bluuebunny/crossref_metadata_2025 huggingface dataset:
“I”-branched Carbohydrates Negatively Regulate Galectin-3-binding and Melanoma Malignancy
Authors: Sweeney Jenna Geddes , Liang Jennifer , Giovannone Nicholas , Schaffer Lana , Head Steven R. , Antonopoulos Aristotelis , Haslam Stuart M. , Widlund Hans R. , Dimitroff Charles J
Published: January 2023

Malignant transformation is often associated with aberrant glycosylation of cell surface proteins. Commonly observed changes in glycan structures include increased presence of sialic acid, altered expression of mucins, and abnormal branching of N-glycans. Increasing evidence supports the idea that the presence of certain glycans correlate with cancer progression by affecting tumor cell invasiveness, ability to bind pro-tumorigenic galectins, and by promoting metastasis to distant organs. Taking this relationship into consideration, we conducted MALDI-TOF mass spectrometry on cell surface glycans as well as glycomic gene profiling from normal human epidermal melanocytes (NHEM) and human metastatic melanoma (MM) cells. We found that a defining feature between NHEMs and MMs was the relative level of “I”-branched and “i”-linear poly-N-acetyllactosamines on N-glycans. While NHEM predominantly expressed “I”-branched structures, MMs expressed “i”-linear modified N-glycans. Furthermore, glycomic gene profiling and confirmatory RT-qPCR data showed that the “I”-branching β1,6 N-acetylglucosaminyltransferase 2, GCNT2, is decreased in MM compared with NHEMs. With regards to its role in malignancy, we found that GCNT2 functioned as a negative regulator of galectin-3 (Gal-3) binding. GCNT2 overexpression in human MM cells inhibited Gal-3 mediated ERK1/2 phosphorylation and significantly reduced primary tumor growth. While GCNT2 knockdown in human MM cells promoted Gal-3 mediated ERK1/2 phosphorylation and significantly enhanced primary tumor growth. These findings highlight new glycobiological insights into melanoma development, implicating GCNT2 as a negative regulator of Gal-3 binding and melanoma malignancy.
Unsaturated fatty acids suppress anti-tumor T cell function.
Authors: Cui Guoliang , Kaech Susan M
Published: January 2023

Remarkable progress in cancer immunotherapy has revealed the power of harnessing immunity to combat cancer. Anti-tumor T cells suppress tumor development by secreting cytotoxic cytokines and activating other immune cells. However, the tumoricidal function of tumor-infiltrating T cells is often suppressed and inefficient to combat tumors. Thus one of the key questions in the field of cancer immunology is to address what causes the immunosuppressive nature of the tumor microenvironment. Using a melanoma mouse model, we found that 1) fatty acids accumulated in the tumor microenvironment, 2) unsaturated fatty acids suppressed T cell effector function through CD36/PPAR pathway and 3) blocking FAs transportation into T cells enhanced T cell cytotoxicity and delayed tumor growth. This study has identified unsaturated fatty acid species as immunosuppressive factors in tumor, which may lead to the development of novel forms of therapy that stimulates anti-tumor immunity by manipulating fatty acid transportation and metabolism.
Detection of HIV Env-mediated B cell receptor aggregation on engineered B cells by flow cytometry.
Authors: Gallaher Brandon L , Stamatatos Leonidas
Published: January 2023

Approximately 20% of individuals infected with HIV-1 produce broadly neutralizing antibodies (bNAbs) against the virus. Understanding how bNAb linked B cell receptors (BCRs) are initially stimulated by the HIV-1 envelope (Env) in the context of natural HIV infection is a major focus of HIV research. While germline-reverted BCR forms of bNAbs do not efficiently recognize Env, recombinant Env proteins have been recently designed to engage and activate some germline BCRs. To better understand how such modified Envs engage BCRs and to further optimize the activation of B cells, we (a) created a stable Ramos cell line that expresses the germline BCR form of a potent and broadly neutralizing antibody, VRC01 and (b) developed a fluorescence-based assay that allows for the monitoring of Env-mediated BCR aggregation. B cells were incubated with different multimeric forms of Env (valency ranging from 1–24) and determined the extent of BCR aggregation and compared it with the extent of B cell activation (measured by Ca2+flux assays). Significant increases in BCR aggregation and B cell-activation were detected in response to heptameric and 24-meric forms of Env. In contrast, lower valency multimeric forms of Env activated B cells inefficiently. No aggregation or calcium flux was detected in response to monomeric Env. A positive correlation was observed between the magnitudes of calcium flux and BCR aggregation. Thus, either BCR aggregation or Ca2+ flux assays can be used to monitor the activation of B cells by recombinant Env proteins that are being evaluated as possible immunogens to elicit bNAbs. Our results are relevant to optimizing the design and formulation of recombinant Env proteins for the elicitation of broadly neutralizing HIV-1 antibodies.
Immune correlates for VEEV replicon vaccine protection against Ebolavirus
Authors: Stonier Spencer W , James Rebekah M , Josleyn Nicole M , Bakken Russell R , Dye John M
Published: January 2023

The progress of filovirus vaccines has advanced greatly due in part to the urgent need to develop countermeasures for the unprecedented Ebolavirus (EBOV) outbreak in West Africa. While a variety of vaccination approaches have been moved into clinical trials, the understanding of what constitutes a protective response after vaccination is still under investigation. Our approach to addressing immune correlates has involved the use of various mouse models to first test whether or not protection is afforded in the absence of a cell population. To this end, wild type (wt), β2m−/− and μMT−/− mouse models were used to assess the protective efficacy of vaccination with a Venezuelan Equine Encephalitis Virus replicon bearing the EBOV glycoprotein (GP). Vaccinated β2m−/− mice, despite lacking CD8 T cells, enjoyed full protection against mouse-adapted (ma) EBOV exposure. Vaccinated μMT−/− mice, on the other hand, succumbed to maEBOV exposure, similar to unvaccinated control mice. A flow cytometry analysis of T cell responses in Wt, β2­m−/− and μMT−/− mice revealed all mice mounted CD4 T cell responses, whereas only wt mice generated CD8 T cell responses. Antibody responses to EBOV GP were predictably lacking in μMT−/− mice after vaccination, but were present at high titers in wt and β2m−/− mice. We were able to achieve protection in vaccinated μMT−/−, but not unvaccinated μMT−/− mice, given serum from vaccinated wt mice before and after maEBOV exposure. These data indicate that T cell responses in this model can contribute to protection when given antibody help. Ultimately, antibody responses appear to be the best protective correlate in this model, though we also provide evidence that T cell responses can be essential in certain conditions.
Meeting the threshold of multifunctional CD8+ TEM by γ-irradiated sporozoite vaccination, essential to ensue sterile protection against Plasmodia liver stage infection
Authors: Patel Hardikkumar Bipinchandra , Yadav Naveen , Parmar Rajesh , Dalai Sarat K
Published: January 2023

People in malaria endemic area develop immunity after several years of exposure to parasite, children particularly take longer to develop protective immunity. Interestingly, the protection is lot more consistent among children living in high transmission area compared to those of low transmission area, suggesting that protection may depend on the frequency of exposure and/or a load of parasite exposure. Thus, we hypothesize children experiencing multiple exposures might develop critical frequencies of anti-parasite lymphocytes ensuring the protection to Plasmodia. CD8+ T cells play a key role in mediating protection against Plasmodia liver-stage infection. In present study, we took whole sporozoite (spz) vaccination strategy and immunized C57Bl/6 mice with multiple doses of γ-spz at different time intervals. We found continuous immunization with 3 and 4 doses of γ-spz, 2 weeks apart, provides ~50% and 100% sterile protection, respectively. However, mice immunized with 3 doses of γ-spz with an interval of 90 days during 2nd – 3rd dose fail to be protected. Upon measuring the CD8+ T cell responses, we found that higher frequencies of liver-stage specific multifunctional CD8+ T cells (CD44hiKLRG-1intCD107+IFN-γ+) were accumulated in mice immunized with 4 doses of γ-spz (180–410 cells/106; Avg. 284), whereas the same among mice receiving 3 doses showed lower frequencies (50–125 cells/106; Avg. 75) before challenge, and mixed frequencies (126–551 cells/106; Avg. 340) following challenge, suggesting that protection against Plasmodia infection could be ensured either by pre-existing multifunctional memory CD8+ T cells before the infection or mobilizing the required frequencies of the same to liver during the course of infection.
ABVIC: an improved serological test to resolve indeterminate diagnoses for herpes simplex virus 2 (HSV-2)
Authors: Sowa Gavin M , Geltz Joshua , Kempa Annie , Bernert Rene , Warren Terri , Halford William P
Published: January 2023

Approximately 50 million Americans are infected with herpes simplex virus type 2 (HSV-2) and may, at any time, transmit infectious virus to sexual partners. Current HSV-2 serological antibody tests are imperfect; 5–6% of those who initiate HSV-2 ELISA IgG screening receive low-positive results, known to include about 50% false-positives compared to HSV Western Blot, the current gold standard test. Even when low-positive samples are run by Western, ~20% obtain indeterminate results. We report a new, flow-cytometry-based method that measures serum antibody-binding to virus-infected cells (ABVIC). Unlike the glycoprotein G-based HerpeSelect assay, ABVIC tests for antibodies to ~30 HSV-2 antigens and appears to be ~100-fold more sensitive than the gG-2-based antibody capture ELISA. We report that when serum was collected from n=17 patients who had previously received “HSV-2 indeterminate” results by Western Blot, the ABVIC test offered a clear answer in every case. Sixteen of 17 were HSV-2 seronegative by ABVIC and only 1 of 17 was weakly HSV-2 seropositive. Most of the “Indeterminate” serum samples exhibited high background, which produces weak reactivity in the HerpeSelect and Western blot assays, but which does not confound the internally controlled ABVIC assay. We conclude that the highly quantitative, flow cytometry-based ABVIC test may be used to resolve the vast majority of “HSV-2 Indeterminate” results that have misled thousands of patients to the erroneous conclusion that they carry HSV-2 and pose a transmission risk to others.
Pathogenic conversion of Foxp3+ T cells into Th1 and Th17 cells in Stromal Keratitis
Authors: Varanasi Siva Karthik , Bhela Siddheshvar , Rouse Barry T
Published: January 2023

Ocular infection with herpes simplex virus 1 (HSV-1) can result in a chronic immuno-inflammatory lesion that is a significant cause of human blindness. IFN-γ producing CD4 + T cells are generally considered the main orchestrators, and lesions are more severe if the regulatory T cell (Treg) response is compromised. Tregs have been shown to lose FoxP3 (Treg lineage factor) and adopt alternate lineage fates in a changing cytokine environment. Moreover, little is known about the stability of Treg cells in an ongoing inflammatory reaction such as is the case of SK. In this study-using fate mapping mice, we were able to demonstrate that the population of ex-Tregs increased at the site of infection that is the cornea and also at the secondary lymphoid organs after HSV-1 infection. In vivo studies showed that these ex-Tregs acquired the Th1 and Th17 phenotype, which may play a destructive role in stromal keratitis. We also demonstrate in vivo that CD25lo subset of Tregs is less suppressive and more prone to conversion than CD25hi Tregs. Additionally, in-vitro studies suggested that Tregs in the presence of IL-12 converted into ex-Tregs, however this plasticity was prevented when Tregs were generated in the presence of DNMT inhibitor (Azacytidine). Thus, plasticity of Tregs might represent a problem during SK, which needs to be controlled with appropriate therapeutic procedures. Overall, our results indicate that Tregs lose FoxP3 expression in ocular inflammatory setting and drugs that reverse Treg plasticity could be useful to restore Treg function and to inhibit ocular immunopathology.
PD-1 inhibition results in reduced dendritic cell (DC) activation of HIV-1-specific de novo CTL and enhancement of DC-induced CTL memory recall responses
Authors: Garcia-Bates Tatiana M , Kim Eun , Gambotto Andrea , Rinaldo Charles R , Mailliard Robbie B
Published: January 2023

Combination strategies consisting of DC vaccines and suppression of immunoregulatory pathways such as PD-1 blockade have been advocated to elicit optimal CTL responses in chronic diseases including cancer and HIV infection. We have shown that inactivated HIV-1 virus loaded, high IL-12 producing, mature, type-1 polarized DC (DC1) can activate CTL from naïve CD8+ T cell precursors that more effectively kill HIV-1-infected cells than CTL derived from memory CD8+ T cells. Here we tested DC1 transfected with an adenoviral (Ad) vector encoding anti-PD-1 antibody (DC.αPD1) enhancement of both primary as well as memory CTL responses to HIV-1. Ad-αPD1-transduced DC1 (DC.αPD1) secreted high levels of functional anti-PD1 Ab without affecting their phenotype or IL-12p70-producing capacity. We next compared HIV Gag peptide epitope-loaded DC.αPD1 to DC1 that were transfected with an empty vector for their ability to activate either purified, autologous naïve or memory HIV-1-specific CD8+ T cells from chronic HIV-1-infected participants of the Multicenter AIDS Cohort Study. When compared to the control DC1, antigen-loaded DC.αPD1 enhanced the overall magnitude of HIV-1-specific CTL responses induced, as determined by the expansion of HIV-1-peptide responsive CD107a and IFNγ expressing T cells. In contrast, the overall number HIV-1 antigen-reactive CTL derived from the naïve CD8+ T cell fraction sharply decreased when using the DC.αPD1-based approach. These results suggest previously unrecognized, opposing roles of the inhibitory receptor PD-1 in DC1-induced primary versus memory recall CD8+ T cell responses to HIV-1.
A large scale screening of HLA-class II-binding peptides from HBs and HBc peptide libraries
Authors: Miyadera Hiroko , Okabe Yuki , Chen Cindy , Tokunaga Katsushi , Mizokami Masashi
Published: January 2023

Objectives

Variations in the human leukocyte antigens (HLA)-DP are associated strongly with susceptibility/protection to chronic hepatitis B virus (HBV) infection. It has been reported previously that HLA-DPB1*05:01 and *09:01, the major HLA-DP alleles in East Asians, are associated with susceptibility, while HLA-DPB1*04:01 and *04:02, the major alleles in Europeans, confer protection against chronic hepatitis B (Kamatani, et al. Nat Genet (2009); Nishida, et al. PLoS One (2012)). To identify the peptides that are potentially involved in immunological responses against HBV infection, we screened the peptide libraries of the HBV surface antigen (HBs) and core antigen (HBc) through the cell-surface HLA expression assay.

Methods

The six major HLA-DP haplotypes that are associated with susceptibility to or protection against chronic hepatitis B were used in this study. Peptide libraries were designed for the entire HBs and HBc antigens, based on the consensus sequence of the genotype C. To identify the HLA-DP-binding region, we expressed HLA-DP in fusion with each peptide, using fibroblast cell line as an expression host.

Result

The level of HLA-peptide interaction was estimated by measuring the level of cell-surface expression of each HLA-peptide fusion construct. Out of >70 peptides that cover the entire HBs and HBc antigens, we found several regions that bound strongly to certain HLA-DP allele products. In this presentation we show an overview of the methodology and discuss potential mechanisms that might underlie association of HLA-DP with chronic hepatitis B.
Dysregulated CD8 T cells resemble Tfh-like cells and promote B cell activation during autoimmunity.
Authors: Hoyer Katrina K , Valentine Kristen , Davini Dan , Sindi Suzanne
Published: January 2023

T follicular helper (Tfh) cells provide essential help for the development of antigen specific antibodies during normal and autoreactive immune responses. IL-2-deficient (KO) mice suffer from excessive lymphoproliferation and spontaneous, systemic autoimmune disease. Depletion of CD4 T cells in IL-2-KO mice eliminates the generation of RBC-specific antibodies, augmenting survival. Surprisingly, depletion of CD8 T cells in IL-2-KO mice also reduces disease and prolongs survival. Several groups have demonstrated a protective role for CD8 T cells in inflammation and autoimmunity, while others have suggested a pathogenic role. However the mechanisms of CD8 T cell influence on autoimmune disease are largely unknown. We have identified a new CD8 Tfh-like population in an autoimmune setting. RNA-sequencing of day 12 IL-2-KO CD8 T cells revealed an increase in the expression of genes associated with the CD4 Tfh lineage (Bcl-6, CXCR5, PD-1, ICOS and IL-21) compared with wild-type CD8 T cells. Flow cytometry confirmed the presence of expanded CD4 Tfh and CD8 Tfh-like cells, and the production of IL-21 in IL-2-KO mice. Germinal center tissue staining revealed localization of both CD4 and CD8 T cells to the germinal center during autoimmunity. In vitro, IL-2-KO CD8 T cells induced proliferation and activation of B cells. Our results indicate that autoreactive CD8 T cells contribute to the autoimmune disease in part through dysregulated follicular-like differentiation and function. NIH funding R00HL090706.
MHC-mismatched but not matched mixed chimerism can tolerize peripheral non-cross-reactive autoreactive T cells in type 1 diabetic NOD mice
Authors: Zhang Mingfeng , Racine Jeremy , Liu Yuqing , Lin Qing , Qi Tong , Wang Tiankun , Riggs Arthur D. , Zeng Defu
Published: January 2023

There are cross- and non-cross-reactive autoreactive T cells in type 1 diabetic (T1D) NOD mice. MHC-mismatched but not matched mixed chimerism reverses autoimmunity with deletion of cross-reactive thymocytes. However, how MHC-mismatched mixed chimerism tolerizes non-cross-reactive autoreactive T cells remains unclear. In the current studies, using Rag-1−/− BDC2.5 and BDC12-4.1 mice that have only non-cross-reactive transgenic autoreactive T cells, we found that MHC-mismatched but not matched mixed chimerism was able to induce anergy of host-type T cells as judged by surface markers (PD-1, CD80, Tim-3 and IL-7Rα), reduce Th1 and Th17 cells, and eliminate insulitis, but had no impact on the percentage of CD4+CD8+ thymocytes, indicating that MHC-mismatched mixed chimerism is required for tolerizing CD4 peripheral non-cross-reactive autoreactive T cells. Although both MHC-mismatched and matched mixed chimerism induced host-type DC upregulation of CD86, MHC-II, and PD-L1 and showed similar percentage of donor-type Foxp3+ Treg cells, only MHC-mismatched mixed chimerism increased the percentage of host-type Treg cells. In addition, although the MHC-mismatched mixed chimeras showed little insulitis, the host-type T cells proliferated vigorously in response to autoantigen stimulation in an in vitro culture, and neither donor- or host-type Treg cells inhibited the proliferation. Co-transfer of host- and donor-type T and Treg cells induced T1D in adoptive NOD-scid recipients but not in NOD-scid recipients with pre-engraftment of donor-type DCs. Therefore, induction of MHC-mismatched mixed chimerism can re-establish a tolerogenic DC and Treg network in the periphery and tolerize non-cross-reactive autoreactive T cells.
Characterizing the intracellular magnesium transporter MagT1 in murine lymphocyte function
Authors: Holmes Derek , Carroll Karen , Brodeur Scott , Pashine Achal
Published: January 2023

Free intracellular magnesium (Mg2+) plays a critical role in diverse immune cell functions. Human individuals with a genetic deficiency in the magnesium transporter MagT1 present with XMEN disease, a X-linked immunodeficiency characterized by uncontrolled Epstein-Barr virus infection and neoplasia, uncovering a critical role for Mg2+ in the regulation of NK and CD8T cell function. To interrogate the role of MagT1 in the immune response, we generated conditional knockout mice utilizing a tamoxifen inducible (Cre-ERT2) system to genetically delete Magt1. Here, we characterized the requirement for MagT1 in immune cell function following in vivo genetic deletion. MagT1 is not required for lymphocyte homeostasis following tamoxifen deletion, as T and B cell populations were unperturbed in the spleen, lymph node and thymus following short and long-term tamoxifan treatment. Magt1-deficient CD4+ T cells were defective in response to antigenic challenge following Ova immunization in vivo, as the numbers of BRDU+ CD4+ T cells were reduced. This observation was not due to a defect in proliferation, as the percentage of BRDU+ CD4+ T cells was similar between wild-type and Magt1-deficient mice. In support of this finding, Magt1-deficient CD4+ T proliferation is normal in multiple in vitro proliferation assays. While this observation is inconsistent with human findings from X-MEN patients, differences in human and mouse physiology might contribute to this discrepancy. Future interrogation of MagT1 function in NK and CD8+ T cells could provide mechanistic insight into the manifestations observed in X-MEN disease.
Absence of the Fyn tyrosine kinase ameliorates T cell induced colitis by inhibiting Th17 differentiation
Authors: Yin Catherine C , Fong Julie , Stein Paul L
Published: January 2023

T helper (Th) 17 cells are a distinct proinflammatory CD4+ T cell subset that protects against fungal and extracellular bacterial infections. Excessive inflammation can be suppressed by Tregs. In autoimmune diseases, such as Crohn’s disease and ulcerative colitis, the Th17/Treg responses are unbalanced. Secretion of IL-17 attracts neutrophils and promotes the production of inflammatory mediators resulting in tissue damage. The Src family tyrosine kinase, Fyn, plays a critical role in regulating IL-17 production. In the absence of Fyn, CD4+ T cells skewed towards the Th17 lineage express RORγt but make little IL-17 and show ectopic expression of the Treg transcription factor FoxP3. We hypothesize that in a T cell transfer model of colitis, the absence of Fyn will decrease IL-17 induced inflammation and reduce disease severity. Rag KO recipients receiving WT CD45Rbhi (naïve) CD4 T cells showed characteristic weight loss associated with colitis. In contrast, recipients receiving Fyn−/− CD45Rbhi CD4 T cells maintained their body weight. Mice receiving either WT or Fyn deficient T cells had similar numbers of T cells in the lamina propria, suggesting that the reduced pathology is not due to proliferative defects. Both groups had similar numbers of IFNγ+ (Th1) and RORγt (Th17) positive cells but the Fyn mutant recipients showed a marked decrease in IL-17 secreting T cells. Neutrophil infiltration was highly reduced as well. Furthermore, IL-17 induced genes associated with neutrophil infiltration (S100A8, S100A9), neutrophil chemoattraction (CXCL1, CXCL2), neutrophil homeostasis (CXCL5) and tissue remodeling (MMP9) were all reduced. Together these data suggest Fyn may serve as a novel target for treating Th17 mediated diseases.
An in vivo functional screen identifies miR-148a as a critical regulator of B cell tolerance and autoimmunity
Authors: Gonzalez-Martin Alicia , Adams Brian D , Lai Maoyi , Shepherd Jovan , Salvador-Bernaldez Maria , Salvador Jesus M , Lu Jun , Nemazee David , Xiao Changchun
Published: January 2023

Autoreactive B cells are major contributors to autoimmune disease pathogenesis, but the molecular pathways responsible for the break of B cell tolerance in these patients remain largely unknown. In this study, we performed an in vivo functional screen of a lymphocyte-expressed miRNA library and identified miR-148a as a key regulator of this process. Elevated miR-148a expression impaired B cell tolerance by protecting immature B cells from BCR engagement-induced apoptosis, through downregulation of its targets Gadd45a, Bcl2l11, and Pten. Moreover, increased expression of miR-148a, which has been consistently found in lupus patients and lupus-prone mice, accelerated the development of lethal autoimmunity in a lupus mouse model. These results demonstrate that miR-148a functions as an important regulator of B cell tolerance and autoimmunity.
Evidence for citrullinated inhibitor of DNA binding 1 as a novel autoantigen in rheumatoid arthritis
Authors: Ohara Ray A , Edhayan Gautam , Remmer Henriette A , Morgan Rachel L , Amin M Asif , Stinson W Alexander , Graham Jerry , Kim Josh S , Campbell Phillip L , Arbab Ali S , Fox David A , Ruth Jeffrey H
Published: January 2023

Inhibitor of DNA binding 1 (Id1) is a nuclear transcription factor that is primarily fibroblast derived with elevated expression in rheumatoid arthritis (RA) synovial tissues (STs) and synovial fluids (SFs). Id1 is potently angiogenic in RA, and targeting Id1 or its signaling pathways may attenuate unwanted inflammatory outcomes. Cell signaling studies showed that Id1 can upregulate Jnk in FLS, human dermal microvascular endothelial cells, and endothelial progenitor cells and that Jnk could be targeted to reduce angiogenic responses in vivo. We also showed Id1 can be citrullinated and serve as an autoantigen by inducing anti-citrullinated protein antibodies (ACPAs) in RA peripheral blood (PB) sera and SFs. ELISA for Id1 was performed on exosomes isolated from RA, osteoarthritis, and normal (NL) FLS supernatants. RA FLS were transfected with a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 plasmid targeting the Id1 gene. Lastly, immunodot blots (IDB) were performed on NL and RA PB sera and SFs to test the presence of ACPAs to citrullinated Id1 (citId1). Exosome analysis showed >80% of Id1 detected in RA FLS supernatants was encapsulated within the exosomes. CRISPR-transfected (Id1 knockout) FLS showed a 75% decrease in cell proliferation compared to sham-transfected cells. Lastly, IDB analysis showed robust signals against citId1 from multiple RA PB sera and SFs. Our data show that Id1 is not only an important nuclear protein but is released from FLS, primarily in exosomes, expanding its role in the orchestration of inflammatory lesions through trans-cellular effects. We also show for the first time the presence of ACPAs with specificity to citId1 in RA PB sera and SFs and propose citId1 as a novel autoantigen in RA.
Deficiencies in myeloid cell populations lead to increased sensitivity of females compared with males to Francisella tularensis infection
Authors: Spencer Charles T , Ramos Muniz Mireya G , Olsen Darlene , Setzu Nicole , Martinez Danielle Y.
Published: January 2023

Preliminary data demonstrated that a greater percentage of female mice infected with F. tularensis succumbed to disease and developed symptoms faster than male mice. Increases in the serum inflammatory cytokine levels corresponded to this enhanced sensitivity in females consistent with a stronger inflammatory. Herein, we sought to understand the cellular basis for this sex difference in the inflammatory response between males and females. We used a combination of in vivo and ex vivo cellular analysis to determine differences in the cellular response to F. tularensis. Multiparametric cytokine analysis was used to quantify changes in serum and supernatant cytokines, real-time RT-PCR was used to measure cytokine mRNA expression in tissues, and multiparametric flow cytometry was used to discriminate cell populations responsive to infection. Despite a tendency for fewer T regulatory (Treg) cells, males had fewer inflammatory macrophage and an increase in regulatory macrophages. This corresponded with an overall decrease in the transcripts of IL-10, IL-5, and IL-4 in the tissues of mice succumbing to disease and an increase in IL-6 transcripts, a marker for the level of inflammation. The functional deficiency of regulatory macrophages and increase of M1 macrophages may enhance the inflammatory response to F. tularensis ultimately leading to decreased survival. Further investigations will focus on understanding the interactions between these myeloid populations with F. tularensis and other cells of the immune system. Development of therapeutics that activate or induce expansion of regulatory macrophages or that suppress the activity/activation of M1 macrophages may prevent death from this disease.
Reprogramming of myeloid compartments supporting tissue repair during DSS-induced colitis recovery
Authors: Tremblay Alexandra V , Bian Zhen , Niu Shuo , Culpepper Courtney , Liu Yuan
Published: January 2023

Myeloid-derived suppressor cells (MDSC) emerging during tumor growth or chronic inflammation play a critical role in regulating T cell function. However, mechanisms governing the generation of these cells remain unclear and need to be further defined. Using a DSS-induced colitis and recovery model, we characterized the dynamic changes within the myeloid compartments and the emerging of MDSC during active and resolution phases of inflammation. We show that the immature myeloid cell population expands and undergoes a functional reprogramming event in bone marrow (BM) and the spleen to form suppressive MDSC not at the active inflammation phase, but specifically at the resolution phase during colitis transition to recovery. In particular, two major functional changes occur when colitic mice were allowed to recover: 1) CD11b+Gr-1+myeloid cells in bone marrow and spleen acquire T cell suppressive functions, and 2) immature granulocytic myeloid cells increase the expression of chemokine receptor CXCR2, which enables their entry into circulation from BM. Additionally we determined enhanced activation of STAT3 and subsequent transcriptional enhancement of Arg1, iNOS, and gp91 in the MDSC isolated from resolution time points. We found that IL-17, the major cytokine associated with active colitis, remains elevated in the serum throughout the wound healing/resolution process. IL-17, together with the enhanced production of tissue repair cytokines, including IL-10, an activator of STAT3, produce a unique cytokine profile, resulting in a switch to induce reprograming and mobilization of the immature myeloid cells to suppress the inflammatory reaction in favor of tissue repair.
IL-7 receptor polymorphisms is related to signaling strength which is associated with autoimmune T cells activation in multiple sclerosis
Authors: Li Wenqing , Hixon Julie A. , Tritapoe Julia , Barata Joao T. , Durum Scott K.
Published: January 2023

IL-7 is required for survival of most T cell subsets and generally promotes T cell immune responses. Loss of functions of IL-7R in human has been known to cause severe combined immunodeficiency. Somatic gain of function mutations of IL-7R have been found in childhood T-cell acute lymphoblastic leukemia (T-ALL), these mutations usually occur in region of residue 244 in the exon 6 and lead to insertion of multiple amino acids. More recently, polymorphisms in IL-7R have been shown be a risk factor for multiple sclerosis (MS) and some autoimmune diseases. The polymorphysims that affects risk to most of these immunopathologies is T244/I, the same region harboring gain-of-function mutations in T-ALL. To investigate the mechanism of IL-7R variants association with MS and seek new therapeutic approaches directed to IL-7R pathway, we hypothesize that the functional significance of the T244/I polymorphism in IL-7R is related to increased signaling. To test our hypothesis, the T244 and I244 alleles of IL7R are transfected into BaF3 cells. The high risk allele T244 induces stronger Stat5 activation and promotes better survival of BaF3 cells. We further examine the strength of signaling in vivo in mice. IL-7R deficient bone marrow hematopoietic progenitors are transduced by retroviral infection with T244 or I244 allele and transferred to Rag1 deficient mice. Human IL-7R rescues T cell development in mice. Consistent with the in vitro study, the CD8 cells harboring the high risk allele T244 display stronger Stat5 activation compared with cells with I244. Our preliminary study suggests that high risk allele T244 of IL-7R signals stronger than allele I244, and this may promote activation of autoimmune T calls in MS and other autoimmune diseases.
PGE2 and IL-27: novel pro-inflammatory mechanisms involving dendritic cells and Tr1 cells
Authors: Hooper Kirsten M , Yen Jui-Hung , Kong Weimin , Ganea Doina
Published: January 2023

Interleukin-27 (p28/EBI3) is an immunomodulatory cytokine expressed by activated antigen presenting cells. Although first discovered to be involved in Th1 cell differentiation, further studies demonstrated the immunosuppressive functions of IL-27 including the inhibition of Th17 cell development and the induction of type 1 regulatory T cells (Tr1) and exhausted CD4+ T cells. The anti-inflammatory effects of IL-27 have been demonstrated in vivo in murine models of parasitic infections and autoimmune disease models such as experimental autoimmune encephalomyelitis and inflammatory bowel disease (IBD). We previously detailed the pro-inflammatory effects of PGE2 in collagen-induced arthritis and IBD through stimulation of IL-23 production leading to development of Th17 cells. Here we describe novel pro-inflammatory functions of PGE2, i.e. inhibition of IL-27 production by murine bone marrow-derived dendritic cells (DC), inhibition of Tr1 differentiation and of IL-10 production by Tr1 cells. We determined that the effect of PGE2 on IL-27 production in LPS-stimulated DC is mediated through EP2/EP4 receptors, induction of cAMP, and partial inhibition of both IRF1 and IRF3. Furthermore, we found that PGE2 treatment of LPS-stimulated DC reduces IRF1 binding to an essential ISRE site within the p28 promoter. In addition to the effects of IL-27 in DC, we also found that PGE2 directly inhibits IL-27-induced differentiation of Tr1 cells through EP4, and inhibits production of IL-10 by Tr1 cells through EP2/EP4. These studies further describe the pro-inflammatory effects of PGE2 and identify possible new targets for treatment of inflammatory diseases.
DCIR is an Important Regulatory Molecule in Allergic Inflammation
Authors: Dembele Marieme , Kaufman Gabriel N. , Xue Di , Beland Marianne , Mazer Bruce D.
Published: January 2023

The dendritic cell immunoreceptor (DCIR) is an inhibitory C-type lection receptor possessing immunoregulatory properties. We have shown that DCIR serves as a receptor for IVIg. In this study, we sought to determine its requirement in modulating IVIg mechanism of action by using a murine model of allergic of airways disease.

Wild type (WT) and DCIR−/− mice were sensitized and challenged with ovalbumin (OVA), and lung histopathology, bronchoalveolar lavage and phenotyping of inflammatory cells were performed. We further studied the effect of IVIg on allergen uptake and processing of CD11c+ cells in vitro. Briefly, bone marrow-derived dendritic cells from WT, DCIR−/− and Fcr2b−/− mice were treated with IVIg prior to exposure to OVA-ALEXA-647 or DQ-OVA.

DCIR−/− mice exhibit exacerbated pulmonary inflammation and eosinophilia compared to WT mice; this phenotype is rescued by IVIg treatment as in WT mice. Furthermore, IVIg significantly impairs the antigen uptake and processing machinery of dendritic cells from WT and DCIR−/− derived BMDCs but not that of Fcr2b−/−-derived BMDCs. In conclusion, DCIR is important in regulating allergic airway inflammation and its presence is not essential for IVIg to function. DCIR appears to be required for normal regulation of pulmonary inflammation.
PLAG (1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol) modulates eosinophil chemotaxis by regulating CCL26 expression from epithelial cells
Authors: Jeong Jinseon , Kim Young-Jun , Yoon Sun Young , Kim Young-Jae , Kim Joo Heon , Kim Myung-Hwan , Sohn Ki-Young , Kim Heung-Jae , Han Yong_Hae , Chong Saeho , Kim Jae Wha
Published: January 2023

Increased number of eosinophils in the circulation and sputum is associated with the severity of asthma. The respiratory epithelium produces chemokine (C-C motif) ligands (CCL) which recruits and activates eosinophils. A chemically synthesized monoacetyl-diglyceride, PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol) is a major constituent in the antlers of Sika deer (Cervus nippon Temminck) which has been used in oriental medicine. This study was aimed to investigate the molecular mechanism of PLAG effect on the alleviation of asthma phenotypes. A549, a human alveolar basal epithelial cell, and HaCaT, a human keratinocyte, were activated by the treatment of interleukin-4 (IL-4), and the expression of chemokines, known to be effective on the induction of eosinophil migration was analyzed by RT-PCR. The expression of IL-4 induced genes was modulated by the co-treatment of PLAG. Especially, CCL26 expression from the stimulated epithelial cells was significantly blocked by PLAG, which was confirmed by ELISA. The transcriptional activity of signal transducer and activator of transcription 6 (STAT6), activated by IL-4 mediated phosphorylation and nuclear translocation, was down-regulated by PLAG in a concentration-dependent manner. In ovalbumin-induced mouse model, the infiltration of immune cells into the respiratory tract was decreased by PLAG administration. Cytological analysis of the isolated bronchoalveolar lavage fluid (BALF) cells proved the infiltration of eosinophils was significantly reduced by PLAG. In addition, PLAG inhibited the migration of murine bone marrow-derived eosinophils, and human eosinophil cell line, EoL-1, which was induced by the addition of A549 culture medium.
Dermal exposure to the antimicrobial triclosan affects the gut microbiome and augments Th2 allergic responses mediated through Toll-like receptor 4
Authors: Marshall Nikki B , Lukomska Ewa , Long Carrie Mae , Anderson Stacey
Published: January 2023

Antimicrobial compounds like triclosan are ubiquitous in our environment; the CDC finds that75% of the general U.S. population has detectable levels in their urine. Curiously, recent epidemiological studies report positive associations between allergic disease and triclosan exposure. Our laboratory recently reported augmented allergic responses to ovalbumin in BALB/c mice with dermal triclosan exposure although triclosan itself was not immunogenic. Here we show that repeated application of triclosan to the skin alters expression of toll-like receptors including adramatic increase in TLR4 expression. In-vivo antibody-blockade of TLR4/MD2 significantly decreased responses in the skin tissue with triclosan exposure including expression of IL-6, TSLP, CXCL2, TLR2 and TLR4. The draining lymph nodes were also significantly affected by TLR4 blockade, including decreased cellularity and production of IL-4 and IL-13 by CD4 T cells. Proposed endogenous ligands of TLR4, S100A8 and S100A9, were also found to be highly expressed in the skin tissue and infiltrating neutrophils thus identifying a possible mechanism of TLR4 activation. Changes in gut microbiome have also been linked to changes in Th2 allergic responses. Here we found that after 28 days of topical application of triclosan, the relative abundance of Lactobacillus was increased in the gut. A specific mechanism of action of triclosan is inhibition of the enoyl-ACP reductase FabI gene involved in bacterial fatty acid synthesis which is not expressed by Lactobacillus. Together these data suggest that triclosan could augment allergic responses in part through activation of the TLR4 pathway and modulating the host microbiome.
Two phase 3 study results of children and adults with mild-to-moderate atopic dermatitis treated with Crisaborole Topical Ointment, 2%, a novel, nonsteroidal, topical, anti-inflammatory, phosphodiesterase 4 inhibitor
Authors: Zane Lee Thomas , Paller Amy S. , Tom Wynnis L. , Lebwohl Mark G. , Blumenthal Robin L. , Boguniewicz Mark , Call Robert S. , Eichenfield Lawrence F. , Forsha Douglass W. , Rees William C. , Simpson Eric L. , Stein Gold Linda F. , Zaenglein Andrea L. , Hebert Adelaide A.
Published: January 2023

Up to 90% of children and adults with atopic dermatitis (AD), a chronic inflammatory skin disease, present with mild-to-moderate disease. Crisaborole Topical Ointment, 2%, is a novel, nonsteroidal, topical, anti-inflammatory, phosphodiesterase 4 inhibitor being studied for the treatment of AD. The efficacy and safety of crisaborole was assessed in 2 identically designed, multicenter, vehicle-controlled, double-blind Phase 3 studies (301 and 302) that enrolled patients ≥2 years old with mild-to-moderate AD affecting ≥5% of body surface area (BSA). Patients were randomized 2:1 to receive crisaborole or vehicle twice daily and evaluated on Days 8, 15, 22, and 29. The primary endpoint defined success in the Investigator’s Static Global Assessment (ISGA) as “almost clear/1” or “clear/0” with ≥2-grade improvement from baseline at Day 29. Secondary endpoints analyzed the time to success and the percentage of patients achieving “almost clear/1” or “clear/0” on ISGA. At Day 29, more crisaborole-treated patients achieved ISGA success than vehicle (301: 32.8% vs 25.4%, P = 0.038; 302: 31.4% vs 18.0%, P < 0.001), with a greater percentage of “almost clear/1” or “clear/0” ISGA scores (301: 51.7% vs 40.6%, P = 0.005; 302: 48.5% vs 29.7%, P < 0.001). Success in ISGA scores was achieved earlier with crisaborole than vehicle (P < 0.001). Treatment-related adverse events (AEs) were usually mild and included upper respiratory tract infection (pooled data, crisaborole vs vehicle: 3.0% vs 3.0%) and application site pain (4.4% vs 1.2%). AE-related discontinuation rates were low for both groups (1.2%). 2 large Phase 3 studies demonstrated crisaborole may represent a novel, safe, and efficacious treatment for patients with mild-to-moderate AD.
Characterization of Allergen Experienced Group 2 Innate Lymphoid Cells: where do they come from, where do they go?
Authors: Mathae Laura , Martinez-Gonzalez Itziar , Takei Fumio
Published: January 2023

We identified group 2 innate lymphoid cells (ILC2s) in mouse lungs. Upon exposure to the protease allergen papain, lung ILC2s produced large amounts of IL-5 and IL-13 and induced eosinophilia and mucus hyperproduction. Intranasal injections of recombinant IL-33 also stimulated lung ILC2s. ILC2s that were previously stimulated by an allergen persisted long after the initial inflammation was resolved and responded to allergen challenge more vigorously than naïve ILC2s. Microarray analyses of the gene expression profiles of ILC2s isolated from naïve and allergen-treated mice showed that allergen-experienced ILC2s have a similar gene profile to that of memory T cells. To further study the process of “memory ILC2” generation, we analyzed the expression of cell surface molecules on ILC2s from untreated mice and those after intranasal IL-33 injections at various time points, which showed differential expression of CD27, CD103, CD41 and the IL-25 receptor (IL-25R). Memory ILC2s expressed higher level of IL-25R than naïve ILC2s, and intranasal injection of IL-25 stimulated memory, but not naïve, lung ILC2s. Intranasal IL-33 injections also increased ILC2 numbers in the lung draining mediastinal lymph node (mLN), peripheral blood, spleen and liver but not in the intestine or skin. Phenotype analysis of those ILC2s suggested that activated lung ILC2s migrated into mLN and the blood, and some settled in the liver. The results indicated that IL-25-responsive memory ILC2s are generated and persist in the lung as well as in mLN while the role of the latter in allergic lung inflammation induced by intranasal allergen challenge in sensitized mice is yet to be determined.
CD4+ T-cell cytokine production within airway mucosal DC-T-cell clusters drives the local recruitment of myeloid effector cells in response to house dust mite allergen.
Authors: Veres Tibor Z , Kopcsányi Tamás , van Panhuys Nicholas , Gerner Michael Y , Liu Zhiduo , Rantakari Pia , Dunkel Johannes , Miyasaka Masayuki , Jalkanen Sirpa , Germain Ronald N
Published: January 2023

Allergic asthma develops in the mucosal tissue of small bronchi. Upon allergen exposure, a TH2-type inflammatory response leads to airflow obstruction and wheezing. Recent studies provided a detailed understanding on the role of various cell types and mediators in the allergic response on the level of the whole organism or tissue. However, less well understood is the micro-anatomical organization of the key cellular players, dendritic cells (DCs) and CD4+ T cells within the airway mucosa and the mechanisms by which local T-DC interactions contribute to the ‘downstream’ inflammatory response (e.g. eosinophil recruitment). Here we utilized advanced in vivo dynamic and in situ static imaging to visualize CD4+ T-cell activation and effector function in the tracheal mucosa.

Upon mucosal sensitization to house dust mite (HDM) allergen, newly recruited CD4+ T cells formed discrete cellular clusters and showed prolonged interactions with DCs in an antigen-specific manner. While forming direct cell-cell contacts with MHC-II+ DCs, a small fraction of CD4+ T cells, but not innate lymphoid cells (ILCs), produced IL-13 or IL-17. As a result of local TH2 cytokine signaling, revealed by STAT-6 phosphorylation, eosinophils were locally recruited into these cellular clusters. Neutrophils also infiltrated these clusters in a T-cell dependent manner; however, their distribution was more diffuse. Our findings reveal the focal nature of antigen-driven allergic responses in the airways and define multiple steps with potential for interference with the progression of asthmatic pathology.

This work was supported in part by the Intramural Research Program of NIAID, NIH.
Pulmonary NK cells prevent allergic airway sensitization: Regulation by CB2 cannabinoid receptors
Authors: Jaffar Zeina , Ferrini Maria , Roberts Kevan
Published: January 2023

Allergic asthma is a chronic inflammatory disease involving the interaction between the innate and adaptive immune response. Endogenously generated cannabinoids, acting via CB receptors play important roles in both homeostatic and inflammatory processes. CB2-active compounds are known to impact innate immunity including responses by monocytes and NK cells. However, the contribution of pulmonary NK cells and CB2-active endocannabinoids to the innate events preceding sensitization of the airways to the common house dust mite (HDM) allergen is unclear. We investigated the significance of CB2 activation during allergen-triggered pulmonary inflammation and NK cell effector function. Allergic airway inflammation was induced in mice by sensitization via the airways by intranasal HDM instillation, and responses in wild type (WT) and CB2-deficient (CB2−/−) mice were compared. Mice lacking CB2 receptor exhibited elevated numbers of pulmonary NK cells yet were resistant to the induction of allergic inflammation and showed diminished airway hyperreactivity, pulmonary eosinophilia, TH2 cytokine production and mucus secretion after allergen inhalation. Cellular depletion and adoptive transfer studies were undertaken to dissect the mechanisms involved. Depletion of NK cells restored HDM responsiveness. Conversely, transfer of CB2−/− NK cells into WT mice suppressed the allergic airway inflammation and was associated with a reduction in monocyte-derived dendritic cells but elevated CX3CL1 release and recruitment of Ly6C−CX3CR1+ monocytes. These studies established a crucial role for CB2 activation in allergic lung disease via regulation of NK cell function and identified novel therapeutic targets for treatment of asthma.