bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancing host defense against bacterial pore-forming toxin by suppressing HMG-CoA reductase pathway in airway epithelial cells (112.13)
Authors: Kao Cheng-Yuan , Hung Li-Yin , Chang Ching-Yun , Lim Jae-Hyang , Li Jian-Dong , Wu Reen
Published: January 2023

Pneumolysin and α-hemolysin are the main pore-forming toxins known to play critical roles in bacterial pneumonia caused by the infections of Streptococcus pneumoniae and Staphylococcus aureus, respectively. Airway epithelia are the initial and the primary targets of these air-borne bacterial infections. Several epidemiological studies have linked the use of statin to the improvement in pneumonia outcomes, but the nature of the mechanism is still unknown. Using primary normal human bronchial epithelial (NHBE) cells and an immortalized normal bronchial epithelial cell line, HBE1, we confirmed the effects of simvastatin pre-treatment in enhancing host defense against these bacterial toxins. The protective effects could be further demonstrated in vivo with simvastatin administration prior to intra-tracheal instillation of these toxins. We further found that the protection requires protein synthesis and is calcium dependent. Using siRNA gene silencing, pharmacological treatment with inhibitors, immuno fluorescence microscopy, Western blot and qRT-PCR approaches, we have delineated the protection mechanisms in airway epithelial cells through HMG-CoA-Reductase pathways. Our results also suggest that the inhibition of HMG-CoA reductase pathway may enhance host defense molecule synthesis in airway epithelium. It is plausible that this study may lead to the development of an adjuvant therapy against pore-forming toxin-related bacterial infections in lung.
IgG keeps virulent Salmonella from evading dendritic cell uptake (112.15)
Authors: Riquelme Sebastián , Bueno Susan , Kalergis Alexis
Published: January 2023

Salmonella enterica serovar Typhimurium (ST) can prevents Dendritic Cells (DCs) from activating antigen-specific T cells. In addition, it has been described that the Salmonella Pathogenicity Island 1 (SPI-1), which promotes phagocytosis of bacteria in non-phagocytic cells, can suppresses this process in DCs in a PI3K-dependent manner. Recent studies have shown that IgG-opsonization of Salmonella can restore the capacity of DCs to present antigenic pMHC complexes and prime T cells in a FcγRIII-dependent manner. This suggests that this receptor could counteract both antigen presentation and phagocytosis evasion by bacteria. Here we show that, despite IgG-coated Salmonella retains its capacity to secrete anti-capture proteins, DCs are efficiently capable to engulf high loads of IgG-coated bacteria. These results suggest that DCs employ another mechanism to engulf IgG-coated ST different from that used for free bacteria. Thus, we noted that DCs do not employ PI3K, actin cytoskeleton or dynamin to capture IgG-coated bacteria. Likewise, we observed that the capture is a FcγR-independent mechanism. Interestingly, these internalized bacteria were rapidly targeted for degradation within lysosomal compartments. Thus, our results suggest a novel mechanism in DCs that does not employ PI3K/actin cytoskeleton/dynamin/FcγRs to engulf IgG-coated Salmonella, is not affected by anti-capture SPI-1-derived effectors and enhance DC immunogenicity, bacterial degradation and antigen presentation.
The Membrane Attack Complex of complement is required for the development of murine Experimental Cerebral Malaria (112.2)
Authors: Ramos Theresa , Darley Meghan , Barnum Scott
Published: January 2023

Cerebral malaria (CM) is a severe complication of P. falciparum malaria with a 15-30% fatality and 10% of CM survivors have permanent neurological sequelae. Since inflammation is key to CM development, we examined the role complement in experimental cerebral malaria (ECM) development using the P. berghei model. Previous studies indicated that C5-/- mice were protected in ECM. To determine the role of complement activation, we examined mice deficient in pathway specific components, C4 and Factor B, as well as C3, which forms the C5 convertase. We found no differences in survival between wild type (WT) and C4 or Factor B deficient mice. We observed moderate protection in C3-/- mice, suggesting that blocking C5 cleavage is important in ECM development. We found that complement anaphylatoxin receptor deficient mice were not protected, suggesting that the mechanism of protection in C5-/- mice is not mediated through C5a-induced inflammation. We next examined C5b indirectly by treating WT mice with anti-C9 antibody and found significant increase in survival. We also treated C3-/- mice with anti-C9 antibody to determine if blocking MAC in these mice could abrogate disease, and found significant protection in treated mice compared to controls. Our results suggest that pathological effects of complement in ECM are primarily mediated at C5 through the terminal pathway and that this is occurring even in the absence of C3 suggesting an alternative mechanism for C5 cleavage in C3-/- mice.
Molecular mechanism for type I interferon induction by atypical strains of Toxoplasma gondii (112.6)
Authors: Melo Mariane , Nguyen Quynh , Saeij Jeroen
Published: January 2023

Host cells infected with Toxoplasma gondii undergo profound physiological and morphological changes as a result of the host cell’s response to the parasite and the parasite’s attempt to convert the host cell into a suitable growth environment and evade the immune system. T. gondii infection is known to affect cytokine induction by host cells through modulation of intracellular signaling pathways, including those controlled by NFκB and STAT3/6. To identify other relevant host cell signaling pathways altered by parasite infection, bone marrow-derived macrophages were infected with 26 different strains of Toxoplasma. Expression profiles of both parasite and host cells were then determined by highthroughput RNA sequencing. Initial data analysis revealed that host cell response varies considerably depending on the strain used for infection. We were able to identify a group of strains that can induce IFNβ production and the subsequent upregulation of interferon regulated genes, including viperin and IRF7. Type I interferons are usually produced upon recognition of nucleic acids by either surface or cytoplasmic pathogen-associated molecular pattern receptors, including Toll-like receptors, and the RNA helicases RIG-I and Mda5, triggering a signaling cascade that activates the transcription factors IRF3, IRF5 and/or IRF7, which induce transcription of the IFNβ gene. We are currently investigating the molecular mechanisms controlling IFNβ production in Toxoplasma-infected macrophages.
Large Scale T Cell Epitope Discovery in Yellow Fever Virus (113.12)
Authors: Kongsgaard Michael , Stryhn Anette , Buus Søren
Published: January 2023

This project involves a comprehensive, large-scale discovery effort directed at identifying T cell epitopes targeted in the acute immune response following vaccination with attenuated yellow fever (YF) virus in an out bred population of vaccinees. WHO recommends revaccination once every 10 years. Volunteers have donated blood before and about 2 weeks after vaccination and divided into groups of naïve and boosted donors. Initially, pre-and post PBMCs from the vaccinees were analyzed using multicolor flowcytometry to determine the frequency of activated T cells using intracellular and surface phenotypic markers. Clearly, boosted donors generated low frequencies of KI67+, Bcl-2-, CD8+ T cells (1.4%) and CD38+, HLA-DR+, CD8+ T cells (2.1%), resembling background levels (1.2% and 1.2%), while naïve donors generated higher frequencies of activated cells (5.1% and 5.1%). YF peptide-specific, HLA-restricted T cell responses were identified by ELISpot with a 30x30 peptide matrix and validated using multicolor flowcytometry to determine the nature of the YF specific T cell response (CD4, CD8 and CD38, functional markers such as IFNγ and TNFα and specificity markers (HLA tetramers)). The number of responses and the frequency of peptide-specific cells are clearly lower in boosted donors, than in naïve donors. This coincides with YF-specific antibody titers in boosted donors.
Modification of immune activation in HIV-1-infected humanized mouse model using TLR7/9 antagonists (113.19)
Authors: Chang J. Judy , Lindsay Robert , Doyle Erin , Vrbanac Vladimir , Seung Edward , Dudek Timothy , Precopio Melissa , Kandimalla Ekambar , Tager Andrew , Altfeld Marcus
Published: January 2023

Background: T cell immune activation is a strong predictor of HIV-1 disease progression, and IFNα production following TLR7 stimulation has been associated with elevated CD8+ T cell activation. We hypothesize that modulation of TLR7 stimulation could be used to manipulate IFNα production and subsequently reduce HIV-1-associated immune activation. Methods: Humanized BLT mice were generated by transplanting irradiated NOD/SCID/IL2Rγc-/- mice with human fetal thymus, liver tissue and human hematopoietic stem cells. After immune reconstitution, human DC, monocyte and T cell populations were detectable in the mice, and in vitro TLR responsiveness of DCs and monocytes reflected those observed in human PBMCs. Humanized mice were infected with HIV-1 and then treated with a TLR7/9 antagonist. The T cell activation markers were examined at each stage by flow cytometry. Results: BLT mice-derived human pDC responses to TLR7 stimulation were significantly blocked by in vitro treatment with the TLR7/9 antagonist (P<0.05). HIV-1 infection of BLT mice led to increased expression of immune activation marker CD38 on human T cells in vivo, and treatment of infected mice with the TLR7/9 antagonist led to a significant reduction in CD38 expression. Conclusion: Treatment of HIV-1-infected humanized BLT mice with TLR7/9 antagonist resulted in significant reduction of HIV-1-associated immune activation, indicating an important role of the TLR7/9 pathway in mediating immune activation in HIV-1.
Eliciting mucosal and systemic CD8+ T-cell responses via pulmonary vaccination with synthetic nanoparticles (113.21)
Authors: Li Adrienne , Moon James , Elkhader Jamal , Yen Mimi , Abraham Wuhbet , Suh Heikyung , Irvine Darrell
Published: January 2023

Mucosal vaccines offer needle-free administration and can stimulate immune responses systemically and at mucosal sites. “Vectorless” vaccines based on antigens packaged in synthetic nanoparticles (NPs) offer potential advantages in manufacturing, enhanced safety, and lack of anti-vector immune responses. To this end, we tested the ability of lipid NPs encapsulating ovalbumin or SIV-gag epitopes with Toll-like receptor agonists to stimulate mucosal immunity following pulmonary vaccination in mice. CD11c+B220+MHC-II+CD205+ DCs and CD11c+MHC-IIloF4/80+CD11b+ macrophages were the major cell types acquiring antigen in the lungs. Following prime and boost, a dramatic expansion of endogenous antigen-specific CD8+ T-cells to ~15% of CD8+ cells in blood was observed-twice the level seen with soluble antigen vaccines and 3 times the level achieved by s.c. immunization. Antigen-specific T-cell expansion was detected in the local lung tissue (~60% of CD8 cells), as well as in the gut and vaginal tract (~40% CD8 cells). These T-cells produced effector cytokines, and lasting CD127hi KLRG1lo effector memory cells were detected at 11wks after priming in both systemic and mucosal compartments. ELISA analysis of serum, fecal pellets and vaginal washes demonstrated that humoral responses were also stimulated. This data provides evidence that mucosal delivery of synthetic NPs can safely and effectively stimulate CD8+ T cells at sites relevant for protection against mucosal pathogens such as HIV.
Identification and surface display on liposomes of neutralizing linear B-cell epitopes as a strategy for biodefense vaccine development (113.25)
Authors: Vance David , Yermakova Anastasiya , Mantis Nicholas
Published: January 2023

The design of subunit vaccines for Category A-B toxins, including anthrax and ricin, has proven largely unsuccessful. Recombinant subunit antigens are minimally protective, difficult to manufacture and/or highly unstable. Thus, there is an urgent need to develop alternative, more effective approaches to vaccine design. With ricin toxin as a model system, we are using a two-tiered approach to address this issue. First, we have identified and validated continuous epitopes on ricin’s A (RTA) and B (RTB) subunits that are recognized by toxin neutralizing monoclonal antibodies (mAbs). In the case of mAb CMA2, pepscan analysis identified residues 190-CLTSDSNIRETVVKI-204 on RTB as being the target of antibody binding. Screening of a phage-displayed peptide library revealed that residues DxNxR are the critical contact points between CMA2 and ricin. An RTA-specific mAb, PB10, bound to peptide 97-NQEDAEAITHLF-108 in pepscan analysis, and phage display identified residues QExA as the likely contact points. The second tier of this project involves attaching these specific peptide epitopes polyvalently to the surface of liposomes (100 nm), with or without the TLR4 agonist MPLA present in the liposome bilayer, and then testing whether these liposomes, when injected into mice, are capable of eliciting protective anti-ricin antibodies. In preliminary studies, we have successfully produced liposomes displaying peptide epitopes from RTA and RTB, and have initiated mouse immunization studies.
Stable incorporation of functionally active purified GPI-anchored immunostimulatory molecules on H5 influenza VLP surfaces by protein transfer. (113.33)
Authors: Patel Jaina , Song Jae Min , Shashidharamurthy Rangaiah , Compans Richard , Kang Sang-Moo , Selvaraj Periasamy
Published: January 2023

H5N1 influenza, a bilayered lipid enveloped virus, is highly pathogenic and can lead to a pandemic threat; therefore, a potent vaccine is required. The use of virus-like particles (VLPs) as a vaccine has recently gained importance as they are safe and highly immunogenic. VLPs are similar in size and structure to their viral counterparts but do not contain the viral genome required for replication. Although VLPs are immunogenic, full protection and cross-reactivity to other influenza strains does not always occur; therefore, we aim to modify H5 influenza VLPs to incorporate immunostimulatory molecules (ISMs) by using a protein transfer method. Previously, we have shown that GPI-anchored ISMs (ICAM-1 and IL-12) are able to incorporate onto H5 influenza VLPs by a simple and easy protein transfer method. In this study, we show that purification of GPI-IL-12 does not alter the functionality of the protein in stimulating proliferation of ConA-activated splenocytes suggesting that purified GPI-ISMs remain functionally active. Furthermore, the detection of incorporated GPI-ISMs onto the surface of H5 influenza VLPs by protein transfer was conducted by using immunogold staining electron microscopy and incorporation occurred uniformly as detected by flow cytometry analysis. Moreover, these GPI-ISMs remain stably expressed on the VLP surface for at least a week at 4oC. This study suggests that H5 influenza VLPs can be modified to stably express ISMs on its surface by protein transfer.
IL-2 and IL-6 cooperate to enhance the generation of effectors from aged human CD8 T cells to live influenza virus (113.34)
Authors: Zhou Xin , Behzad Hayedeh , Haynes Laura , Reed Steven , Swain Susan , McElhaney Janet
Published: January 2023

An aAge-related decline in cytolytic activity has been mainly described in CD8+ T cells and , with relative sparing of CD4+ T cells. wWe have shown that the poor CD8+ effector T cell response to influenza A/H3N2 challenge is due to a decline in the proportion and function of these cytolytic T lymphocytes (CTL). Hee we report In this study, we showed that therethis response corresponds to a significant decline in IL-2 levels but increased IL-6 levels in influenza-stimulated PBMCs from vaccinated older compared to young adults. We find that addition of exogenous Supplementation of PBMC with IL-2 and IL-6 to PBMC cultures with restoresd the proliferative response and reducesd the proportion of apoptotic effector CD8+ T cells in older adult PBMC to resemble that of younger adults. To further elucidate the mechanism, Wwe finshowed that exogenous IL-2 and IL-6 resulted in the differentiation of naïve CD4+ T cells into effector T helper cells which express IL-2 in response to influenza virus stimulation. Further, use of a TLR4 ligand combined with influenza vaccine ,whichto respectively stimulate IL-6 and IL-2 production, resultsed in an enhanced granzyme B response of aged CD8 T cells to a live influenza virus challenge. These results suggest provide a mechanism that could be targeted in the development of influenza vaccines to potentially with enhance theird efficacy in older adults.
NLRC5 dependent activation of the inflammasome in response to bacteria (114.8)
Authors: Davis Beckley , Roberts Reid , Barker Brianne , Duncan Joseph , Ting Jenny
Published: January 2023

The nucleotide-binding domain leucine-rich repeat-containing proteins, NLRs, are intracellular sensors of pathogen-associated molecular patterns and damage-associated molecular patterns. A subgroup of NLRs can form inflammasome complexes, which facilitate the maturation of procaspase 1 to caspase 1, leading to IL-1β and IL-18 cleavage and secretion. NLRC5 is predominantly expressed in hematopoietic cells and has not been studied for inflammasome function. RNA interference-mediated knockdown of NLRC5 nearly eliminated caspase 1, IL-1β, and IL-18 processing in response to bacterial infection, pathogen-associated molecular patterns, and damage-associated molecular patterns. This was confirmed in primary human monocytic cells. NLRC5, together with procaspase 1, pro-IL-1β, and the inflammasome adaptor ASC, reconstituted inflammasome activity that showed cooperativity with NLRP3. The range of pathogens that activate NLRC5 inflammasome overlaps with those that activate NLRP3. Furthermore, NLRC5 biochemically associates with NLRP3 in a nucleotide-binding domain-dependent but leucine-rich repeat-inhibitory fashion. These results invoke a model in which NLRC5 interacts with NLRP3 to cooperatively activate the inflammasome
mTORC2 controls T-cell receptor processing and surface expression during thymocyte development (115.18)
Authors: Werlen Guy , Chou Po-Chien , Oh Won , Wu Chang-Chih , Ruegg Markus , Hall Michael , Jacinto Estela
Published: January 2023

An efficient immune response relies on the presence of T-cells expressing a functional T-cell receptor (TCR). While the mechanisms generating TCR diversity for antigenic recognition are well defined, what controls its surface expression is less known. The mammalian target of rapamycin (mTOR) forms two structurally and functionally distinct protein complexes, mTOR complex 1 (mTORC1) and mTORC2. While mTORCs were found to regulate T lymphocyte activation and lineage commitment, their role in early thymocyte development remains to be defined. Here we found that mTORC2 modulates T-cell ontogeny and activation by controlling the quantity of TCR that reaches the cell surface of developing thymocytes as well as transformed T-cells. Deletion of the mTORC2 component rictor at early stages of T cell ontogeny (DN2 and onward) led to aberrant maturation and increased proteasomal degradation of nascent TCR polypeptides. Consequently, the levels of TCR as well as other receptors, including CD4, CD8 and CD147 were significantly attenuated on the surface of rictor-deficient thymocytes. Since CD147 was also defectively glycosylated and expressed in SIN1-deficient fibroblasts, our findings suggest that mTORC2 is involved in the co/post-translational processing of membrane receptors. Thus, we uncover a novel function for mTORC2 that provides rationale to target this complex in disorders caused by deregulated expression of surface receptors.
Post-translational regulation of RhoH via the proteasomal pathway (115.2)
Authors: Tamehiro Norimasa , Oda Hiroyo , Suzuki Harumi
Published: January 2023

RhoH, an atypical GTPase-deficient Rho GTPase, is exclusively expressed in hematopoietic lineage cells and is required for preTCR- and TCR-dependent signal transduction during T cell development in the thymus. In the current study, we defined tyrosine residues critical for stability of RhoH protein. HA-tagged mutant RhoH with the substitution of all five tyrosines to phenylalanine was not stable when expressed in 293 cells. The mutant RhoH degraded more quickly and was more highly ubiquitinated than wildtype RhoH, and also its degradation was proteasome dependent. We also found that anti-CD3 plus -CD28 stimulation in T lymphocytes enhanced ubiquitination of RhoH in primary lymphocytes, indicating that TCR-stimulation could induce active RhoH degradation. From analysis of each single mutant of five tyrosines, we determined that three tyrosine residues (Y33, Y73 and Y148) were critical for proteasome-dependent protein degradation, whereas the other two (Y38 and Y83) were not. Furthermore, both Y38 and Y83 mutants showed association with ZAP-70, like wildtype RhoH, suggesting that phosphorylation of these two tyrosines were not important for the binding of RhoH to ZAP-70. From these results, we propose that stability of RhoH protein is regulated by tyrosine phospholyation-dependent ubiquitin-proteasomal degradation, as well as the lysosomal degradation pathway reported before.
Eri1 regulates microRNA homeostasis and mouse natural killer cell development and anti-viral function (115.9)
Authors: Ansel K Mark , Thomas Molly , Abdul-Wajid Sarah , Panduro Marisella , Babiarz Joshua , Rajaram Misha , Woodruff Prescott , Lanier Lewis , Heissmeyer Vigo
Published: January 2023

Natural killer (NK) cells play a critical role in early host defense to infected and transformed cells. Here we show that mice deficient in Eri1, a conserved 3’-to-5’ exoribonuclease that represses RNA interference, have a cell-intrinsic defect in NK cell development and maturation. Eri1-/- NK cells displayed delayed acquisition of Ly49 receptors in the bone marrow and a selective reduction in Ly49D and Ly49H activating receptors in the periphery. Furthermore, Ly49H+ NK cells deficient in Eri1 failed to expand efficiently during mouse cytomegalovirus (MCMV) infection. Consequently, Eri1 was required for immune-mediated control of MCMV. We identified miRNAs as the major endogenous small RNA target of Eri1 in mouse lymphocytes. Both NK and T cells deficient in Eri1 displayed a global, sequence-independent increase in miRNA abundance. Ectopic Eri1 expression rescued defective miRNA expression in mature Eri1-/- T cells. Thus mouse Eri1 regulates miRNA homeostasis in lymphocytes and is required for normal NK cell development and anti-viral immunity.
Fatal pneumococcal infection in mice deficient for the actin-bundling protein L-plastin (117.26)
Authors: Morley Sharon Celeste , Todd Elizabeth , Deady Lauren , Davis Chris , Muenzer Jared
Published: January 2023

L-plastin (LPL) is a leukocyte-specific actin-bundling protein that increases the stability of actin-based structures such as podosomes. Fewer lymphocytes and dendritic cells populate the lungs of LPL-/- mice, presumably due to defective motility of these cell types. To determine whether defective immunity in LPL-/- mice would result in increased susceptibility to infection, we challenged mice intra-tracheally with S. pneumoniae. Less than 20% of LPL-/- mice survived pneumococcal infection, compared to 80% of wild-type mice. Rapid mortality of LPL-/- mice correlated with a 100-fold higher bacterial burden in the blood and bronchoalveolar lavage fluid 24 hours following infection, indicating a significant failure to control and limit bacterial growth in vivo. Defective clearance of pneumococcus was apparent as early as six hours after intra-tracheal challenge, and comparison to Rag1-/- mice indicated defective innate immunity. Preliminary experiments demonstrated defective phagocytosis of pneumococci by LPL-/- alveolar macrophages, providing an explanation for the early clearance defect observed. No requirement for LPL in macrophage function has been previously reported. Delineation of the molecular pathways critical to early innate pulmonary defense against pneumococci through the use of mice deficient for LPL will support research efforts aimed at reducing the global health burden of pneumococcal pneumonia.
Genetic polymorphism in the human LILRB3/LILRA6 locus (118.13)
Authors: Bashirova Arman , Apps Richard , Vince Nicolas , Yu Xu , Carringotn Mary
Published: January 2023

LILRB3 is an orphan receptor expressed on the surface of myeloid cells. It is structurally distinct from HLA class I binding LILRs and extremely polymorphic in the extracellular domain. LILRB3 can mediate negative signaling through its ITIM-bearing cytoplasmic tail. LILRA6 is identical to LILRB3 in the extracellular part but is potentially an activating receptor. The molecules are encoded by two neighboring genes on human chromosome 19. We have analyzed polymorphism in the LILRB3/A6 locus and found that LILRA6 exhibits a level of polymorphism comparable to LILRB3 with similar polymorphic sites. Moreover, we identified copy number variation (CNV) in the LILRA6 locus with 33% of healthy individuals possessing duplication of LILRA6 and 4% bearing deletion of the gene. Duplications and deletions of LILRA6 and a constant number of LILRB3 seem to be a consequence of unequal crossing-over between the two genes resulting in exchange of the extracellular domains between the receptors. Such exchange explains the maintenance of structural similarity of the two receptors and identity of polymorphic sites. Analysis of mRNA expression in the major PBMC fractions shows that LILRA6 is primarily expressed on monocytes, similarly to LILRB3, and its expression level correlates with the CNV. We suggest that the LILRA6 CNV may influence the ratio of inhibitory vs activating versions of this receptor on the cell surface, potentially affecting the biological consequence of LILRB3/A6 ligation.
IgE antibody-mediated degranulation of cutaneous mast cells induces thermal pain in mice (118.8)
Authors: Chatterjea Devavani , Mack Madison , Wetzel Abigail , Shabani Estela , Allen Juliann
Published: January 2023

Pain is a key component of inflammation and a public health issue of significance. Mast cell activity has been implicated in pain responses associated with irritable bowel syndrome, migraine headaches, interstitial cystitis, postoperative pain, and endometriosis, but the mechanisms underlying mast cell involvement in pain pathologies are not clear. We show that degranulation of plantar mast cells caused by passive local antibody (IgE anti-DNP/HSA) sensitization and systemic antigen (DNP/HSA) challenge, induces thermal hyperalgesia as well as significant edema, neutrophil influx, and upregulation of IL-1beta and IL-6 proteins in the plantar tissue. Pre-treatment with histamine receptor H2 and H3/4 antagonists significantly abrogates pain, but not edema, associated with IgE/antigen-mediated mast cell degranulation This suggests that while pain has a multifactorial pathophysiology, tissue-resident sentinel mast cells may be a critical initiators of allergen-invoked hyperalgesia in mice.
Increased bacterial colonization and delayed wound closure in aged mice is associated with altered leukocyte recruitment (118.9)
Authors: Brubaker Aleah , Rendon Juan , Choudhry Mashkoor , Kovacs Elizabeth
Published: January 2023

We developed a murine model of cutaneous wound infection to elucidate the role of innate immune dysfunction in aberrant responses to wound infection with advanced age. Young (10-14 weeks) and aged (18-20 months) BALB/c mice were subjected to 3mm dorsal punch wounds inoculated with ~10^3 CFU Staphylococcus aureus (S.aureus) per wound. At days 1, 3, 7 and 10, aged mice exhibited heightened bacterial colonization (F=0.0648; Age, p=0.0002; Time, p=0.002 by two-way ANOVA) and increased wound size compared to young mice (F=0.7666; Age, p<0.0001; Time, p<0.0001 by two-way ANOVA). At day 1, there was no difference in wound neutrophil (PMN) or macrophage (MΦ) recruitment between young and aged mice despite higher levels of the chemokines KC, MIP-2 and MCP-1 in aged animals (p<0.05). Conversely, at day 3, fewer numbers of PMN and MΦ were recruited to wounds in aged mice (p<0.05) though chemokine levels in aged mice were comparable to young. The frequency or expression of TLR2+ wound PMN or MΦ was not affected by age. No differences in S. aureus phagocytosis by wound PMN or MΦ were observed. Interestingly, in circulation, aged mice had a higher frequency of CXCR2+ PMN and elevated CXCR2 expression compared to young mice at early time points (p<0.05). Together, these data suggest that early dysregulation of leukocyte recruitment impairs resolution of cutaneous wound infection with advanced age.
Administration of anti-FcER1 antibody injections daily for 10 consecutive days delays type 1 diabetes onset in NOD mice (119.11)
Authors: Torrero Marina , Hübner Marc , Shi Yinghui , Mitre Edward
Published: January 2023

Helminth infections protect against autoimmune diseases. In this study, we investigated whether mimicking the immune response induced by helminths could protect against autoimmunity in the absence of actual infection. As helminths cause chronic IgE-mediated activation of basophils and mast cells, we tested the hypothesis that continuous activation of these cells would delay the onset of diabetes in NOD mice. Twelve week old NOD mice were injected i.p. daily with 5ug of anti-FcER1 antibody or isotype control for 10 days and onset of diabetes assessed. In vitro effects of anti-FcER1 were studied by measuring IL-4 production in basophils by flow cytometry and in vivo effects were assessed by measuring plasma histamine and IL-4 levels 30 min and 18 h after i.p. injection of antibody. Cellular immune responses were evaluated by measuring cytokine production from splenocytes by ELISA and assessing frequencies of natural T and B regulatory cells by multicolor flow cytometry. Injections of anti-FcER1 resulted in release of IL-4 and histamine into the bloodstream, consistent with in vivo basophil and mast cell activation. Anti-FcER1 or isotype treatment increased production of IL-10 from splenocytes. By 20 weeks of age, 80% of mice in the control and isotype groups had developed diabetes, compared to only 20% of mice treated with anti-FcER1. These results demonstrate that anti-FcER1 therapy can delay the onset of Type I diabetes in NOD mice.
Vitamin C insufficiency aggravates Dextran Sodium Sulfate (DSS)-induced colitis in Gulo(-/-) mice (120.20)
Authors: Kim Hyemin , Bae Seyeon , Yu Yeonsil , Kim Yejin , Kim Hang-Rae , Hwang Young Il , Kang Jae Seung , Lee Wang Jae
Published: January 2023

Intestinal mucosal damage in the inflammatory bowel diseases (IBD) involves the dysfunctional immunoregulation of the gut. Among the immunoregulatory factors, reactive oxygen species (ROS) are produced in abnormally high levels in IBD and, their destructive effects may contribute to the initiation or propagation of the disease. Vitamin C not only scavenges free radicals as an antioxidant but also has anti-inflammatory effects. Therefore, we investigated the effect of vitamin C on DSS-induced colitis in gulo(-/-) mice which cannot synthesize vitamin C like human. Vitamin C-insufficient gulo(-/-) mice showed decreased survival and lowering recovery efficacy. It accompanied with more severe colitis such as epithelial erosion, infiltration of inflammatory cells and contraction of colon. The production of pro-inflammatory cytokine, IL-6 was remarkably higher in DSS-treated vitamin C-insufficient gulo(-/-) mice than vitamin C-sufficient gulo(-/-) mice and wild type mice. In contrast, the production of anti-inflammatory cytokine, IL-22 was significantly decreased and IL-22 producing NKp46(+) cells were lower in vitamin C-insufficient gulo(-/-) mice. The phosphorylation of STAT3, a downstream signaling of IL-6 and IL-22, was increased in both epithelial cells and lamina propria cells. Taken together, it suggests that vitamin C represents a protective effect on DSS-induced colitis by regulating the production of cytokine and the induction of inflammation.
Effect of spa spring water on cytokine expression in human keratinocyte HaCaT cells and on differentiation of CD4+ T cells (120.26)
Authors: Lee Hye Jin , Cho Kyung-Ah , Woo So-Youn , Lee Kyung-Ho , Kim Jin-Wou
Published: January 2023

Skin contributes to the first line of defense against foreign materials outside of our body. In inflammatory skin disease, the pathogenesis is due to an immune reaction in the keratinocytes, immune cells and soluble mediators. Spa therapy is widely used for the treatment of inflammatory skin disease but the mechanisms are only partly understood by immune regulation. Spa therapy or balneotherapy in dermatologic disease can affect the secretion of pro-inflammatory cytokines, IL-1alpha and tumor-necrosis factor (TNF) from keratinocytes and possibly affect T cell differentiation. In this study, we evaluated the effect of spa spring water from Yong-Goong-Oun-Chun on cells and investigated the skin immune reaction. The treatment of spa spring water from Yong-Goong-Oun-Chun decreased the expression of pro-inflammatory cytokines under TLR stimulation to HaCaT cells and APCs. In addition, spa spring water attenuated the differentiation process of subsets of CD4+ T cells, i.e., Th1, Th2 and Th17 cells. All these immune parameters can be used to evaluate the efficacy of spa spring water in Korea in terms of immune modulatory effect.
Selective autophagy of Bcl10 modulates T cell receptor activation of NF-κB (121.16)
Authors: Schaefer Brian , Kashyap Anuj , Paul Suman
Published: January 2023

The adaptor protein Bcl10 is a critically important mediator of T cell receptor (TCR)-to-NF-κB signaling. Bcl10 can be degraded by TCR-dependent proteolysis, but the molecular mechanism of degradation is poorly understood. We show that TCR engagement triggers the degradation of Bcl10 in primary effector T cells, but not in naïve T cells. TCR engagement promotes K63-polyubiquitination of Bcl10, causing Bcl10 and its signaling partner Malt1 to associate with the autophagy adaptor protein, p62. Confocal microscopy shows that cytosolic aggregates of Bcl10, Malt1, and p62 also co-localize with the autophagosome membrane protein, LC3. Biochemical analysis shows that LC3 co-precipitates in a TCR-dependent manner with Bcl10 and p62, but not with Malt1. Inhibition of autophagosome formation prevents Bcl10 degradation, leading to long-term association between Bcl10 and Malt1. Thus, the physical separation of Bcl10 and Malt1 is a direct consequence of Bcl10 autophagy, and the autophagy of Bcl10 is a highly selective process. Finally we show that inhibition of Bcl10 autophagy enhances TCR activation of NF-κB. Together, these data demonstrate that selective autophagy of Bcl10 is a homeostatic mechanism that modulates TCR signaling to NF-κB in effector T cells. To our knowledge, our data represent the first example of selective autophagy as an intrinsic homeostatic mechanism modulating a signaling cascade.
STAT5 controls Tfh development (121.19)
Authors: Nurieva Roza , Podd Andrew , Chen Yuhong , Alekseev Andrei , Wang Junmei , Dong Chen , Wang Demin
Published: January 2023

Recent work has identified a new subset of CD4+ T cells named as T follicular helper (Tfh) cells that are localized in germinal centers and critical in germinal-center formation. Tfh cell differentiation is regulated by IL-6 and IL-21, possibly via STAT3 factor, and Bcl6 is specifically expressed in Tfh cells and required for their lineage specification. In current study, we characterized the role of STAT5 in developmental regulation of Tfh cells. We found that a constitutively active form of STAT5 effectively inhibited Tfh differentiation by suppressing the expression of Tfh-associated factors CXCR5, c-Maf, Bcl6, Batf and IL-21, whereas STAT5 deficiency greatly enhanced Tfh differentiation. In addition, deletion of STAT5 in CD4+ T cells in vivo resulted in enhanced development of Tfh cells and germinal center B cells, and led to an impairment of B cell tolerance, suggesting that STAT5 signaling controls the humoral immunity. This knowledge may help us to find ways to treat antibody-mediated autoimmune diseases.
CD1d expression and NKT cells in a haploinsufficiency model of NF1 (121.20)
Authors: Liu Jianyun , Gallo Richard , Khan Masood , Renukaradhya Gourapura , Brutkiewicz Randy
Published: January 2023

Neurofibromin 1 (NF1) is a tumor suppressor gene encoding a Ras GTPase that negatively regulates Ras signaling pathways. Mutations in NF1 are linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia (JML) and Watson syndrome. CD1d-dependent natural killer T (NKT) cells play an important role in anti-tumor immunity. We have shown that CD1d-mediated antigen presentation is regulated by cell signaling pathways. To study whether a haploinsufficiency in NF1 would affect CD1d-mediated activation of NKT cells, we analyzed the NKT cell population as well as CD1d expression levels from NF1+/- mice. Our results suggest that NF1+/- mice have similar levels of NKT cells as WT littermates. However, but interestingly, CD1d expression on BMDCs from NF1+/- mice was found to be lower than those from WT littermates. There was also a slight but statistically significant decrease in the splenic B220+CD1dhi population from NF1+/- mice. On the other hand, NF1+/- mice survived better than WT littermates when challenged with a T-cell lymphoma in vivo. NKT cells from NF1+/- mice were found to be more responsive to CD1d-mediated antigen presentation in vitro, which may partly explain the increased antitumor activity observed in NF1+/- mice. In conclusion, our data suggest that NF1 plays a role in negatively regulating CD1d-mediated activation of NKT cells, and this is translated in enhanced antitumor activity in mice with a haploinsufficiency in NF1.
Choline Acetyltransferase-Expressing T cells are rare in young mice (122.7)
Authors: Olofsson Peder , Rosas-Ballina Mauricio , Ochani Mahendar , Lu Ben , Chavan Sangeeta , Diamond Betty , Tracey Kevin
Published: January 2023

Neural circuits regulate cytokine production to prevent potentially damaging inflammation. A prototypical vagus nerve circuit, the inflammatory reflex, inhibits tumor necrosis factor production in spleen by a mechanism requiring acetylcholine signaling through the α7 nicotinic acetylcholine receptor on macrophages. Nerve fibers in spleen lack the machinery necessary for acetylcholine production. Instead, an acetylcholine-producing, memory phenotype T cell relays the neural signal. These choline acetyltransferase+ (ChAT+) T cells are required for inhibition of cytokine production by vagus nerve stimulation. Thus, action potentials originating in the vagus nerve regulate T cells, which in turn produce the acetylcholine required to control innate immune responses. The understanding of the phenotype of ChAT+ T cells is incomplete. Using flow cytometry, we detected TCRβ+ but no TCRγδ+ ChAT+ T cells. Since acetylcholine-producing T cells are memory cells, we hypothesized that this relay function is acquired, and investigated the splenic ChAT+ T cell population early in life. Indeed, splenic ChAT+ T cells were rare in 2 to 25 days old mice (2%±0.3 of ChAT+ cells) while B cells constituted the majority of the ChAT+ splenocytes (86±5%). In contrast, frequencies of ChAT+ T cells varied considerably in adults and could constitute 1/4 of the ChAT+ population. The infrequency of ChAT+ T cells early in life suggests that the inflammatory reflex may be impaired in young mice.
Th17/Th1 axis polarization is mediated by IL-1 receptor in type 1 diabetes murine model (123.13)
Authors: Carlos Daniela , Bonfá Giuliano , Ramos Simone , Cunha Fernando , Cardoso Cristina , Ryffel Bernhard , Silva João
Published: January 2023

Introduction: Autoimmune diseases including type 1 diabetes (T1D) are thought to have a Th1/Th17 bias, but the underlying mechanisms driving the differentiation and activation of these pathogenic T cells has been investigated. Recently, the IL-1beta has been shown to regulate Th17 cell differentiation through of the expression of the transcription factor RORγt. Objective: For this reason, we addressed the respective IL-1 receptor (IL-1R) role in the TD1. Methods: IL-1R deficient mice and their wild-type (C57BL/6) were inoculated intraperitoneally with 40mg/Kg of streptozotocin for 5 consecutive days. Blood glucose levels and body weight were monitored weekly. The pancreatic lymph nodes (PLN) were removed to assess the Th17 and Th1 frequency by flow cytometry. Results: Diabetic IL-1R deficient mice developed lower hyperglycemia with only 50% of mice becoming diabetic. In agreement, mice lacking IL-1R have reduced inflammatory infiltrate (insulitis) and augmented insulin content into pancreatic islets by second week. In addition, the IL-1R deficiency caused a decrease in IL-17 and IFN-γ-producing CD4+ T cell population in pancreatic lymph nodes (PLN). The IL-17, IL-6, TNF-α, IFN-γ levels were not altered, but IL-10 and IL-4 levels were higher in pancreatic tissue of diabetic IL-1R deficient mice. Conclusion: These results suggest that IL-1receptor promotes the Th17/Th1 skewing in the PLN and possibly contributes to pancreatic islet damage during the T1D onset.
The T cell response to interleukin 10 alters cellular dynamics and paradoxically promotes CNS autoimmunity (123.16)
Authors: Geiger Terrence , Liu Xin , Alli Rajshekhar , Steeves Meredith , Nguyen Phuong , Vogel Peter
Published: January 2023

IL10 is a critical anti-inflammatory cytokine, deficiency of which leads to spontaneous autoimmunity. Yet therapeutically administered or ectopically expressed IL10 may either suppress or promote autoimmunity. Distinct lineage-specific activities may explain these contradictory effects. To dissect the T cell-specific response to IL10 during organ-specific autoimmunity, we generated mice with a specific deletion of IL10Rα in T cells and analyzed its impact in experimental allergic encephalomyelitis (EAE). Surprisingly, the T-cell response to IL10 increased EAE severity. This did not result from altered T cell functional potential; T cell cytokine profile and migratory capacity were preserved. IL10 diminished the proliferation of T cells in situ, both at the site of induction and within the target organ, actions that should restrain disease. However, this decreased proliferation was not associated with diminished cell numbers. IL10 acted cell autonomously to sustain the autoreactive T cells essential for immunopathogenesis, promoting their accumulation and distorting the regulatory and effector T cell balance. Therefore T cell specific actions of IL10 can support autoimmune inflammation, and this appears to result from an overall increase in the long term fitness of pathologic T cells. Lineage-restricted, disease-promoting activities of IL10 should be considered in its therapeutic application.
Absence of CD28 promotes development of severe thyrocyte epithelial cell hyperplasia and proliferation (TEC H/P) in IFN-γ-/- NOD.H-2h4 mice (123.20)
Authors: Kayes Timothy , Mullen Helen
Published: January 2023

60-70 % of IFN-γ-/- NOD.H-2h4 mice given NaI in their drinking water for 6-7 mo develop TEC H/P, a CD8-mediated thyroid specific autoimmune disease characterized by uncontrolled TEC proliferation, lymphocytic infiltrates, autoantibody production, and fibrosis, resulting in destruction of TEC and loss of thyroid function. CD28-/- IFN-γ-/- NOD.H-2h4 mice develop very severe TEC H/P at an accelerated rate with nearly 100 % of CD28-/- IFN-γ-/- NOD.H-2h4 mice developing severe TEC H/P 5-8 weeks after exposure to NaI. Thyroids have extensive fibrosis and severe TEC H/P correlates with low serum T4 levels. CD8+ T cell numbers are increased in thyroid, LN, and spleen, and CD8 cells highly express PD-1 and NKG2D. CD28-/- IFN-γ-/- NOD.H-2h4 mice have extensive lymphocyte infiltrates in salivary glands and pancreatic islets, and a few develop diabetes at 4-6 mo of age. CD28-/- IFN-γ-/- NOD.H-2h4 mice with TEC H/P have reduced numbers of peripheral FoxP3+ T reg compared to IFN-γ-/- NOD.H-2h4 mice with TEC H/P. In mice with severe TEC H/P, thyroids from CD28-/- IFN-γ-/- NOD.H-2h4 mice have reduced numbers of FoxP3+ T reg compared to IFN-γ-/- NOD.H-2h4 mice. The accelerated development of TEC H/P in CD28-/- IFN-γ-/- mice may be a result of reduced T reg number or function as preliminary data indicates that development of TEC H/P in CD28-/- IFN-γ-/- NOD.H-2h4 mice is inhibited by transfer of FoxP3+ T reg from CD28 sufficient IFN-γ-/- NOD.H-2h4 mice.
Overexpression of β-catenin in dendritic cells from type 1 diabetes patients and NOD mice may drive induction of T cells involved in β-cells destruction (123.34)
Authors: Alard Pascale , Zirnheld Arin , Parnell Sarah , Kosiewicz Michele
Published: January 2023

Type 1 diabetes (T1D) is characterized by the destruction of insulin producing β-cells in the pancreas mediated by IFNγ+ T cells. Non-obese diabetic (NOD) mice and T1D patients share similar characteristics of disease progression and the immune defects associated with disease, e.g., their dendritic cells (DC) produce elevated levels of pro-inflammatory cytokines such as IL-12, a cytokine that drives differentiation of IFNγ+ T cells. We have identified a new defect in DC in NOD mice, i.e., an abnormal accumulation of the transcription factor, β-catenin, which appears to be associated with the production of pro-inflammatory cytokines. Moreover, we found that DC isolated from T1D patients also expressed elevated levels of β-catenin compared to healthy individuals. To examine the function of β-catenin in DC, we either transfected human or murine DC with β-catenin siRNA or cultured them with an β-catenin inhibitor, and found that the production of pro-inflammatory cytokines (IL-12, IL-6, TNF) and subsequent production of IFNγ by T cells added to the DC culture was abrogated. Finally, treatment of NOD mice with an β-catenin inhibitor delayed onset and decreased incidence of disease. These data suggest that elevated expression of β-catenin in T1D may regulate the pro-inflammatory cytokine production in DC that subsequently drives induction of β-cell destroying T cells. Manipulation of β-catenin may, therefore, be a novel strategy for preventing and/or treating T1D.
Conditional deletion of tumor necrosis factor receptor-associated factor 6 (TRAF6) in medullary thymic epithelial cells leads to the development of classical autoimmune hepatitis in mice (123.40)
Authors: Bonito Anthony , Aloman Costica , Fiel Maria , Danzl Nichole , Alexandropoulos Konstantina
Published: January 2023

Autoimmune hepatitis (AIH) is an enigmatic disease in which loss of tolerance towards self-antigens is suspected among the causes; animal models are limited, however. TRAF6 is an important signaling adaptor protein that plays a critical role in T cell tolerance by regulating medullary thymic epithelial cell (mTEC) development. mTECs in turn regulate central tolerance by ectopically expressing self-antigens and eliminating autoreactive T cells during development. In this report, conditional knockout mice lacking TRAF6 expression in thymic epithelial cells (TRAF6ΔTEC) were generated using FoxN1-cre transgenic mice (FoxN1 expression is confined to mTECs). TRAF6 deletion inhibited mTEC development and the production of mature mTECs in the thymus. This correlated with spontaneous liver-specific autoimmunity and pathognomonic features of chronic autoimmune hepatitis including interphase and lobular hepatitis, T cell and plasma cell infiltration, elevated serum transaminases and IgG, and production of anti-nuclear autoantibodies against the livers of TRAF6ΔTEC animals. A significant increase in fibrosis was also detected in TRAF6ΔTEC mice compared to littermate controls. Our data indicate that mTECs play an intrinsic role in central tolerance, and their depletion leads to AIH. This model of spontaneous AIH represents an exciting new tool to investigate autoantigen-specific T and B cell responses and regulatory mechanisms underlying the disease.