Th2 cytokines including IL-4 and IL-13 are strongly associated with development of atopic dermatitis. IL-4 and IL-13 have been demonstrated to play a regulatory role in structural molecules and antimicrobial peptides expressed by keratinocytes at the final stage of differentiation in the stratum corneum. Since a regulatory role of IL-4 and IL-13 in structural molecules of keratinocytes at earlier stages has remained unknown, we investigated the role of Th2 cytokines using primary murine and HaCaT human keratinocytes. Our study revealed that IL-4 or IL-13 was capable of downregulating keratins and desmosomal cadherins expressed in the spinous layer of murine keratinocytes but not in the basal layer. The identical results were obtained in differentiating HaCaT human keratinocytes. The attenuated expression of these molecules did not occurred in Stat6- or Il4ra-deficient murine keratinocytes, and the overexpression of an active form of STAT6 in keratinocytes without stimulation did not change the expression of keratins in the spinous layer. All suggest that STAT6 and unknown IL-4- or IL-13-induced transcription factor(s), possibly p44/42 MAPK signaling molecules, might synergistically downregulate the expression of structural molecules of keratinocytes in the spinous layer. The attenuated expression of the structural molecules of keratinocytes in both the spinous layer and the stratum corneum in the presence of IL-4 or IL-13 may thus cause the instability of the epidermis.
Human mesenchymal stem cells constitutively express the serine protease granzyme B. (P1097)
Authors: Schüll Marion , Beyer Thamara , Lindner Stefanie , Kaltenmeier Christof , Rojewski Markus , Schrezenmeier Hubert , Jahrsdörfer Bernd
Published: January 2023

Human mesenchymal stem cells (MSC) are known for their immunoregulatory potential and are allegedly involved in tolerance induction. Several immune cells have been shown to exhibit their regulatory effects via expression of the cytotoxic serine protease granzyme B including regulatory T cells and plasmacytoid dendritic cells. Since MSC were recently found to express the GrB inhibitor PI-9, which is primarily expressed by cytotoxic cells, we hypothesized that MSC may also express GrB. Using various methods including Western immunoblotting, ELISpot, FACS analysis we found that MSC freshly isolated from healthy donor bone marrow biopsies indeed express considerable amounts of GrB protein. ELISpot assays demonstrated that GrB was not only expressed but also secreted by MSC. Moreover, a colorimetric GrB activity assay confirmed that GrB was secreted in its enzymatically active form. Importantly, when testing the effect of various cytokines we found that G-CSF, IFNg and TPO were capable of further enhancing GrB expression in MSC. Future studies have to elucidate which aspects of the suppressive activity of MSC possibly depend on GrB and which culture conditions may support a suppressive phenotype of these cells including GrB expression. Our results may open novel approaches to the use of MSC as immunoregulatory cellular therapeutics in inflammatory conditions such as graft-versus-host disease.
Ubiquitin E3 ligase deltex1 promotes PKCθ degradation for full T cell anergy (P1109)
Authors: Lai Ming-Zong , Hsu Tse-Sheng , Wu Pei-Jung , Hsiao Huey-Wen
Published: January 2023

The generation of T cell anergy is associated with upregulation of ubiquitin E3 ligases including Cbl-b, Itch, and GRAIL. Both Cbl-b and Itch contribute to the downregulation of PKCθ in anergic T cells. Recently, we identified deltex1 (DTX1) as another E3 ligase that is upregulated in T cell anergy. DTX1-deficiency prevents T cell anergy production and leads to autoimmunity. In the present study, we found that DTX1 also targeted to PKCθ for degradation. DTX1 overexpression stimulated the degradation of PKCθ. There was a direct interaction between DTX1 and PKCθ. Because DTX1 also promotes Cbl-b expression, we showed that Cbl-b knockdown did not affect the ability of DTX1 to downregulate PKCθ, excluding an involvement of Cbl-b in DTX1-mediated PKCθ degradation. We also found that DTX1-induced PKCθ degradation was not prevented by proteasome inhibitor. Instead, DTX1 directed PKCθ into lysosomal pathway for degradation. In addition, T cell anergy-induced proteolysis of PKCθ was prevented in Dtx1-/- T cells, supporting the essential role of DTX1 in PKCθ downregulation. Our results suggest that DTX1 acts together with Cbl-b and Itch to assure a complete silencing of PKCθ for T cell anergy and immune tolerance.
Sublingual immunization with M2-based vaccine induces broad protective immunity (P4301)
Authors: Song Manki , Shim Byoung-Shik
Published: January 2023

Background: M2e-based vaccine constructs have been shown to be more protective when administered by the intranasal (i.n.) route than after parenteral injection. However, i.n. administration of vaccines poses rare but serious safety issues associated with retrograde passage of inhaled antigens and adjuvants through the olfactory epithelium. In this study we examined whether the sublingual (s.l.) route could serve as a safe and effective alternative mucosal delivery route for administering a prototype M2e-based vaccine. The mechanism whereby s.l. immunization with M2e vaccine candidate induces broad protection against infection with different influenza virus subtypes was explored. Methods and Results: A recombinant M2 protein with three tandem copies of the M2e (3M2eC) was expressed in Escherichia coli. Parenteral immunizations of mice with 3M2eC induced high levels of M2e-specific serum Abs but failed to provide complete protection against lethal challenge with influenza virus. In contrast, s.l. immunization with 3M2eC was superior for inducing protection in mice. In the latter animals, protection was associated with specific Ab responses in the lungs. Conclusions: The results demonstrate that s.l. immunization with 3M2eC vaccine induced airway mucosal immune responses along with broad cross-protective immunity to influenza. These findings may contribute to the understanding of the M2-based vaccine approach to control epidemic and pandemic influenza infections.
The B cell response to influenza infection is limited by CD4 T cell help (P4305)
Authors: Knowlden Zackery , Alam Shabnam , Sant Andrea
Published: January 2023

Seasonal influenza and the risk of widespread influenza pandemics are an ongoing major world health concern. In the United States alone, influenza infection results in more than 200,000 hospitalizations and up to 41,000 deaths each year. The current vaccine strategies focus on eliciting neutralizing antibodies against hemagglutinin and neuraminidase, the major surface glycoproteins involved with viral entry and exit from a host cell. Because of the highly mutable nature of influenza, the vaccine must be reformulated each year - highlighting the necessity for an alternate approach that provides broad and long-lasting protection. We have approached this problem by asking if CD4 T cell memory, elicited by peptide priming, is sufficient to accelerate T cell and B cell responses to influenza infection. After immunization with immunogenic peptides derived from A/New Caledonia/20/99 followed by infection with the same virus, we observed an increased frequency of virus specific CD4 T cells 7 days post infection. Additionally, there was an enhanced B cell response both kinetically and in magnitude. We have also observed a strong correlation between the frequencies of follicular T helper cells and germinal center B cells in the peptide primed/infected cohorts that are not seen in the control primed/infected group. Our results suggest that T cell help for a primary B cell response to infection is limiting, and that peptide priming is sufficient to generate a functional memory CD4 pool.
Cross-reactive neutralizing antibody responses to enterovirus 71 infections in young children (P4319)
Authors: Lee Min-Shi , Huang Mei-Liang , Chiang Pai-Shan , Chia Min-Yuan , Luo Shu-Ting , Chang Luan-Yin , Lin Tzou-Yien , Ho Mei-Shang
Published: January 2023

Recently, enterovirus 71 (EV71) has caused life-threatening outbreaks involving neurological and cardiopulmonary complications in Asian children with unknown mechanism. EV71 has one single serotype but can be phylogenetically classified into 3 main genogroups (A, B and C) and 11 genotypes (A, B1~B5 and C1~C5). In Taiwan, nationwide EV71 epidemics with different predominant genotypes occurred in 1998 (C2), 2000-2001 (B4), 2004-2005 (C4), and 2008 (B5). In this study, sera were collected to measure cross-reactive neutralizing antibody titers against different genotypes. We collected historical sera from children who developed an EV71 infection in 1998, 2000, 2005, 2008, or 2010 and measured cross-reactive neutralizing antibody titers against all 11 EV71 genotypes. In addition, we aligned and compared the amino acid sequences of P1 proteins of the tested viruses. Serology data showed that children infected with genogroups B and C consistently have lower neutralizing antibody titers against genogroup A (>4-fold difference). The sequence comparisons revealed that five amino acid signatures (N143D in VP2; K18R, H116Y, D167E, and S275A in VP1) are specific for genogroup A and may be related to the observed antigenic variations. In conclusion, this study documented antigenic variations among different EV71 genogroups and identified potential immunodominant amino acid positions. Enterovirus surveillance and vaccine development should monitor these positions.
Endogenous specialized proresolving mediators enhance antigen-specific antibody response against influenza virus: A new class of adjuvant (P4285)
Authors: Ramon Sesquile , Serhan Charles , Topham David , Phipps Richard
Published: January 2023

Inflammation is a highly regulated process triggered by the presence of pathogens, injury or trauma. Specialized proresolving mediators (SPMs) are endogenous lipid-derived molecules, which regulate the resolution of inflammation. Resolving inflammation is critical for maintaining homeostasis while assuring immune protection against pathogens, such as influenza virus. We previously showed that the SPM 17-hydroxydosahexaenoic acid (17-HDHA) increased human B cell antibody production in vitro and promoted differentiation towards an antibody-secreting phenotype. Herein, we used a pre-clinical influenza virus immunization mouse model to study the effects of 17-HDHA in vivo. Mice immunized with influenza-derived hemagglutinin (HA) protein plus 17-HDHA have enhanced HA-specific IgM and IgG production compared to mice immunized with HA alone. Enhanced HA-specific antibody production was due to plasma cell increase in the bone marrow. HA-specific antibodies were protective against live influenza viral infection as they decreased morbidity and mortality following viral challenge. Our study shows that DHA-derived SPMs play an important role in enhancing B cell-mediated immunity. An enhanced antigen-specific immune response promotes faster pathogen clearance, and thus return to homeostasis. In addition, these new findings highlight the potential applications of SPMs as new endogenous and non-toxic adjuvants or vaccine “boosters”.
The anatomy of a protective CTL response in influenza virus infected lungs (P3204)
Authors: Cauley Linda , Wu Tao , Hu Yinghong
Published: January 2023

Cytotoxic T lymphocytes (CTL) can provide immunity between serologically-distinct strains of influenza virus, known as heterosubtypic immunity. Although the kinetics of the CTL response have been examined in detail by flow cytometry, changes in the distribution of different subsets of virus-specific CTL within the infected lungs are largely undocumented. We previously used parabiosis experiments to show that some virus-specific CD8 T cells, which maintain stable CD69 and CD103 expression, colonize the lungs and local lymph nodes during primary viral infection and remain sequestered from the circulation long after most symptoms of inflammation have resolved. Fluorescence microscopy has been used to follow changes in the distribution of these virus-specific Trm cells as they respond to secondary viral infection. We show that robust transforming growth factor-beta production selectively eliminates KLRG1+ effector CD8 T cells from the lung tissues, while CD103+ Trm cells persist in the airways. When the immune animals are reinfected with a new strain of influenza virus the CTL response is focused on the tissues around the infected airways. Circulating central memory CD8 T cells begin to divide in the local lymph nodes a few days later, but are insufficient to accelerate viral clearance in non-immune animals.
Intestinal CX3CR1+ macrophages induce and maintain unique CD103+ regulatory T cells (P3206)
Authors: Jang Min Seong , Yang Bo Gie , Lee Eun-Jung , Kim Jung-Hwan , Hong Chun Pyo , Jeun Eun Ji , Doh Junsang , Jang Myoung Ho
Published: January 2023

In the gut, there are tremendous antigens existed such as food proteins, commensal bacteria and even self-antigens. For this reason, maintenance of intestinal tolerance is important to regulate excessive inflammations. Recently, Foxp3+ Tregs are required to maintain intestinal tolerance. However, their phenotypes and functions in immune regulation are heterogeneous, thus it is required to find which subpopulations of Tregs in the gut suppress inflammatory cells effectively to prevent inflammatory diseases. Here we found that majority of the Tregs in the small intestinal lamina propria highly express CD103 molecule on their surface compared to Tregs in other tissues such as thymus, spleen, mesenteric lymph nodes and colon. We next investigated which antigen presenting cells from small intestinal lamina propria can induce CD103+ Tregs in vitro. We found that intestinal CX3CR1+ macrophages, which produce immunoregulatory cytokine IL-10 highly, induce CD103+ Tregs more efficiently, but not dendritic cells. These CD103+ Tregs had effect on reducing occurrence of diarrhea in food allergy model. Further, intestinal CX3CR1+ macrophages induced helios+ Tregs which have been recently suggested to be stable Tregs, under inflammatory stimulations. These findings suggest that CD103+ Tregs induced by intestinal CX3CR1+ macrophages raise the possibility of improvement in therapeutic potential of Tregs for inflammatory diseases such as graft versus host diseases and autoimmune diseases.
The dissemination of Listeria monocytogenes shuttled by Batf3-dependent DC from intestine to mesenteric lymph node following oral infection (P3226)
Authors: Xu Daqi , Sheridan Brian , Freitag Nancy , Schubert Wolf-Dieter , Lefrançois Leo
Published: January 2023

Listeria monocytogenes (L.m.) is a food borne disease-causing bacterium. The natural infection route in humans is through consumption of contaminated food. However, the rules governing L.m. dissemination and the subsequent innate and adaptive immune responses in the intestinal mucosa following oral infection have not been clarified. Thus, this study focused on immune responses to L.m. after oral infection using a murinized L.m. to mimic L.m. pathogenicity in humans. Initially, infection was limited to the gut mucosa, and antigen presentation predominantly occurred within mesenteric lymph nodes (MLN) and Peyer’s patches (PP). However, PP were dispensable for bacterial dissemination and clearance. L.m., but not Salmonella, was shuttled intracelluarly by Batf3-dependent DC from the intestine to the MLN, entering through the sub-capsular sinus into the interfollicular zones to eventually converge in the T cell zone. An organized architecture rapidly developed in the MLN with Ly6G+ neutrophils surrounding L.m., and with an outer zone of DCs and T cells, indicating a potential antigen presentation site. In addition, Batf3-dependent DC were also required for dissemination of irradiated L.m. to the MLN, suggesting the existence of a receptor-mediated system for L.m. acquisition. In sum, our data demonstrated organized immune responses to a unique L.m. dissemination pattern in the gut mucosa.
The cathelicidin LL-37 exerts its mucosal adjuvant activity via enhancing germinal center formation and dendritic cell maturation (P3234)
Authors: Jang Yong-Suk , Kim Sae-Hae , Lee Ha-Yan , Lee Hojun , Kim Ju , Kim Dae-Hyuk , Lee Kyung-Yeol
Published: January 2023

Oral mucosal vaccine confers feasibility and efficacy in vaccination. However, it imposes restrictions such as difficulty in antigen delivery and poor immunogenic environment. Consequently, many studies have been concentrated to develop effective mucosal adjuvants. We determined mucosal adjuvant activity of cathelicidin LL-37 based on the previous report of its activity in parenteral vaccination, although it has not been studied in mucosal system. In oral mucosal vaccination using EGFP protein, LL-37-conjugated antigen effectively evoked the antigen-specific systemic and mucosal immunities. Notably, when the LL-37 was applied to pathogenic antigen, an EDIII of dengue virus, antibody responses with high neutralization activity was induced. Further studies on chemokine profiling induced by LL-37 in Peyer’s patch lymphocytes were well accordance with the increase in the number of germinal centers in Peyer’s patch and mesenteric lymph node by chemotactic effect of LL-37. In addition, oral priming with LL-37-conjugated antigen induced Th1- and Th17-skewed immune responses through CD11c+ CD70+ cell activation in Peyer’s patch. More interestingly, we found the expression of formyl peptide receptor-2, one of the receptors for LL-37, in subepithelial dome of Peyer’s patch and M cells. Collectively, we conclude that LL-37 can play a role as mucosal adjuvant through enhanced antigen delivery, dendritic cell maturation, and chemotactic effect by FPR-2 on M cells and subepithelial dome.
Pseudomonas aeruginosa biological functions are inhibited by normal airway epithelial secretions: implications for cystic fibrosis (P3074)
Authors: Bankers-Fulbright Jennifer , Sorum Alexander , Hinz Sandra , Weitz Anna , Zank Allison
Published: January 2023

P. aeruginosa does not colonize healthy individuals, but it is the leading cause of death in patients with cystic fibrosis (CF). The ability of P. aeruginosa to readily colonize CF-lungs is at least partially related to alterations in airway secretions caused by the disease. In addition to being dehydrated, these secretions have reduced antimicrobial activity compared to that found in normal airways. Thus, we proposed that normal airway secretions contain one or more molecules that reduce P. aeruginosa virulence whereas CF airway secretions do not. To test this hypothesis in vitro, we collected apical secretions from polarized CFTR+ and CFTR- Calu-3 cell monolayers. We applied these secretions at various concentrations to the P. aeruginosa strain PA14 and examined the effect of the secretions on two key biological activities associated with P. aeruginosa virulence: motility and biofilm formation. Secretions from CFTR+ (“normal”) cells dose-dependently inhibited both PA14 flagellar motility and biofilm formation. However, secretions from CFTR- (“CF-like”) cells failed to inhibit either biological function. Our preliminary identification experiments suggest that the inhibitory agent(s) in the CFTR+ secretions is a heat-labile, large (>50 kDa) molecule, probably a protein. Thus, it is likely that normal epithelial secretions play a direct role in inhibiting P. aerugionsa virulence and that these secretions are dysfunctional in patients with CF.
Eosinophils promote immune responses that preserve parasitic nematode larvae (P3086)
Authors: Huang Lu , Gebreselassie Nebiat , Gagliardo Lucille , Lee Nancy , Lee James , Appleton Judith
Published: January 2023

We have shown that eosinophils protect, rather than attack Trichinella spiralis larvae during primary infection in the skeletal muscle. Accumulation of Th2 cells at sites of infection is also impaired in the absence of eosinophils. Here we further elucidated the mechanism underlying this effect. Intravenous infection with T. spiralis newborn larvae specifically and rapidly induced the recruitment of eosinophils to skeletal muscle. The presence of eosinophils only during the earliest stages of muscle infection was sufficient to support parasite growth and survival, suggesting that eosinophils are required to be present at the time newborn larvae first arrive in the muscle, when the local immune response is first engaged. Transfer of MHCII-/- eosinophils also supported parasite growth and survival, showing that antigen presentation by eosinophils is not required. Furthermore, the numbers of myeloid dendritic cells in cervical lymph nodes and skeletal muscles were reduced in eosinophil-ablated, infected mice and improved when eosinophils were restored. The reduction in myeloid dendritic cells correlated with decreased numbers of proliferating Th2 cells in cervical lymph nodes, indicating that eosinophils promote accumulation of myeloid dendritic cells, which may promote Th2 responses. Taken together, our findings suggest that eosinophils exert their influence at the very early stages of the immune response that preserves T. sprialis larvae.
Sublingual vaccination using alpha-galactosylceramide adjuvant induces mucosal and systemic adaptive immunity for protection against lung tumors (P4446)
Authors: Singh Shailbala , Yang Guojun , Schluns Kimberley , Sastry Jagannadha
Published: January 2023

Sublingual (SL) route enables safer and convenient administration of vaccines in comparison to other systemic and mucosal strategies. Here we present evidence demonstrating the effectiveness of SL immunization employing the synthetic glycolipid alpha-galactosylceramide (aGC) for harnessing the adjuvant potential of invariant natural killer T (NKT) cells for the induction of cellular and humoral immune responses to co-administered Ovalbumin (OVA) antigen. Compared to OVA alone, co-administration of aGC with OVA by SL route resulted in the generation and persistence of strong antigen specific CD8+ T cell responses along with serum IgG and vaginal IgA responses. Only in the presence of aGC adjuvant, could a booster administration of the antigen by SL route recall adaptive immune responses effectively. Multiple doses of aGC delivered by SL route resulted in repeated NKT cell activation s in the draining lymph nodes and lungs of immunized mice concurrent with the progressively improving adaptive immune responses. This is different compared to systemic intravenous delivery of aGC that induced anergy of NKT cells and subsequent doses of aGC led to dampening of adaptive immune responses. SL vaccination with OVA antigen admixed with aGC, but not OVA or aGC alone, was significantly effective against challenge of B16 melanoma tumors expressing OVA in the lungs supporting clinical utility of aGC-mediated NKT cell activation by SL route for stimulating protective adaptive immunity.
Varicella zoster virus decreases transcription and translation of STAT1 and blocks translocation of pSTAT1 to the nucleus in cerebrovascular adventitial fibroblasts. (P6149)
Authors: James Stephanie , Choe Alexander , Wyborny Ann , Tratkinskiy Igor , Gilden Don , Nagel Maria
Published: January 2023

Varicella zoster virus (VZV) is the only human virus known to replicate in cerebral arteries and produce stroke. After reactivation, virus travels transaxonally from ganglia and initially infects the arterial adventitia. To analyze the virus-host relationship, we infected primary human cerebrovascular adventitial fibroblasts with VZV and studied the interferon-α initiated STAT pathway, which is commonly activated after virus infection. Quantitative PCR showed that expression of STAT1, STAT2 and Mx1 mRNA were significantly decreased in VZV-infected, but not uninfected adventitial fibroblasts (p<0.05). Western blotting confirmed decreased STAT1 and Mx1 expression in VZV-infected fibroblasts compared to uninfected cells. Although Western blotting did not reveal phosphorylated STAT1 (pSTAT1) in VZV-infected cells, interferon-α is known to trigger STAT1 phosphorylation; thus, we treated uninfected adventitial fibroblasts with interferon-α, after which pSTAT1 expression was readily seen on Western blots. Furthermore, immunocytochemical staining detected pSTAT1 exclusively in the cytoplasm of VZV-infected cells. In interferon-α -treated uninfected cells, pSTAT1 was found exclusively in the nucleus, suggesting that VZV infection blocks egress of pSTAT1 from the cytoplasm to the nucleus in cerebral adventitial fibroblasts.
MicroRNA-155: regulator of HSV-1 encephalitis but promoter of stromal keratitis (P6108)
Authors: Bhela Siddheshvar , Mulik Sachin , Rouse Barry
Published: January 2023

Herpes simplex virus (HSV) infection of humans can lead to life threatening herpes simplex encephalitis (HSE) and sometimes blinding ocular lesion, stromal keratitis (SK). Here, we show that mice with a deficiency of miR-155 are highly susceptible to HSE with a majority of mice (75-80%) dying after ocular infection with HSV. Acyclovir treatment (a day after virus reaches brain) reduced brain viral levels and protected miR-155 KO mice from HSE thus supporting the role for virus replication in brain as the cause of encephalitis. Furthermore, HSV infected miR-155 deficient mice developed compromised virus specific CD8 T cell responses. Moreover, miR-155 KO survivors developed attenuated ocular lesions and revealed significant reduction in pathogenic Th1 cells in corneas and lymphoid organs. Local delivery of antagomir-155 did not increase the incidence of HSE but led to profound reduction in pro-inflammatory milieu and significantly diminished SK lesions. In conclusion, we have discovered dual role for miR-155, a regulator of brain damage while a promoter of ocular immunopathology after HSV infection.
Interaction of mesenchymal stem cells with fibroblast-like synoviocytes via cadherin-11 promotes the angiogenesis by enhanced secretion of placental growth factor (P5113)
Authors: Cho Chul-Soo , Park Su-Jung , Kim Ki-Jo , Kim Wan-Uk
Published: January 2023

Bone marrow derived mesenchymal stem cells (MSC) exist in synovium of patients with rheumatoid arthritis (RA), yet the role of MSC in RA was elusive. Placental growth factor (PlGF) was known to be increased in RA synovial fluids, and blocking of PlGF attenuated the arthritis progression in mice. Herein, we investigated the migration of MSCs by PlGF and interaction of MSC with RA fibroblast-like synoviocytes (FLS). We observed that PlGF induced the chemotaxis of MSC in a dose-dependent manner. PlGF-mediated chemotaxis was inhibited by anti-flt-1 hexapeptide (GNQWFI) and by inhibitors of PI3-kinase (LY294002) and p38 MAPK inhibitor (SB203580), respectively, but not by ERK1/2 inhibitor (PD98059). MSC exposed to PlGF elicited increased phosphorylation of Akt and p38 MAPK. FLS constitutively produced PlGF, while MSC released negligible amounts of PlGF. However, coculture with MSC significantly increased the PlGF production in FLS, and this increase was depedent on the number of added MSC. As revealed by transwell experiments, direct cell contact was required to increase the PlGF production in coculture. Cadherin-11 was expressed both in FLS and MSC, and siRNA knockdown of cadherin-11 in MSC significantly abrogated the enhanced PlGF production. These data indicate that PlGF in synovial cavity could induce the migration of MSC to synovium, and homophilic interaction of MSC with FLS by cadherin-11 contribute to the progression of synovitis by enhancing the secretion of PlGF.
Human neutrophil peptides induce inflammatory atherosclerotic events (P5115)
Authors: Zhang Haibo
Published: January 2023

Atherosclerosis includes complex inflammatory events that leafs to leukocyte recruitment, adhesion and migration, foam cell formation, and platelet aggregation. Human neutrophil peptides (HNP), the major protein content in the azurophilic granules, are released upon PMN activation, and have been found in the lesion area of coronary artery diseases. We hypothesized that HNP play an important role in PMN-mediated inflammatory cardiovascular responses in atherosclerosis. Human coronary artery endothelial cells were stimulated with HNP at a clinical relevant concentration for 4 h and 8 h, followed by cocultured with human peripheral monocyters for 2 h. The monocyte adhesion to the endothelial cells and transmigration increased in the HNP-treated conditions than in the controls. Stimulation of human monocyte-derived macrophages with HNP resulted in oxidative stress that accelerated foam cell formation, which was attenuated by the administration of superoxide dismutase. Stimulation of platelets with HNP resulted in platelet aggregation and deletion of LDL receptor-related protein8 (LRP8), the only LRP phenotype expressed in platelets had no response to HNP. The HNP-induced production of reelin, a LRP8 ligand, by the arterial endothelial cells was required for platelet aggregation. In conclusion, HNP exert pro-atherosclerotic properties mediated through reelin-LRP8 signaling pathways.
Antigen-specific TCR-pMHC catch bond enables force to trigger T cell signaling (P5079)
Authors: Liu Baoyu , Chen Wei , Evavold Brian , Zhu Cheng
Published: January 2023

A long-standing fundamental question in immune-biology is how T cells recognize, discriminate, and respond to pMHC molecules expressed by antigen presenting cells. The current study aims to address this question from the perspective of TCR-pMHC binding kinetics, focusing on the role of mechanical force. Particularly, we investigated how force regulates dissociation of single TCR-pMHC bonds and how such regulation impacts T cell responses. We used a single-bond mechanical assay to demonstrate that force unexpectedly stabilizes TCR-pMHC interaction on live T cells via an agonist-specific catch bond, where bond lifetime is prolonged by force. Concurrent Ca2+ imaging and binding measurement showed that a 10-piconewton durable force applied via agonist pMHC to a TCR is both sufficient and necessary to trigger intracellular Ca2+ flux. Triggering occurs after serial TCR-pMHC engagements to accumulate bond lifetimes above a threshold in the first minute of repeated force application. Furthermore, Ca2+ signaling strength negatively correlates with the number of short lifetimes. Our data support a T cell triggering model, where a mechanical force applied to the TCR delivers either an activating or inhibitory signal depending on the bond lifetime, such that agonists form catch bonds with the TCR to rapidly accumulate long lifetimes to activate the T cell, whereas antagonists form short-lived slip bonds that attenuate activation.
Altered adhesion receptor expression impairs recruitment of aged T cell to the nasal mucosa in response to influenza virus infection. (P5085)
Authors: Baaten Bas , Douglass Mia , Wong Lily
Published: January 2023

Influenza viruses cause significant annual illness and death in the elderly with recurrent seasonal epidemics and sporadic devastating pandemics. CD8 T cells are crucial for resolving influenza virus infection, can protect from divergent viruses, and are highly correlated with protection in the elderly. Due to its anatomical location, the nasal-associated lymphoid tissue (NALT) is crucial for the induction of the immune response, yet little is known regarding the requirements for optimal immunity at this mucosal site. We demonstrated that the number of antiviral CD8 T cells in the NALT was reduced in the aged. Phenotypic analyses of nasal T cells revealed that the carbohydrate-dependent rolling receptors L-Selectin, P-Selectin glycoprotein ligand-1, and CD44 were differentially expressed on aged T cells. We addressed the hypothesis that the reprogramming of adhesion receptor expression in the aged functionally impacts the migration of virus-specific CD8 T cells to the infected nasal mucosa. We demonstrated that rolling receptors, especially L-Selectin, were crucial for CD8 T cell recruitment to the NALT. Thus, the differential adhesion receptor expression profile on aged CD8 T cells may provide a mechanism that contributes to defective mucosal immunity in the elderly and our provides crucial information for novel strategies for improved nasal vaccines.
CD73 regulates nitric oxide production and inflammatory responses in Salmonella infected RAW 264.7 macrophages (P4223)
Authors: Costales Matthew , Alam Mohammad Samiul , Williams Kristina
Published: January 2023

Surface enzymes CD39 & CD73 mediate synthesis of extracellular adenosine that can regulate immune responses. Innate response to Salmonella infection involves efficient macrophage (MΦ) function. CD73 is expressed by variety of cell types & mediates anti-inflammatory responses. We investigated the role of CD73 in RAW 264.7 MΦ functions involving nitric oxide (NO) & proinflammatory responses after Salmonella infection. Macrophage mRNA expression of CD39 & CD73 were measured by qPCR after Salmonella antigen (ST-Ag) treatment. Proinflammatory cytokine mRNA & NO production were tested in ST-Ag treated MΦ with or without CD73-inhibitor (APCP) treatment. Phagocytosis & Salmonella-killing ability of RAW MΦ were also evaluated after GFP-Salmonella infection, in the presence of APCP. CD73 & CD39 mRNA were expressed in RAW MΦ & ST-Ag treatment significantly reduced their expression. ST-Ag treated & CD73-inhibited MΦ produced more NO & proinflammatory cytokines (e.g. IL1-β, TNF-α). Phagocytosis of GFP-Salmonella was much higher in APCP treated, infected MΦ compared to untreated infected cells. Phagocytosed Salmonella were more efficiently cleared from MΦ in the absence of CD73 as observed by fluorescence-microscopy. CD73-inhibition of MΦ during Salmonella infection can enhance proinflammatory cytokines & NO responses. Extracellular adenosine synthesized by CD73 suppresses anti-bacterial responses in macrophage which may impair immunity against intracellular Salmonella infection.
Complex role of immature myeloid cells in Salmonella Typhimurium infection (P4225)
Authors: Tam Jason , Kullas Amy , Mena Patricio , van der Velden Adrianus
Published: January 2023

Immature myeloid cells are a heterogeneous population of cells that, under normal conditions, provide tissues with protective cells (e.g. macrophages, dendritic cells). Under certain pathological conditions, myeloid cell differentiation is overridden and immature forms of these cells accumulate in tissues. Immature myeloid cells that express CD11b and either Ly6C or Ly6G (isoforms of Gr-1) have been associated with immunosuppression in cancer and, more recently, infection. We report that large numbers of CD11b+Ly6Chi Ly6G- and CD11b+Ly6CintLy6G+ cells accumulate and persist in tissues of mice infected with Salmonella Typhimurium. The CD11b+Ly6Chi Ly6G- cells exhibited a mononuclear morphology whereas the CD11b+Ly6Chi Ly6G- cells exhibited polymorphonuclear and ring-shaped nuclear morphologies. The CD11b+Ly6Chi Ly6G- cells expressed surface MHC-I and II, and differentiated into macrophage-like cells following ex vivo culture. The CD11b+Ly6Chi Ly6G- cells were capable of inducing T cell proliferation in vitro, but only when their ability to produce nitric oxide was blocked. The CD11b+Ly6Chi Ly6G- cells also were capable of presenting antigen to T cells in vivo, albeit less efficiently than macrophages. These findings indicate that CD11b+Ly6Chi Ly6G- cells have a complex role in S. Typhimurium infection, providing a balance of immunosuppressive and protective functions where the tipping of this balance may be an important factor influencing the outcome of infection.
Understanding altered toll-like receptor function in the elderly (P4251)
Authors: Canan Cynthia , Rajaram Murugesan , Turner Joanne
Published: January 2023

As the number of elderly individuals increases, infectious diseases, such as influenza, pneumonia and tuberculosis have become a significant public health concern. Recent interest in pathogen recognition via toll-like receptors (TLR) and the ability of TLR agonists to generate enhanced vaccine immunogenicity has led to questions regarding altered immune function in the elderly. In contrast to young mice, recent studies in our lab have shown that in old mice, Mycobacterium tuberculosis infected pulmonary macrophages can utilize a TLR2 independent pathway to generate cytokine secretion. This new development of specific altered TLR function in the elderly requires more analysis before vaccines can be generated to increase protective immunity. In this study, we will elucidate the differences in cytokine profile in the presence and absence of TLR2 by siRNA knockdown in old mice. This will allow us to determine changes that impair protection against infectious disease in old age. We will also examine pattern recognition receptors that compensate for cytokine production in the absence of TLR2. Understanding the compensatory mechanism could give us insight into targeting alternate receptors as adjuvants in vaccines development. Investigation into these age-related alterations in TLR function is of utmost importance in order to boost vaccine’s potential to generate strong immunity in the elderly.
Regulation of neutrophil-mediated anti-microbial immunity by type 2 cytokines (P4253)
Authors: Krieg Carsten , Guglietta Silvia , Bouchaud Grégory , Boyman Onur
Published: January 2023

Neutrophils are innate immune effectors primarily acting as a first line of defense against bacterial infections. Interestingly, acute type 2 inflammation, as seen in allergic disorders or during parasitic infections, displays a striking reduction in neutrophils. Moreover, allergic patients show an increased susceptibility towards certain bacteria usually contained by neutrophils. Here, we show that both elevated and physiologic levels of distinct type 2 cytokines dominantly inhibit several functional aspects of murine and human neutrophils, thus impairing neutrophil-mediated bacterial immunity. In particular, type 2 cytokines were found to inhibit egress of neutrophils from bone marrow as well as their anti-bacterial cytotoxicity and migration. Notably, mice lacking type 2 cytokine receptor signaling or receiving blocking type 2 cytokine antibodies survived a lethal infection with different bacteria. Based on the afore-mentioned results, we postulate a prototypic role of certain type 2 cytokines in regulating neutrophil-mediated anti-bacterial immunity.
The facultative intracellular pathogen Cryptococcus neoformans is susceptible to in vivo clearance by alveolar macrophages (P4211)
Authors: Calverley Matthew , Harmsen Allen
Published: January 2023

Cryptococcus is a yeast commonly found in soil, decaying wood and bird excreta. Normally characterized as an opportunistic human infection, cryptococcosis is a leading cause of meningitis and death in HIV/AIDS patients in Sub-Saharan Africa. Recent Cryptococcus gattii outbreaks within healthy, immunocompetent populations in the Pacific Northwest are calling this characterization into question. Recent studies support the ability of Cryptococcus to replicate intracellularly, particularly in macrophages, leading to classification as a facultative intracellular pathogen. However, in vivo analysis of this capacity for intracellular replication in the context of a disease model for pulmonary infection has been lacking. Here we employ a GFP expressing strain of Cryptococcus neoformans and present novel dye labeling strategies to assess whether intracellular replication is productive in a mouse model of pulmonary cryptococcosis. We show that, while limited intracellular replication occurs, cryptococcal clearance by alveolar macrophages within the first forty-eight hours of pulmonary infection predominates. Cryptococcus may correctly be characterized as a facultative intracellular pathogen; however, alveolar macrophages possess the capacity to clear this pathogen, keeping early infection in check. Our finding, showing the importance of the alveolar macrophage in early cryptococcal clearance, opens the possibility of novel treatment strategies for a clinically important human pathogen.
The role of c-Myc in phagocytosis of mycobacteria in human macrophages (P4213)
Authors: Wang Liangjie , Li James C. B. , Lau Allan S. Y.
Published: January 2023

Tuberculosis remains a major cause of morbidity and mortality worldwide as a result of Mycobacterium tuberculosis infection. Macrophages are the major immunocytes to initiate host immunity against mycobacteria. Among the multiple strategies employed by macrophages to inhibit mycobacteria growth or kill intracellular mycobacteria, phagocytosis is the first step. Phagocytosis involves recognition, internalization and finally digestion of pathogens at phagolysosome. c-Myc is a transcription factor that regulates a variety of target genes. It can form a complex with Max and bind to the enhancer box sequences of the promoter to mediate the transcription. Recently, our group revealed that c-Myc has a potential role in regulating the antimicrobial responses in macrophages. Here, we further revealed that c-Myc may play a positive role in phagocytosis and contribute to host defense to mycobacteria. We demonstrated that mycobacteria could induce c-Myc expression in primary human macrophages. Using colony forming unit assay and cell imaging assay, we showed that pretreatment of c-Myc inhibitor, 10058-F4, could significantly reduce the amount of mycobacteria internalized by macrophages. In addition, the acidification of phagolysosome in mycobacteria infected macrophages was shown to be inhibited by 10058-F4. However, the action of c-Myc in the process of phagocytosis is independent of Rho family GTPases. In conclusion, c-Myc may play a role in phagocytosis of mycobacteria.
C1q promotes recognition and engulfment of H. pylori infected AGS cells and modulates inflammatory cytokine production in macrophages. (P4019)
Authors: Fox Sarah , Ryan Kieran , Das Soumita , Crowe Shiela , Ernst Peter
Published: January 2023

H pylori resides in the gastric lumen and is a significant risk factor for gastritis and gastric cancer. This organism promotes apoptosis of gastric epithelial cells that are subsequently cleared by local phagocytes. C1q, the recognition component of the complement cascade, is an important molecule in immune regulation where it promotes uptake of apoptotic cells and modulates inflammation. We demonstrated that H pylori infection induces apoptosis of gastric AGS cells in vitro and that apoptotic epithelial cells are recognized and engulfed by macrophages independent of the method of cell death. Further, C1q binds to apoptotic cells and is important in the interaction between apoptotic cells and macrophages. We show that the absence of serum reduced attachment of AGS cells to phagocytes by up to 90% and the addition of 40μg/ml of C1q was sufficient to restore normal binding. A comparable effect of C1q on internalization of AGS cells was observed using a flow cytometric assay. C1q, in the absence of serum, attenuated IL-6, TNF-α and IL-1β production induced by 100 ng/ml of LPS or 100 MOI H. pylori compared to controls (45-60% reductions). In conclusion, we show that C1q is important in the resolution of inflammation by facilitating the engulfment of apoptotic cells and inhibiting the pro-inflammatory cytokine responses. Further, the inhibition of host responses attributed to the engulfment of apoptotic cells may be induced by extracellular factors including C1q.
CAR-dependent intestinal epithelial wound repair is inhibited by JAML released from migrating neutrophils (P4047)
Authors: Parkos Charles , Weber Dominique , Sumagin Ronen , Neumann Philipp-Alexander , Leoni Giovanna , McCall Ingrid , Andargachew Rakieb , Brazil Jennifer , Nusrat Asma
Published: January 2023

Junctional adhesion molecule-like protein (JAML) expressed on neutrophils (PMN) has been shown to bind to the epithelial tight junction protein cocksackie-adenovirus receptor (CAR) and partially mediate neutrophil transepithelial migration. Since CAR has also been implicated in regulating epithelial barrier function, we investigated the contribution of JAML-CAR interactions to CAR-mediated mucosal epithelial homeostasis during inflammation. Here we report that JAML is shed from activated/migrating PMN and monocytes in a stimulus and dose-dependent manner that is mediated by zinc-metalloproteases. Using complementary in-vitro and in-vivo approaches, we demonstrate that CAR ligation by shed JAML as well as by adenoviral fiber knob protein-5, an analogous CAR ligand that binds to the same site as JAML, inhibits intestinal epithelial barrier function and impairs epithelial wound healing. Inhibitory effects of soluble JAML on CAR-mediated regulation of wound repair were secondary to downregulation of ERK-dependent epithelial cell proliferation. Lastly, an anti-JAML mAb that blocks JAML-CAR binding, reversed the inhibitory effects of JAML released by migrating PMN on epithelial wound repair. These findings represent a new mechanism by which transmigrating myelomonocytic cells actively shed TJ-binding ligands that alter epithelial homeostasis and contribute to disease pathophysiology.
Evaluation of vaccines against P. salmonis: analysis of acquired versus innate immune response (P4055)
Authors: Yañez Alejandro , Pontigo Juan , Carcamo Juan
Published: January 2023

The Salmonid Rickettsial Septicaemia (SRS) is one of the most damaging syndrome that affects salmon farming in Chile. The causal agent of this pathology is the bacterium called, Piscirickettsia salmonis and vaccination remains the best way to reduce the economic impact caused by this disease. Several vaccines are used based in bacterins and recombinant proteins. However, the modulation of the innate and humoral immune response has not been determined. Thus 3 prototypes vaccines were used to evaluate the functionality of cellular components of P. salmonis in the modulation of immune response. In order to evaluate the immunogenicity produced by the prototype AUSTTRAL-SRS vaccine in Atlantic salmon. We designed a trial where we injected three groups of fish with three different formulations in two doses. After three weeks, the fish of each group were challenged with a lethal dose (LD70) of P. salmonis. The result obtained by studying the interleukin gene expression, ROS and lisosyme analysis let us to conclude that the three prototype vaccines were able to largely stimulate the innate immune response. However, only one prototype was efficient to stimulate the specific acquire immune systems in fish and also was highly efficient to protect the vaccinated salmon against SRS. These results indicate that the used marker of the innate immune system were not a good vaccine markers compared with acquire immune response. INNOVA 11IDL2-10523
IL-36γ induction in the human female reproductive tract is microbe-dependent (P4009)
Authors: Herbst-Kralovetz Melissa , Radtke Andrea
Published: January 2023

The interleukin-1 (IL-1) family consists of pleiotropic cytokines that play a role in the initiation of immune and inflammatory responses. IL-36γ (IL-1F9) is a newly identified IL-1 family member that functions as an agonist. IL-36γ is highly expressed in epithelial cells of the intestine, lungs, and skin, however IL-36γ has not previously been characterized in the human female reproductive tract (FRT). Upon pathogen infection or during inflammatory disorders, IL-36γ is induced and signals through the IL-1R-related protein 2 receptor to trigger NF-κB expression and IL-6 and IL-8 production. Here, we demonstrate for the first time the expression and regulation of IL-36γ in human FRT epithelial cells. Utilizing a human 3-D FRT epithelial cell model previously shown to express functional TLRs similar to in vivo tissue, we demonstrate a unique IL-36γ expression signature upon microbial product stimulation. Treatment of the FRT cells with pathogenic microbial products resulted in a significant increase in IL-36γ expression by quantitative RT-PCR, however exposure of FRT cells to commensal bacteria showed no significant increase in IL-36γ. Induction of IL-36γ by microbial products appears to be TLR dependent. From this data we show for the first time that human 3-D FRT cells express IL-36γ and respond in a microbe-dependent manner. In conclusion, our work begins to elucidate a novel innate immune response mechanism involving IL-36γ induction during FRT mucosal infection.