bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a high throughput cell migration assay using the SRU BIND reader (102.13)
Authors: McAllister Kathryn , Abodeely Marla , Shamah Steven , Zhang Qin
Published: January 2023

The chemotactic ability of non-adherent cells can be assessed by a variety of techniques, but is typically performed using transwell plates in which a chemoattractant induces the migration of cells through a porous membrane. Throughput and robustness are limitations to this assay format. The aim of this study was to develop a novel, higher throughput, robust, cell migration assay using the human monocytic derived U937 cell line and the SRU BIND® label free detection platform. C5a is a powerful chemoattractant which is generated by all 3 pathways of the complement system. C5a binds to the C5a receptor which is expressed on many different inflammatory cell types, including neutrophils, monocytes, and macrophages, and it mediates migration of these cells under pro-inflammatory conditions. Differentiated U937 cells express the C5a receptor and migration of these cells can be induced by C5a. These cells were seeded on a 384-well BIND® biosensor and following an attachment period, collagen was added and allowed to form a solidified layer. Following the addition of C5a the cells migrated up off the biosensor toward the chemoattractant stimulus producing a bell shaped concentration response curve, as typically observed for C5a in classical chemotaxis assays. The response was inhibited by reference C5a receptor antagonists. This assay format could also be developed to evaluate the migratory ability of primary cells such as monocytes or neutrophils to inflammatory chemoattractants.
ECM1 controls Th2 cell egress from lymph nodes through re-expression of S1P1 (102.3)
Authors: Sun Bing , Li Zhenghu , Zhang Yuan
Published: January 2023

The T helper 2 (Th2) subset of T cell is critically involved in allergy and asthma. In this study, we demonstrate that the Extra-Cellular Matrix protein 1 (ECM1), a secreted protein, is highly and selectively expressed in Th2 cells. ECM1 deficiency caused impaired Th2 responses and reduced allergic airway inflammation in vivo. Functional analysis demonstrated that although Th2 polarization was unimpaired, ECM1 deficient Th2 (but not Th1) cells exhibited a defect in migration and were retained in peripheral lymphoid organs. This was associated with reduced expression of the transcription factor klf2 and its downstream target, the chemokine receptor S1P1 and could be corrected by retroviral transduction of individual members of the pathway. We also found that ECM1 can directly bind to IL-2 receptor to inhibit IL-2 signaling and activate S1P1 expression. Our data identify a novel function of ECM1 in regulating Th2 cell migration through controlling of klf2 and S1P1 expression.
TNF-α produced by inflammatory DCs as a molecular switch in Th17/Th2 and neutrophilic/eosinophilic responses during persistent fungal infection (103.2)
Authors: Fei Mingjian , Bhatia Shikha , Oriss Timothy , Yarlagadda Manohar , Khare Anupriya , Akira Shizuo , Iwakura Yoichiro , Ray Prabir , Kolls Jay , Ray Anuradha
Published: January 2023

Aspergillus fumigatus is associated with allergic bronchopulmonary aspergillosis, a pulmonary allergic disorder, in which neutrophilia predicts a poor outcome. In our efforts to recapitulate fungus-induced airway neutrophilia in the allergic setting, we found that similar exposure to the fungus resulted in a neutrophil-dominated response in BALB/c mice but an eosinophil-biased response in C57BL/6 mice. By performing a comparative study, we identified TNF-α as the key mediator that skewed the response towards neutrophilia but away from eosinophilia. Between the two strains, TNF-αhigh CD11b+ Ly6C+ DCs were more abundant in the lungs of WT BALB/c mice which was compromised in MyD88-/-, dectin-1-/- and TNF-α-/- mice. As compared to TNF-αhigh Balb/c-DCs, TNF-αlow C57BL/6-DCs contained more NF-B p50 homodimers as strong repressors of TNF-α transcription. Lower TNF-α accounted for lower TLR2 levels on C57BL/6 DCs impairing collaboration between TLR2 and Dectin-1 in augmenting TNF-α via a positive feedback loop. Functionally, in Balb/c mice, the high level of TNF-α enhanced Th17 responses and in collaboration with IL-17A upregulated the neutrophil chemoattractants KC and MIP-2. Conversely, the lungs of C57BL/6 or TNF-α-/- mice showed higher IL-5 levels and therefore an eosinophil-rich response. Our study for the first time highlights a central role of TNF-α as a molecular switch that regulates the neutrophil/eosinophil balance in airway inflammation.
Role of specific dendritic cells in inducing Tregs in response to inhaled allergen. (103.5)
Authors: Khare Anupriya , Qi Zengbiao , Fei Mingjian , Ray Prabir , Ray Anuradha
Published: January 2023

Foxp3+ Regulatory T cells (Treg), both naturally occurring thymus-derived nTregs and peripherally induced Foxp3+ induced Tregs (iTreg), play a critical role in suppressing immune responses to self as well as external antigens, ensuring protection from autoimmune and allergic diseases such as asthma. Recent investigations have established a role for iTregs in the regulation of chronic allergic lung inflammation. However, the cellular and molecular mechanisms mediating induction of Tregs in the lungs are still unclear. In this study, we explored the role of dendritic cells (DCs) in Treg induction in response to inhaled allergen. Using an adoptive transfer model we demonstrate that exposure to low doses of allergen results in the induction of Foxp3+ Tregs (tolerance) in the lungs which requires the presence of CD11c+ DCs. Amongst the various resident DC subsets in the lungs, CD103+ lung DCs were the most potent inducers of iTregs. The tolerogenic property of the CD103+ DCs was compromised under inflammatory conditions and the DCs did not induce Tregs as efficiently. Finally, the induction of Tregs by CD103+ DCs was dependent on retinoic acid and TGF-β and was promoted in the absence of IL-4 signaling. Taken together, we show that despite the presence of multiple DC subsets in the lung, CD103+ DCs selectively mediate airway tolerance to inhaled allergens. Thus, strategies to improve the function of CD103+ DCs in the lung would help ameliorate allergic airways disease.
The protective role of TLR6 during experimental fungal asthma is mediated by IL-23 and IL-17A. (103.6)
Authors: Moreira Ana , Cavassani Karen , Hullinger Rikki , Knight Darryl , Kunkel Steven , Uematsu Satoshi , Akira Shizuo , Hogaboam Cory
Published: January 2023

Toll-like receptors (TLRs) mediate pathogen recognition in the respiratory system, but TLR activation has both beneficial and deleterious effects during asthma. Clinical data demonstrates that TLR6 is an example of a pattern recognition receptor that exerts protective effects in clinical asthma. To explore the mechanism through which TLR6 expression by immune and inflammatory cells mediates this effect, we investigated its role in a murine model of Aspergillus fumigatus-induced chronic asthma. TLR6-/- mice with fungal asthma exhibited significantly increased airway hyperresponsiveness, inflammation, and remodeling compared with WT asthmatic groups. Surprisingly, whole lung levels of IL-23 and IL-17 were significantly lower in TLR6-/- versus WT asthmatic mice. TLR6-/- DCs generated significantly less IL-23 upon activation with either lipopolysaccharide or zymosan. Impaired IL-23 generation in TLR6-/- mice also corresponded with significantly lower dectin-1 expression and Th17 expansion both in vivo and in vitro. Exogenous IL-23 treatment of asthmatic TLR6-/- mice in vivo restored IL-17A production and significantly reduced airway hyperresponsiveness, inflammation, and lung fungal burden compared with untreated asthmatic TLR6-/- mice. Together, we demonstrate that TLR6 activation is critical for IL-23 and Th17, and both regulate the allergic inflammatory response during fungal asthma.
Co-expression of CD8α and CD8β defines a subset of tolerogenic plasmacytoid dendritic cells able to induce the differentiation of Foxp3+ regulatory T cells. (103.9)
Authors: Lombardi Vincent , Szely Natacha , Akbari Omid
Published: January 2023

Recent findings suggest that different subsets of dendritic cells (DCs) control the balance between protection against pathogens and tolerance to innocuous or self antigens. Latest data propose that plasmacytoid DCs (pDCs) act as antigen-presenting cells during adaptive immune responses and promote peripheral tolerance. In this study, we demonstrate for the first time that mouse pDCs can be segregated into three distinct populations according to their surface expression of CD8α alone or combined with CD8β. These subsets not only differ in phenotype but are also functionally distinct since CD8α+ CD8β− and CD8α+ CD8β+ pDCs are more potent inducers of CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) compared to CD8α− CD8β− pDCs. Our results indicate that, in a mouse model of allergic asthma, adoptive transfer of CD8α+ CD8β− or CD8α+ CD8β+ prevents the development of airway hyperreactivity. By comparing the gene expression profile of the three subsets of pDCs described in this study, we established that the expression of CD98hc, a receptor for the Galectin-3, was significantly up-regulated in both CD8α+ CD8β− and CD8α+ CD8β+ subsets. Adding Galectin-3 to sorted CD8α+ CD8β− or CD8α+ CD8β+ pDCs in vitro, enhances the conversion of naive CD4+ T cells into Tregs. Thus, our data unveil for the first time, subsets of pDCs with the capacity to induce regulatory functions that may contribute to the establishment of immunological tolerance.
Determining the role of basal NF-κB in the survival of quiescent naïve T cells (104.14)
Authors: Miller Michelle , Mashayekhi Mona , Chen Luqiu , Liu Xindong , Michelotti Monica , Tramontini Nicole , Powers Sarah , Zhu Xiaoping , Alegre Maria-Luisa , Molinero Luciana
Published: January 2023

NF-κB is an essential transcription factor for the survival of T cells following antigen stimulation, but whether it plays a role in maintaining the survival of unstimulated naïve T cells is not known. We have detected basal NF-κB activity in naïve quiescent T cells and investigated whether it is important in cell survival in the absence of T cell activation. NF-κB-impaired naïve CD4+ T cells showed decreased lifespan in vivo. IL-7 has been shown to be important for naïve T cell survival prompting investigation into whether basal NF-κB activity could regulate IL-7/IL-7R signaling. Our results indicate that NF-κB-impaired naïve T cells are resistant to the pro-survival effects of IL-7 in vitro, and that these T cells express less surface IL-7Rα than wild type T cells. To determine whether NF-κB could regulate IL-7Rα expression by transcription, a putative NF-κB-binding site 3.5 kB upstream of the IL7r translational start site was tested. Nuclear NF-κB from naïve T cells was capable of binding an oligonucleotide probe containing this sequence, as determined by EMSA. A ChIP assay confirmed that the p50 and p65 subunits of NF-κB bound this region in live wild type but not NF-κB-impaired naïve T cells. Finally, re-expression of IL-7Rα after IL-7-mediated degradation was RNA polymerase- and NF-κB-dependent. Together, these results support the idea that basal NF-κB activity plays a role in the survival of quiescent naïve T cells by controlling transcription of the IL7r gene.
Virus-specific T cell clonal expansions can limit recall responses to secondary respiratory virus infections (104.4)
Authors: Connor Lisa , Ryan Lynn , Kohlmeier Jacob , Cookenham Tres , Roberts Alan , Quinn Adam , Woodland David
Published: January 2023

Respiratory virus infections are a major health problem in the elderly. This is due to declining immune responsiveness in the elderly and the development of CD8+ T cell clonal expansions (TCE) that perturb the memory T cell pool. We recently showed that TCE can arise over time from memory CD8+ T cells established by an acute respiratory viral infection. Virus-specific TCE are phenotypically identical to conventional memory cells and do not express the characteristics of exhausted cells. Since TCE can dominate the memory T cell pool, it is essential to determine the rate at which they develop, and their ability to mount a recall response to secondary pathogen challenge in vivo. In the current study, we developed an assay to detect developing TCE in a cohort of mice following Sendai virus infection and found a progressive increase in the appearance of TCE within the first year following infection. Using a dual adoptive transfer approach to address the recall potential of virus-specific TCE, we demonstrate that the majority of TCE examined are poorly responsive to a secondary infection. Taken together, the data suggest that the development of TCE is a common occurrence due to the progressive dysregulation of the virus-specific memory T cell pool with age, and many TCE are profoundly defective in their ability to mediate recall responses.
An influence of galectin-9/Tim-3 interaction on Herpes simplex virus-1 latency (105.14)
Authors: Jagadeesh Reddy Pradeep Babu , Sehrawat Sharvan , Suryawanshi Amol , Rajasagi Naveen , Rouse Barry
Published: January 2023

In this report, we show that the majority of CD8+T cells in the trigeminal ganglion (TG) of mice ocularly infected with HSV-1 express Tim-3, a molecule that delivers negative signals to CD8+T cells when it engages its ligand, Galectin 9 (Gal-9). Curiously Gal-9 glycoprotein is also up regulated in the TG both during the time when replicating virus is present as well during early viral latency. Accordingly it is conceivable that the efficiency of ganglionic CD8+T cell function could be influenced by Gal-9/Tim-3 interaction. In support of this we show that the activity of peptide stimulated CD8+T cells recovered from the TG of WT mice produced less IFN-γ, TNF-α and Granzyme B molecules in comparison to TG CD8+T cells of Gal-9KO mice. Differences in the number of productively and latently infected neuronal cells were also evident between WT and Gal-9KO mice. In vitro studies demonstrated that TG cell cultures of WT HSV-1 infected mice exposed to recombinant Gal-9 in the latent phase caused apoptosis of some CD8+T cells and induce viral reactivation. Our results demonstrate that the host homeostatic mechanism mediated by Gal-9/Tim-3 interaction on CD8+T cells can influence the outcome of latent HSV-1 infection. Neuronal cell function is likely to be preserved when host Gal-9 levels are low, since inhibitory effects on latency controlling CD8+T cells will be minimal.
The role of GPI-anchored proteins in the preferential binding and infection of poxviruses with primary human monocytes (105.19)
Authors: Byrd Daniel , Hu Ningjie , Amet Tohti , Hu Sishun , Grantham Ayslinn , Yu Qigui
Published: January 2023

The poxviruses including vaccinia virus (VV) and canarypox virus (ALVAC) have shown a strong preference for binding and infection of human cells from the monocytic lineage, particularly primary human monocytes (PHMs). In this study, we investigated whether specific molecules on the surface of PHMs regulate the preferential binding and infection of VV and ALVAC to this cell population. We found that the cleavage of glycosylphosphatidylinositol-anchored proteins (GPI-APs) on primary human monocytes by phosphatidylinositol-specific phospholipase C (PI-PLC) and the blockage of GPI-APs with mouse sera raised against GPI-APs significantly reduced the binding of VV and ALVAC to the cell surface, and subsequently inhibited viral infection. This reduction is not related to abatement of phagocytosis as internalization of latex beads by PHMs was not affected by PI-PLC digestion. As well, the human monocytic cell lines THP-1, U937 and U1 express high, moderate and low levels of surface GPI-APs, respectively. The susceptibility of these cells to VV and ALVAC infection correlated to levels of surface expression of GPI-APs. In addition, THP-1 cells showed reduced susceptibility to VV and ALVAC infection when they were mutated by ethyl methanesulfonate (EMS) or aerolysin (AE) to express low or undetectable surface GPI-APs. Thus, specific GPI-APs on the surface of monocytes are involved in poxvirus binding and infection, and may act as monocyte-specific surface receptors for poxviruses.
A mouse model of T cell dysfunction in chronic Hepatitis B (105.24)
Authors: Tao Mi-Hua , Huang Ya-Hui , Chou Ho-Yuan , Fang Cheng-Chieh
Published: January 2023

The virus-host interactions responsible for dysfunctions of hepatitis B virus (HBV)-specific T cells and the subsequent development of severe liver diseases are poorly understood. Mice are not naturally infected by HBV, presumably due to a lack of HBV receptors on mouse hepatocytes. To bypass this entry step of HBV infection, we used adeno-associated viral vectors to introduce the HBV genome (AAV/HBV) into the liver of immunocompetent mice. We confirmed the presence of HBV virions and proteins in the serum and liver of all AAV/HBV-transduced mice. These mice were negative for HBV surface (HBs) and e protein (HBe)-specific Abs, but positive for HBV core (HBc)-specific Ab, a profile similar to that observed in chronic HBV patients. AAV/HBV transduction induced HBs- but not HBc-specific IFN-γ-producing CD8+ T cells, preferentially enriched in the liver. The number of HBs-reacting CD8+ T cells decreased with time and their effector functions were severely impaired as suggested by their failure to respond to HBs immunization. Interestingly, PD1 expression on hepatic CD8+ T cells increased gradually after AAV/HBV transduction, suggesting an important role of PD-1 in constraining T cell activity and maintaining HBV chronicity. Nevertheless, six months after transduction these AAV/HBV-transduced mice manifested evidence of hepatic inflammation and regeneration. Our AAV/HBV-transduced murine model is a unique instrument for studying the immunological events in human chronic HBV.
Genetic similarities and differences in the anti-HIV-1 Env antibody responses between humans and rhesus macaques (105.40)
Authors: Guan Yongjun , Sajadi Mohammad , Robinson James , Reid William , Lewis Roberta , Gorny Miroslaw , DeVico Anthony , Lewis George
Published: January 2023

We previously described a method for rapid cloning human monoclonal antibody (mAb) from memory B cell (Bmem). Here, we have put this method in practice for isolating a large panel of mAbs against HIV-1 envelope (Env) protein and have extended it as a general strategy to isolate monkey mAbs against HIV-1 Env from Bmem of a SHIV infected rhesus macaque. Ninety-six new mAbs were cloned from 5 HIV-1 infected humans, of which 64 are clonally unique Env mAbs. Thirty-five mAbs against HIV-1 Env were cloned directly from Bmem or from immortalized Env B cell lines both derived from SHIV infected rhesus macaques. Genetic analyses showed that humans and rhesus macaques can use similar VH genes for generating Abs against similar Env epitopes. For example, they both can use VH1-69, VH1-24 and VH2-70 genes for mAbs against CD4 induced (CD4i) Env epitopes, and use VH5-51 gene for V3 mAbs. However, the strong bias usage of VH1-69 gene for human CD4i Abs was not seen in rhesus macaques. In addition, the biased usage of kappa gene for anti-Env Abs in HIV-1 infected people was not seen in rhesus macaques. Instead, SHIV infected rhesus macaques tends to use lamda light chain gene to encode mAbs against the HIV-1 Env protein. This information on the similarities and differences of anti-Env Ab responses between human and rhesus macaques should help to more judiciously employ the non-human primates model to advance HIV vaccine candidates into clinical trials.
T cell epitopes were identified in HTNV structural protein (105.9)
Authors: Jin Boquan , Ma Ying , Wang Jiuping , Zhuang Ran , Wang Meiliang , Xu Zhuwei , Yi Jing , Yan Guolin , Zhang Yusi
Published: January 2023

Objectives: To investigate the cellular immune response to Hantaan virus (HTNV) infection by identifying T cell epitopes of HTNV structural protein (nucleoprotein, NP and glycoprotein, Gn/Gc) and their HLA-restriction. Methods: The HLA-restriction of HTNV-NP T cell epitopes were identified by determining T cell response to peptide-loaded B lymphoblastoid cell lines which were HLA partially matched. T cell responses to an array of overlapping peptides derived from HTNV-Gn/Gc was quantitated by ex vivo IFN-γ ELISPOT analysis. Results: In the HTNV-NP, one HLA-A*02-, one HLA-B*07-, one HLA-A*11-, two HLA-A*33-, and three HLA-B*35-restricted T cell epitopes were identified. 86.6% hemorrhagic fever with renal syndrome (HFRS) patients (71/82) showed T cell response to 61.9% HTNV-Gn/Gc 15-mer peptides (174/281), in which, 11 potential dominant epitopes were screened out. Moreover, the response frequencies of HTNV-Gn/Gc-specific T cells at the earliest available time point were significantly higher in patients who had mild or moderate HFRS than in those who had severe or critical HFRS (P=0.007), suggesting that T cell response to these epitopes may affect HFRS disease progression in patients. Conclusion: T cell immunty plays a role in immunoprotection upon HTNV infection. Identification of T cell epitopes of HTNV structural protein and their HLA restriction may advance our understanding of immunoprotection and/or pathogenesis of HFRS, thereby improve the design of novel HTNV vaccine.
Enhanced in vivo immunity and protection provided by cationic lipid-DNA complex adjuvanted influenza vaccination is independent of Toll-like receptor 9 (106.16)
Authors: Botham Crystal , Hong David , Giffon Thierry , Draghi Monia , Fairman Jeff , Lewis David
Published: January 2023

As current licensed influenza vaccines provide little or no cross-protective immunity, we have pursued using cationic lipid-DNA complex (CLDC) adjuvant to boost adaptive immunity to inactivated influenza vaccines. CLDC consists of a non-coding plasmid DNA, rich in immunostimulatory CpG motifs, which is encapsulated in cationic lipids. Previous murine and non-human primate studies showed that CLDC adjuvanted influenza vaccination induced more robust immunity and cross-protection. We hypothesized that the CLDC plasmid mediates these effects, at least in part, in a TLR9-dependent manner. Bone-marrow derived dendritic cells (BMDCs) incubated in vitro with CLDC secreted high levels of interferon-alpha (IFN-α), and this induction required CLDC’s DNA component. CLDC-induced IFN-α expression was significantly reduced using TLR9-/- BMDCs. We next vaccinated wildtype and TLR9-/- mice with CLDC-adjuvanted inactivated influenza vaccines. Surprisingly, CLDC’s immunostimulatory responses in vivo were TLR9-independent in that there was no significant difference between wildtype and knockouts for either vaccine immunogenicity (influenza-specific antibody and T-cell responses) or for cross-protection after viral challenge. These studies suggest that the DNA moiety of CLDC enhances immune responses in vivo by a novel, TLR9-independent mechanism. Determining this mechanism will likely be important in the optimal application of CLDC as an adjuvant to human vaccination.
Colonization factor antigen I expression by Salmonella induces CREB-dependent stimulation of CD39+CD4+ T cells responsible for protection against collagen-induced arthritis (107.5)
Authors: Kochetkova Irina , Thornburg Theresa , Callis Gayle , Pascual David
Published: January 2023

Salmonella vaccine vector expressing enterotoxigenic E. coli CFA/I fimbriae has been shown to protect against EAE and CIA. Vaccine-induced CD25+CD4+ and CD25-CD4+ T cells were potent against CIA, and such protection was reversed by anti-TGF-β treatment. We demonstrate, Salmonella-CFA/I-stimulated CD39+CD4+ T cells correlate with accelerated hydrolysis of extracellular ATP, a mechanism of immune suppression. Since vaccine-induced FoxP3-CD39+CD25-CD4+ T cells are shown to be the source of TGF-β, we hypothesized TGF-β could be important in regulating CD39 expression and consequential suppression of CIA. Salmonella-CFA/I-induced CD4+ T cells featured enhanced activation of cAMP-response element-binding protein (CREB), essential for transcriptional regulation of CD39. CREB phosphorylation was suppressed by in vivo treatment of Salmonella-CFA/I-immunized mice with anti-TGF-β mAb. Decrease in phosphorylated CREB correlated with 2-fold less CD39+CD4+ T cells. Although Salmonella-CFA/I-induced FoxP3-CD4+ T cells protected against CIA, as well as CD39+CD4+ T cells upon adoptive transfer, subsequent FACS analysis of recipients’ CD4+ T cells revealed conversion of FoxP3-(GFP-) cells to FoxP3+. Thus, Salmonella-CFA/I stimulates TGF-β production by FoxP3-CD39+CD4+ T cells, promoting the CREB-dependent expansion of CD39+CD4+ T cells becoming regulatory FoxP3+CD4+ T cells. Supported by AT004312.
The characterization of thymus-derived natural Th17 cells and their response to inflammation. (108.9)
Authors: Nowyhed Heba , Bertino Sarah , Tao Jian , Marks Benjamin , Choi Jin-young , Craft Joseph
Published: January 2023

CD4 T helper (Th) cells that secrete interleukin (IL)-17 (Th17) complement the function of Th1 and Th2 cells as a third effector lineage, with a distinct role in neutrophil recruitment necessary for extracellular pathogen clearance and the promotion of certain autoimmune conditions. We have previously demonstrated that natural Th17 (nTh17) cells develop within the thymus in a manner similar to that of natural T regulatory (nTreg) cells (Marks et al., Nat. Immunol. 2009), with activation in response to an increase of self-antigen due to tissue destruction and subsequent host protection via IL-22 secretion. In this study, we report microarray analysis of lymph node nTh17 cells, revealing specific patterns of gene expression compared to naïve, Th1, and nTreg cells. We identified 317 genes uniquely over- or under-expressed in nTh17 cells compared to controls. These results give new insights into the differences among naïve, nTreg, nTh17, and Th1 cells. We further identified two receptors -- IL-1R1 and IL-17RC -- that are expressed on nTh17 cells and play a role in their development and function. Development of nTh17 cells is hindered in the absence of IL1R1, and augmented in response to an increase of IL-1β. IL17-RC appears to be functional on nTh17 cells, in that it offers a way for the cells to regulate the levels of secreted IL-17A and IL-22.
The effect of B cell receptor affinity on lipid mediated B cell receptor antigen microclustering (109.1)
Authors: Yu-Yang Paul , Reddy A. , Raychaudhuri Subhadip
Published: January 2023

After encountering antigens (Ags), B-cell receptors(BCRs) with bound Ags assemble into microclusters then into a macrocluster. BCR recognition of antigen results in a monotonically increasing graded signaling response with increasing affinity for Ag, a robust feature that seems to be characterizing BCR-Ag affinity discrimination. Recent experiments indicate an important role of very early (within seconds) signaling events, such as formation of BCR-Ag microclusters, in this affinity discrimination process. Our current studies improve upon our most recent biophysical models for BCR-Ag microcluster and macrocluster formation by incorporating lipid-mediated BCR attraction factors. We examine the effect of (a) lipid and BCR concentration, and (b) varying attractive forces between different lipid and BCR pairings. Our studies indicate that a weak attraction between pairs of BCR molecules does not lead to microclusters. However, strong coupling between BCR and lipid (sphingolipids) molecules, that possibly arises upon antigen binding, generates microclusters of size similar to what has been observed in experiments. Our results indicate that microcluster size and number increase with increasing affinity while the formation times of such early BCR-Ag clusters decrease. These features can have important implications for the B cell affinity discrimination problem.
Dependence of TCR triggering on ligand three-dimensional freedom (109.19)
Authors: Ma Zhengyu , Janmey Paul , Finkel Terri
Published: January 2023

T lymphocytes (T cells) recognize antigens through the binding of T cell receptors (TCRs) to peptide ligands presented by MHC molecules (pMHCs) on antigen presenting cells (APCs). The mechanism of how pMHC-TCR binding triggers a signal from TCR is still unclear. One unique aspect of TCR triggering is that surface-anchored pMHC triggers TCR highly efficiently in a two dimensional setting, while soluble monomeric pMHC ligands with full three dimensional freedom have no activity. Here, we investigate the dependence of TCR triggering on the 3D freedom of pMHC ligands using highly flexible hetero-bifunctional poly(ethylene glycol) (PEG) polymer linkers. Fluorescence resonance energy transfer assay showed increased 3D freedom of ligands with increased linker length. As measured by IL2 production, T cells responded equally well to pMHCs anchored on surfaces with 0.56 nm, 3.1 nm and 12.7 nm linkers. T cell response started to decrease, however, when the linker was 22 nm long, and decreased further for the 32 nm linker. pMHCs anchored with the 48 nm linker still stimulated T cells IL2 production, although with only about one tenth the potency. Therefore, pMHC ligands with 3D freedom of up to 22 nm are well tolerated by TCR, but larger 3D freedom gradually reduces potency. Our findings argue against the kinetic segregation of TCR triggering but are in support of receptor deformation, a model of TCR triggering that tolerates increased ligand 3D freedom.
β-arrestin-1 negatively regulates inflammatory response to polymicrobial sepsis in mice (110.11)
Authors: Parameswaran Narayanan , Gonipeta Babu , Parvataneni Sitaram , Packiriswamy Nandakumar , Sharma Deepika
Published: January 2023

β-arrestins are scaffolding proteins that regulate a number of receptor signaling pathways including Toll-like receptors. We recently demonstrated that mice lacking either β-arrestin-1 or β-arrestin-2 are protected from lipopolysaccharide-induced lethality and have a markedly reduced inflammatory response. To assess the role of β-arrestin-1 in a clinically relevant model of sepsis, we subjected wild type and β-arrestin-1 knockout mice to cecal-ligation and puncture (CLP) to mimick septic peritonitis and polymicrobial sepsis. Surprisingly, we found that, mortality of β-arrestin-1 knockout mice was significantly enhanced compared to the wild type mice after CLP. Consistent with lethality, β-arrestin-1 knockout mice had markedly elevated inflammatory cytokine levels in the plasma, peritoneal cavity, and bronchoalveolar fluid. Enhanced systemic inflammatory response of β-arrestin-1 knockout mice was associated with significantly enhanced infiltration of immune cells into the peritoneal cavity after induction of septic peritonitis. Together, these results demonstrate that, contrary to its role in lipolysaccharide-TLR4 signaling in vivo, β-arrestin-1 is a negative regulator of inflammation induced by polymicrobial sepsis and that the phenotype of the mice may be related to a potentially aberrant immune response from excess infiltration of immune cells. These results also suggest that the role of β-arrestin-1 in this model is likely independent of its role in TLR4 signaling in vivo.
SerpinB1 inhibition of cathepsin G is critical for neutrophil survival and the maintenance of the bone marrow reserve of mature neutrophils in vivo (110.14)
Authors: Benarafa Charaf , Baumann Mathias
Published: January 2023

The bone marrow reserve of neutrophils is required for a rapid and effective mobilization to infected tissues. We recently reported that this reserve is considerably reduced in serpinB1-/- mice due to a survival defect of mature neutrophils. SerpinB1 inhibits the three neutrophil serine proteases (NSPs): elastase (NE), cathepsin G (CG) and proteinase-3 and is expressed at high levels in neutrophils but also in non-hematopoietic cells. Here, we found that, in bone marrow chimera, serpinB1 deficiency in hematopoietic cells was necessary and sufficient to reproduce the bone marrow neutropenia observed in serpinB1-/- mice. In addition, when irradiated mice were reconstituted with a 1:1 mixture of serpinB1-/- and wild type bone marrow cells, only a small percentage of the mature neutrophils were from the serpinB1-/- donor, whereas B cells of the two genotypes were found in equal proportions. Mice lacking serpinB1 as well as active NSPs due to deficiency in dipeptidyl peptidase I (serpinB1-/-.dppi-/-) had a normal neutrophil reserve indicating that one or more NSPs was involved in the bone marrow defect. Deficiency in CG alone, but not NE, was sufficient to fully restore a normal number of bone marrow neutrophils in serpinB1-/- mice. Collectively, these findings demonstrate that serpinB1 protects the neutrophil reserve in a cell autonomous manner and indicate a novel cell death pathway in neutrophils mediated by serine protease cathepsin G and regulated by serpinB1.
The TRPM2 ion channel is required for early innate immunity against Listeria infection (110.20)
Authors: Li Yuan , Knowth Heather , Heizer Justin , Mogan Jenny , Perraud Anne-Laure
Published: January 2023

TRPM2 is a non-selective cationic channel, which can mediate Ca2+ fluxes in response to H2O2, indicating a role in signaling as a sensor of oxidative stress. TRPM2 is functionally expressed in many immune cell types, and has been reported to be required for neutrophil chemotaxis and production of the chemokine CXCL2. In order to investigate the function of TRPM2 in response to a pathogen, we infected TRPM2-/- mice with Listeria monocytogenes (Lm). We found that TRPM2-/- mice show higher mortality and strongly elevated bacterial burdens already at an early stage of Lm infection. This susceptibility to Lm infection is not caused by defects in the recruitment of neutrophils, as neutrophil migration remains intact in Lm-infected TRPM2-/- mice. Because the production of IFNγ and IL-12 is reduced in TRPM2-/- mice, we conclude that TRPM2 can enhance the production of some cytokines. Bone marrow transfer experiments suggest that the susceptibility of TRPM2-/- mice to Lm infection might originate from both immune cells and nonhematopoietic cells. In sum, we have demonstrated the importance of TRPM2 in enhancing the crucial production of cytokines during early immunity against Lm.
Strong antibody responses to influenza vaccination are associated with cell survival/proliferation and pro-apoptotic immune traits (110.27)
Authors: Furman David , Kidd Brian , Shen-Orr Shai , Maecker Holden , Dekker Cornelia , Davis Mark
Published: January 2023

Vaccination is the most effective strategy to prevent and control infectious diseases through induction of antibodies that neutralize the pathogen. However, little is known on what immune traits underlie an effective antibody response. With the aim of finding correlates of vaccine response that predict the antibody titers against an influenza vaccine, we studied the antibody responses to the seasonal trivalent influenza vaccine in a young and older individuals, and assayed a comprehensive set of immune parameters that included whole blood gene expression, determination of serum cytokine levels, leukocyte subset frequencies and phosphorylation of signaling proteins upon stimulation of various peripheral blood cells. We have found that rise in antibody titers to influenza strains after vaccination is strongly associated with high pro-apoptotic and low cell survival/proliferation levels of diverse immune traits and genes that could be exploited to improve vaccine responses in those less protected.
Histone deacetylase inhibition facilitates GM-CSF-mediated expansion of myeloid-derived suppressor cells in vitro and in vivo (111.10)
Authors: Rosborough Brian , Castellaneta Antonino , Natarajan Sudha , Thomson Angus , Turnquist Heth
Published: January 2023

Recent studies have demonstrated anti-inflammatory and tolerance-promoting properties of chromatin-modifying histone deacetylase inhibitors (HDACi) through their ability to modulate dendritic cells and regulatory T cells. However, the influence of these agents on rare, immunoregulatory myeloid-derived suppressor cells (MDSC) has not been examined. C57BL/6 (B6) bone marrow (BM) cells were cultured with GM-CSF and the HDACi trichostatin A (TSA) for 5d. Cell lineage was identified by flow cytometry and putative MDSC purified by anti-Gr1 immunomagnetic bead selection. For in vivo experiments, mice received hydrodynamic infusion of GM-CSF or control enhanced green fluorescent protein plasmid then TSA for 5d. Splenic MDSC were isolated by Gr1 positive selection, and suppressive function was tested in allogeneic mixed leukocyte cultures. Exposure of GM-CSF-stimulated BM cells to TSA led to expansion of suppressive MDSC (CD11b+Gr1+) that correlated with increased numbers of hematopoietic stem and progenitor cells (HSPC; Lin-Sca-1+c-Kit+). TSA enhanced mobilization of splenic HSPC in vivo following GM-CSF administration and increased CD11b+Gr1+ cells in the BM and spleen. Increased numbers of Gr1+ cells that suppressed effector T cell proliferation were isolated from spleens of TSA-treated mice given GM-CSF. In conclusion, HDACi enhance GM-CSF-mediated expansion of MDSC in vitro and in vivo, suggesting a novel mechanism of the anti-inflammatory action of HDACi.
Characterization of Vibrio parahaemolyticus serotype O3:K6 activation of murine macrophage. (111.27)
Authors: Waters Stephanie , Parent Michelle , Blumerman Seth , Whitaker Brian , Joerger Torsten , Boyd Ethna
Published: January 2023

Vibrio parahaemolyticus is the leading cause of seafood-related bacterial gastroenteritis in the United States. There is a dearth of literature regarding the host response to, and subsequent elimination of, infection with this pathogen. Here, we have developed an in vitro model using the virulent clinical isolate V. parahaemolyticus RIMD2210633, an O3:K6 serotype. We determined that this isolate could infect RAW264.7 cells, a murine macrophage cell line and bone marrow derived macrophage (BMDM), resulting in the production of the early proinflammatory cytokines interleukin-1 alpha (IL-1α), interleukin 6 (IL-6) and interleukin 10 (IL-10). Additionally, infected RAW264.7 macrophage displayed increased surface CD86, CD40 and class II MHC expression while displaying a decrease in TLR4. Concurrently, we investigated the production of reactive oxygen species (ROS) by both RAW264.7 and BMDM infected with this virulent clinical strain. We report that in response to infection, both types of macrophage demonstrate increased production of ROS. We have determined that infection of macrophage by a virulent clinical isolate of Vibrio parahaemolyticus results in immune activation as demonstrated by increased cell surface molecules and proinflammatory cytokine production. Characterization of this response will provide the foundation to understand elimination of this organism from the infected host.
COX-2 inhibition represses the generation of M2 macrophages and slows tumor progression (111.31)
Authors: Ho Victor , Rauh Michael , Krystal Gerald
Published: January 2023

Macrophages (mΦs) are very heterogeneous and include immune-activating (M1) and immune-suppressive (M2) subsets. In response to cytokine or TLR activation, M1-mΦs (killer mΦs) express iNOS, which produces tumoricidal and bacteriocidal nitric oxide (NO). M2-mΦs (healer mΦs), in contrast, express arginase I, which competes with iNOS for L-arginine, to promote cell proliferation and fibrosis. SHIP (SH2-containing inositol-5’ phosphatase), a hematopoietic-specific negative regulator of the PI3K pathway, prevents M2-mΦ development. Thus, in SHIP-/- C57BL/6 mice, all the mΦs are M2 skewed. As a result, subcutaneously injected Lewis lung carcinoma (LLC) tumors grow faster in SHIP-/- than in +/+ mice, and this rapid tumor growth correlates with high tumor-associated arginase. We therefore hypothesize that high PI3K activity skews bone marrow (bm) progenitors to become tumor-promoting M2-mΦs. Herein we show that PGE2 also skews SHIP-/- , but not +/+, bm progenitors to M2 mΦs and that COX-2 inhibitors repress this skewing. Specifically, the COX-2 inhibitor SC-58125 reduces arginase levels and IL-10 production from in vitro derived SHIP-/- bmmΦs. Furthermore, another COX-2 inhibitor, Celebrex, slows tumor growth in vivo, and this correlates with reduced arginase in tumor-associated mΦs. This is consistent with COX-2 inhibitors slowing tumor growth, at least in part, by preventing M2-skewing of tumor-associated mΦs.
Actions of novel inflammation-resolving lipid mediators on human B cells (112.12)
Authors: Ramon Sesquile , Serhan Charles , Phipps Richard
Published: January 2023

The resolution of inflammation is an active and dynamic process. Newly identified lipid mediators are now recognized as key players during the process of inflammation resolution. These lipid-derived molecules constitute four families of endogenous mediators (lipoxins, resolvins, protectins and maresins) derived from essential fatty acids. New findings also demonstrated that these lipid mediators regulate aspects of the immune response, including inhibition of neutrophil infiltration, reduction of T cell cytokine production and stimulation of macrophage phagocytic activity. Their actions on B lymphocytes are not known. Our results indicate that the lipoxin B4 and 17-HDHA, used at 100nM, increase the ability of normal human B cells to produce IgM and IgG when activated with CpG plus anti-IgM. The two lipid mediators also enhance B cell differentiation towards antibody secreting cells. In addition, resolvin D1 and aspirin-triggered resolvin increase antibody production in CpG-stimulated B cells. None of these inflammation resolution lipid mediators affect proliferation and are non-toxic to B cells. Increase of plasma cell differentiation and antibody production coincides with the known involvement of pro-resolving mediators during the late stages of inflammation and pathogen clearance.
LFA-1 (CD11a/CD18) is required for T-cell homing to the mesenteric lymph nodes and gut but not for T-cell activation during the induction of chronic colitis (112.19)
Authors: Koboziev Iurii , Ostanin Dmitry , Gray Laura , Jackson Sherry , Kosloski-Davidson Melissa , Grisham Matthew
Published: January 2023

We have demonstrated that adoptive transfer of naïve LFA-1-deficient (CD11a-/-) T-cells into RAG-1-/- mice fails to induce chronic colitis whereas transfer of wild type T-cells induces chronic and unrelenting colonic inflammation. The objective of this study was to define the role that LFA-1 plays in T-cell activation and trafficking during the induction of chronic colitis in RAG-1-/- mice. In the first series of studies, we found that enteric antigen (EAg)-pulsed dendritic cells (DCs) induced a concentration-dependent increase in wild type (WT) and CD11a-/- CD4+ T-cell proliferation that was quantitatively very similar between the two groups whether we used bone marrow- or spleen-derived DCs, suggesting that LFA-1 is not critical for EAg-induced activation of T-cells in vitro. Short term trafficking studies (<24 hrs) revealed a major defect in the accumulation of CD11a-/- T-cells within the MLNs compared to WT T-cells. When the observation period was extended to 3 and 7 days post T-cell transfer, we observed approximately 2-3-fold more WT T-cells within the MLNs and colon than CD11a-/- T-cells however, cell analysis revealed similar losses of CFSE fluorescence in both the WT vs. CD11a-/- T-cells suggesting that both populations of T-cells proliferated to same extent in vivo. Taken together, our data suggest that LFA-1 is important for T-cell trafficking to the MLNs and colon but is not required for EAg-induced T-cell activation in vitro or in vivo.
R-spondins protect from ischemia/reperfusion-induced tissue damage (112.25)
Authors: Kannan Lakshmi , Kis-Toth Katalin , Yoshiya Kazuhisa , Thai To-Ha , Tsokos George
Published: January 2023

Local and systemic inflammatory responses triggered by ischemia/reperfusion (IR) represent a leading cause of morbidity and mortality in a number of clinical settings including shock and transplantation. R-Spondins play a key role in wound healing and tissue regeneration through the modulation of the wnt pathway. We hypothesized that R-Spondins could prevent tissue injury caused by mesenteric IR by decreasing the local inflammatory response to ischemia. C57BL/6J mice were pretreated with vehicle or R-Spondins before inducing intestinal IR by clamping the superior mesenteric artery for 30 min. After this period of occlusion, the clamps were removed and the intestine was reperfused for 180 min. Intestinal injury was quantified by histologic scoring. Treatment with R-Spondins significantly decreased local intestinal injury. mRNA and protein analyses revealed that this effect was achieved through the modulation of the expression of inflammatory cytokines. Specifically, R-Spondins suppressed the levels of the IR-enhanced IL-6, IL-1β, TNFα, IL-10/CCL2 and CXCL5 cytokines/chemokines. Our results demonstrate that R-Spondins can mitigate the IR-induced local tissue damage induced by decreasing the local inflammatory cytokine response to ischemia and imply that they may be useful in the prevention of IR-related tissue damage.
The actin-bundling protein L-plastin is required for marginal zone B cell development (112.4)
Authors: Morley Sharon Celeste , Davis Chris , Muenzer Jared , Todd Elizabeth
Published: January 2023

B cell development is exquisitely sensitive to location within specialized niches in the bone marrow and spleen. Location within these niches is carefully orchestrated through chemotactic and adhesive cues. Here we demonstrate the requirement for the actin-bundling protein L-plastin (LPL) in B cell motility towards the chemokines CXCL12 and CXCL13, chemokines that guide normal B cell development. Impaired motility of B cells in LPL-/- mice correlated with diminished splenic maturation of B cells, with a profound (>80%) loss of marginal zone B cells. Entry of LPL-/- B cells into lymph nodes and bone marrow was also impaired. Furthermore, LPL was required for the integrin-mediated enhancement of transwell migration but was dispensable for integrin-mediated lymphocyte adhesion. These results suggest that LPL participates in signaling that enables lymphocyte transmigration. In support of this hypothesis, the phosphorylation of Pyk-2, a tyrosine kinase that integrates chemotactic and adhesive cues, is diminshed in LPL-/- B cells. Furthermore, a well-characterized role of marginal zone B cells is the generation of a rapid humoral response to polysaccharide antigens. LPL-/- mice exhibited a defective antibody response to Streptococcus pneumoniae, and LPL-/- mice succumb rapidly to intra-tracheal infection with Streptococcus pneumoniae. LPL is thus critical for effective immunity against a significant human pathogen.