bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Type I IFN signaling in induction of humoral response induced by viral vectored vaccines
Authors: Liu Fengliang , Niu Qingli , Liang Yuejin , Sun Jiaren , Hu Haitao
Published: January 2023

HIV vaccine candidates based on different viral vectors (adenovirus and poxvirus) have been tested in human efficacy trials but were associated with distinct vaccine outcomes. In particular, the vaccine regimen tested in RV144 trial involving poxvirus (ALVAC) vectored HIV vaccine provided a modest protection, which was associated with vaccine-induced HIV envelope-specific IgG antibody response. Our recent study examining host innate immunity to different viral vectors demonstrated that distinct from Ad5, the poxvirus vector can stimulate strong type-I interferon (IFN) response. In this study, we aimed to understand the impact of type I IFN signaling on generation of HIV vaccine-specific humoral immune response induced by poxvirus vectored vaccines. Here we used poxvirus vectored vaccine (MVA-HIV) that is capable of triggering type-I IFN response as a vaccination model. Prime-boost immunization of MVA-HIV in mice demonstrated that compared to wildtype mice, type I IFN receptor-deficient mice produced significantly lower levels of total and HIV gp120-specific IgG after vaccination. Consistent with the antibody response, type-I IFN signaling deficiency also resulted in lower levels of memory B cells as compared to those in WT mice, although the levels of germinal center B cells were comparable between WT and KO mice. Furthermore, our study also provided evidence that type-I IFN signaling may be involved in the generation of vaccine-specific CD4 T-cell response, including induction of HIV gp120-specific T follicular helper (Tfh) cells. Altogether, our data suggest that type I IFN signaling plays an important role in viral vector vaccination by promoting the generation of vaccine-specific humoral response and Tfh response.
Nmp4-deficiency protects mice from influenza A virus infection
Authors: Yang Shuangshuang , Adaway Michele , Sun Jie , Bidwell Joseph P. , Zhou Baohua
Published: January 2023

Severe influenza A virus infection typically triggers excessive and detrimental lung inflammation with massive cell infiltration and hyper-production of cytokines and chemokines. Discovering research models to alleviate lung damage without compromising viral control is critical. Here we identify that the lack of transcription factor-nuclear matrix protein 4 (Nmp4) protects mice from acute influenza virus infection by modulating the early stage inflammation. Upon infection, Nmp4-deficient mice lose only 5% body weight compared to 20% for the wild type (WT) mice. Furthermore, Nmp4-deficiency significantly reduces the recruitment of neutrophils and macrophages in the bronchoalveolar lavage fluid and lung parenchyma. Consistent with the fewer innate cells in the airways, influenza infected Nmp4-deficient mice have significantly decreased expression of cytokines and chemokines, such as IL-1β, TNF, IFN-γ, and MCP-1. However, Nmp4-deficient mice show similar viral control and adaptive immune responses to the WT mice with comparable virus specific CD8 T cells, CD4 T cells and anti-viral antibodies. Taken together, our data suggest that loss of Nmp4 protects mice from immune-driven lung damage without affecting viral clearance and may serve as a therapeutic target for severe influenza infection.
Nod2 is a negative regulator of arthritogenic T cell responses in SKG mice
Authors: Napier Ruth J. , Lee Ellen J. , Vance Emily E. , Snow Paige E. , Dawson Clare , Stenzel Peter , Davey Michael P. , Sakaguchi Shimon , Rosenzweig Holly L.
Published: January 2023

Nod2 is a pattern recognition receptor known for its role in antimicrobial immunity. Intriguingly, NOD2 appears to have a role outside of infection, as a single point mutation in this molecule leads to 100% incidence of an inflammatory arthritis called Blau Syndrome. Here we investigate the role of Nod2 in regulation of arthritogenic CD4+ T cells in SKG mice. SKG mice have a mutation in ZAP-70 that results in altered thymic selection and spontaneous production of arthritogenic T cells. Nod2−/− SKG mice have exacerbated arthritis and increased numbers of CD4+IL-17+ (Th17) cells that co-produce TNF and GM-CSF compared to SKG mice. Additionally, Nod2−/− SKG mice have augmented levels of IL-17A in their joint synovial fluid. Exacerbated disease in Nod2−/− SKG mice is independent of sex or SKG-mediated dysbiosis as shown by co-housing studies, but dependent on the skg-mutation. Naïve (non-arthritic) Nod2−/− SKG mice have similar numbers of conventional CD4+ T cells with unaltered Treg frequency or function compared to SKG mice. However, CD4+ T cells isolated from naïve Nod2−/− SKG mice produce increased levels of pro-inflammatory cytokines (IL-17, IFNγ) in response to TCR-dependent or -independent stimulus. Furthermore, transfer of CD4+ T cells isolated from naïve Nod2−/−SKG mice into lymphopenic (Nude) recipients results in worsened arthritis compared to an analogous transfer of SKG-CD4+ T cells. These data suggest a previously unappreciated function of Nod2 as a negative regulator of the proinflammatory capacity and pathogenesis of autoreactive CD4+ T cells in SKG mice. Understanding how Nod2 participates in immune tolerance mechanisms will contribute to our knowledge of the pathophysiology of autoimmunity and arthritis.
Reduction of autoantibody in SLE by probiotic exopolysaccharide-induced inhibitory dendritic cells
Authors: Kalinina Olga , Knight Katherine L.
Published: January 2023

Exopolysaccharide (EPS) secreted by a commensal bacterium, Bacillus subtilis, induces alternatively-activated, anti-inflammatory M2 macrophages that protect mice from T cell-dependent inflammatory disease induced by the enteric pathogen Citrobacter rodentium. We tested the activity of EPS on dendritic cells (DCs), and found that in vitro, EPS not only induced the expression of inhibitory molecules, PDL1 and PDL2, but also that these DCs inhibited alloreactive T cell activation and proliferation in mixed lymphocyte reactions (MLR). We also found that in vivo injection of EPS resulted in the generation of DCs expressing PD-L1 and PD-L2. To test if the EPS-induced DCs are inhibitory in vivo, we used a model that mimics Systemic Lupus Erythematosus (SLE) and tested if the level of autoantibody was decreased. We injected Bm12 cells into EPS-treated C57BL/6 mice to induce SLE-like symptoms, and found that the titer of autoantibodies was significantly decreased in the EPS-injected mice compared to control mice. We conclude that EPS can generate inhibitory DCs both in vitro and in vivo, and we suggest that these DCs have the potential to protect from SLE and other autoimmune diseases.
Arginine methylation drives inflammatory T cell responses and autoimmunity
Authors: Webb Lindsay M. , Savardekar Himanshu , Jablonski Kyle A. , Amici Stephanie A. , Li Linsen , Li Chenglong , Baiocchi Robert A. , Guerau-De-Arellano Mireia
Published: January 2023

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system that is the leading cause of disability in 2 million young adults aged 20–40 worldwide. Heightened pro-inflammatory T helper (Th) 1 and Th17 cell responses and deficient Th2 and regulatory T (Treg) cell responses drive myelin sheath damage and disability in MS. Thus, therapeutic strategies that modulate inflammatory T cell responses would be beneficial to the field. Epigenetic modifications are well known to provide control over cellular phenotype, and pan-methyltransferase inhibitors have been shown to suppress Experimental Autoimmune Encephalomyelitis (EAE), the mouse model of MS. However, the methylation enzyme responsible for this therapeutic effect remains unknown. Protein arginine methyltransferase 5 (PRMT5) is the only enzyme known to catalyze symmetric dimethylation (SDM) on arginine residues of histones, mediating transcriptional repression. Although PRMT5 is a known cancer driver, until recently, there was no known role for PRMT5 in T cells. We found that PRMT5 protein was up-regulated during re-activation of memory human Th1 cells, and that PRMT5-selective inhibitors suppressed inflammatory T cell proliferation. Further, we developed a conditional PRMT5 knockout mouse model in which PRMT5 knockout can be induced in CD4+ T cells. With this model, we genetically validated our findings that PRMT5 drives CD4+ T cell proliferation. Importantly, we translated our findings in vivo by showing that PRMT5 inhibitors suppressed established EAE disease severity. Overall, these data support a role for PRMT5 in pathogenic memory T cell responses and suggest its promise as a potential therapeutic target for autoimmune diseases such as MS.
Level of self-reactivity modulates functional potential of effector T cells in autoimmune diabetes.
Authors: Kong Yuelin , Sprouse Maran , Jing Yi , Bettini Matthew L. , Bettini Maria
Published: January 2023

T cell infiltration and accumulation in pancreatic islets during autoimmune diabetes is largely dependent on TCR specificity for beta cell antigens. However, it is not fully clear whether the level of self-reactivity is an important determinant of T cell function in autoimmunity. We observed that CD5 expression is proportional to the level of self-reactivity based on Nur77-GFP reporter of TCR signaling and insulin tetramer staining of islet infiltrating T cells. Using RNAseq profiling, TCR sequencing, and cell transfer diabetes model we show that CD5 segregates functionally and transcriptionally distinct effector T cells. Transfer of CD5hi islet infiltrating T cells resulted in accelerated diabetes development compared to CD5lo cells (8 weeks vs. 11 weeks post transfer), suggesting that both T cell populations are able to re-infiltrate the pancreas and cause beta cell destruction, but CD5hi T cells exhibit increased pathogenicity. RNAseq analysis showed an increase in expression of Lag3 and FR4 in CD5hi cells, consistent with their increased activation and subsequent induction of regulatory and tolerogenic pathways. However, cell intrinsic negative regulators did not suppress IFNg production in CD5hi T cells, which was significantly higher compared to CD5lo cells. Our findings show that the strength of TCR reactivity to beta cell antigens is an important determinant of T cell pathogenicity in autoimmune diabetes.
Rapamycin suppresses T cell responses in recoverin-induced autoimmune retinopathy and prevents disease progression in the eyes
Authors: Nikoopour Enayat , Lin Cheng-mao , Heckenlively John R. , Lundy Steven K.
Published: January 2023

Autoimmune retinopathy (AIR) causes acute and progressive vision loss in patients. We have established a murine model using the retinal autoantigen, recoverin, to better understand the immunopathology of AIR. Immunization of C57BL6 mice with recombinant mouse recoverin resulted in systemic recoverin-specific Th1 and Th17 cell responses and infiltration of CD4+ and CD8+ T lymphocytes, B lymphocytes, and CD11b+Ly6C+ inflammatory monocytes into the eyes. Treatment of mice with rapamycin at the time of immunization suppressed the induction of recoverin immunity and reduced inflammation in the eyes compared to the untreated group. Rapamycin dampened Th1 and Th17 T cell responses in draining lymph nodes and lowered serum antibody titers toward recoverin. There was a significant decrease in the number of CD4+ T cells and CD11b+Ly6C+ inflammatory monocytes infiltrating the eyes of rapamycin-treated mice. A higher number of CD103+CD8+ tissue resident memory T cells were present in the eyes of rapamycin treated mice. Treatment resulted in a significant decrease in the frequency of CD122+CD69−PD-1−CD8+ and the total number of CD44+CD62L+ memory T cells in lymphoid organs. While adoptive transfer of T cells from recoverin-immunized mice into naïve mice led to pathological changes in the eye, adoptive transfer of T cells from a rapamycin treated group did not result in eye pathology in the recipient. Treatment of mice with rapamycin at later time points after disease induction did not prevent disease, however, transfer of recoverin-specific T cells from those mice into recipient mice failed to induce disease. Our findings indicate rapamycin may be an effective drug for suppression of autoimmune retinopathy in some circumstances.
Identification of highly preserved gene co-expression module groups among multiple isolated CD4+ cellular sample datasets contribute to the elucidation of cell-type specific systemic lupus erythematosus bio-signatures in humans
Authors: Geraci Nicholas S. , Catalina Michelle , Heuer Sarah , Robl Robert , Lipsky Peter E. , Grammer Amrie C.
Published: January 2023

Gene expression profiles from three CD4+ T cell lupus datasets were assessed by weighted gene co-expression network analysis (WGCNA) to identify a consistent 65 gene module. Enrichr assessment of functional molecular pathways indicates that this module includes TCR signaling, DNA synthesis, proliferation and apoptosis. GAGE evaluation of this CD4+ T cell gene signature compared with SLE whole blood, PBMC, kidney, synovium, and skin revealed that this CD4+ T cell gene signature was consistently enriched in all datasets tested. Since the mechanisms underlying tissue pathology in SLE are not completely understood, gene expression profiles from lupus affected skin, synovium, and kidney were analyzed for commonalities. Notably, the majority of lupus tissue DE genes were detected in more than one tissue and 439 were differentially expressed in all tissues. Curated STRING-based interaction analysis of these 439 DE genes identified a number of functions and pathways that were commonly activated in all four tissues such as T cell signaling and Interferon Signaling Genes. Curated STRING-based interaction analysis of the up-regulated genes in at least three of the four tissues identified several additional cellular and molecular processes, including T cell adhesion, DNA damage response and cell proliferation. These results indicate that common cellular and molecular pathways involving T cells can be identified in all affected lupus tissues, implying that related processes might be involved in the pathology of multiple organs in this autoimmune disease.
Targeting transcription cofactor OCA-B specifically blocks pancreatic T cell infiltration, cytokine production and elevated glucose in the NOD mouse model of type-1 diabetes (T1D)
Authors: Tantin Dean R. , Kim Heejoo
Published: January 2023

Type 1 Diabetes (T1D) is an autoimmune disease in which the host immune system is misdirected towards antigens associated with beta cells of the endocrine pancreas. In CD4 T cells, OCA-B (Pou2af1/OBF-1/Bob.1) promotes memory T cell function via the maintenance of poised gene expression states at multiple direct immunomodulatory target genes (e.g. Il2, Il21, Ifng, and Il2ra). OCA-B ensures poised gene expression states by recruiting the histone lysine demethylase Jmjd1a/Kdm3a. OCA-B is not expressed in naïve CD4 T cells, but becomes stably expressed upon stable antigen exposure. Memory T cell phenotypes can promote autoimmunity (including T1D), even in cases of persistent self-antigens. Consistently, OCA-B levels are strongly elevated in central memory CD4 T cells, but are even more elevated in islet-reactive, pancreas-infiltrating CD4 T cells in T1D. GWAS studies associate human polymorphisms in binding sites for Oct1, the transcription factor with which OCA-B docks, in autoimmunity generally and T1D in particular. Using a rationally-designed peptide inhibitor of OCA-B downstream effector function, we observe robust improvement in glucose levels in NOD mice genetically pre-disposed to T1D. In contrast, control peptides have no effect. The therapeutic benefit is associated with reductions in pancreatic-infiltrating CD4 and CD8 T cell numbers and cytokine production. In contrast, T cell numbers in pancreatic lymph nodes (PLNs) are normal, suggesting that non-self-directed immune function is largely intact. Our results show that OCA-B promotes pro-inflammatory T cell phenotypes needed in T1D and that it can be targeted pharmacologically.
Autoreactive and immunization-induced follicular helper T cells have opposite glucose and glutamine requirements
Authors: Choi Seung-Chul , Titov Anton , Abboud Georges , Morel Laurence
Published: January 2023

Both glycolysis and mitochondrial oxidative metabolism are elevated in CD4+ T cells from lupus-prone mice, and metabolic inhibitors targeting both of these pathways normalized lupus T cell functions in vitro and reverted disease in mice. Here, we showed that inhibiting glucose metabolism with 2-deoxy-D-glucose (2DG) results in a drastic reduction of follicular helper T (TFH) cell frequency in lupus-prone B6.Sle1.Sle2.Sle3 (TC) mice. However, treatment of TC mice with 2DG had little effect on the production of antibodies or the expansion of TFH cells following immunization with a nominal antigen of T-cell-dependent (TD) humoral responses, and no effect on the frequency of influenza virus-specific TFH cells induced by immunization with PR8 influenza virus. Thus, TFH cells supporting the production of autoantibodies are different from TFH cells providing protective humoral immunity against pathogens. The specific metabolic signature from autoimmune TFH cell is significantly different compared with exogenous antigen-specific TFH cells. Further results obtained with gene expression analysis and treatment with metabolic inhibitors indicated that autoreactive TFH cells depend on glucose metabolism, while exogenous antigen-specific TFH cells are more glutamine-demanding. Overall, our results predict that targeting TFH cellular metabolism provides an effective and safe therapeutic approach for systemic autoimmunity by eliminating autoreactive TFH cells.
Stromal cell-derived factor-1 (CXCL12) activates integrins by direct binding to an allosteric ligand-binding site (site 2) of integrins without CXCR4.
Authors: Takada Yoshikazu , Fujita Masaaki , Davari Parastoo , Takada Yoko K.
Published: January 2023

Leukocyte arrest on the endothelial cell surface during leukocyte extravasation is induced by rapid integrin activation by chemokines. We recently reported that fractalkine induces integrin activation without its receptor CX3CR1 through binding to the allosteric site (site 2) of integrins. Peptides from site 2 bound to fractalkine and suppressed integrin activation by fractalkine. We hypothesized that this is not limited to membrane-bound fractalkine. We studied if stromal cell-derived factor 1 (SDF1), another chemokine that plays a critical role in leukocyte arrest, activates integrins through binding to site 2. We describe here that (1) SDF1 activated soluble integrin αvβ3 in cell-free conditions, suggesting that SDF1 can activate αvβ3 without CXCR4; (2) site 2 peptide bound to SDF1, suggesting that SDF1 binds to site 2; (3) SDF1 activated integrins αvβ3, α4β1, and α5β1 on CHO cells (CXCR4-negative) and site 2 peptide suppressed the activation; (4) A CXCR4 antagonist AMD3100 or pertussis toxin did not affect the site 2-mediated integrin activation by SDF1; (5) Cell-surface integrins were fully activated in 1 min (much faster than activation of soluble αvβ3) and the activation lasted at least for 1 h. We propose that the binding of SDF1 to cell-surface proteoglycan facilitates the allosteric activation process; (6) Mutations in the predicted site 2-binding site in SDF1 suppressed integrin activation. These results suggest that SDF1 (e.g., presented on proteoglycans) can rapidly activate integrins in an allosteric manner by binding to site 2 in the absence of CXCR4 or other GPCR. The allosteric integrin activation by SDF1 is a novel target for drug discovery.
Interleukin-33 modulates human natural killer cell responses
Authors: Ohayon David E. , Ali Ayad , Alarcon Pablo C. , Krishnamurthy Durga , Osterburg Andrew R. , Borchers Michael T. , Waggoner Stephen N.
Published: January 2023

Interleukin-33 (IL-33) promotes type 2 cytokine responses in CD4 T cells, granulocytes, and innate immune lymphocytes. Although type 1 (IL-12) and type 2 (IL-33) cytokines are classically thought to counterbalance one another, a combination of IL-12 and IL-33 paradoxically promotes profound expression of type 1 effector cytokines, interferon gamma (IFN-γ), tumor necrosis alpha (TNF-α), and granulocyte-macrophage colony-stimulating factor (GM-SCF), when administered to human natural killer (NK) cells. Mechanistically, IL-33 stimulates potent mitogen-activated protein kinase (MAPK) p38 in human NK cells, but has no impact on IL-12-induced signal transducer and activator of transcription 4 (STAT4) phosphorylation. Importantly, pharmacological inhibition of p38 MAPK significantly reduced IFN-γ and TNF-α release by IL-12 and IL-33 stimulated NK cells. Even in the absence of IL-12, IL-33 could synergize with other type 2 cytokines like IL-27 or IL-4 to trigger high expression levels of IFN-γ. The altered sensitivity of NK cells to type 1 and 2 cytokines in the presence of IL-33 may have important consequences in diseases associated with mixed cytokine milieus, including cases of severe asthma exacerbated by respiratory viral infection.
Defining IL-2 mediated regulation of Il9 activation using knock-in reporter mice
Authors: Kharwadkar Rakshin P. , Ulrich Benjamin Joseph , Kaplan Mark H.
Published: January 2023

T cells are critical regulators of inflammatory disease and immunity. CD4+ T helper cells develop into various subsets that are specialized in secretion of particular cytokines and mediate restricted types of inflammation. One of these subsets, IL-9 secreting Th9 cells, have proven to be effective in clearing helminthic infections and in tumor immunotherapy. Inappropriate Th9 activity has also been associated with colitis and atopic inflammation. While IL-4 and TGF-β are primary cytokines required for Th9 differentiation, other stimuli, including an IL-2/STAT5 pathway, are necessary for acute activation. Recent studies have reported a reduction in IL-9, when differentiated Th9 cells are stimulated in the absence of exogenous IL-2. This implies that IL-2 plays an important role in modulating IL-9 expression in Th9 cells, in a paracrine manner. Using two distinct IL-9 reporter mice, one with a reporter knocked-in to the first Il9 exon and the other with a downstream IRES-reporter, we noted that exon 1 disruption results in reduced allele expression. Specifically, the allele is no longer responsive to IL-2. This is correlated with diminished STAT5 binding near the promoter, suggesting novel effects of the first exon on cytokine responsiveness of the allele. In ongoing studies we will more precisely map STAT5 binding across the promoter and the 5-prime end of the Il9 gene to define how gene regulation and cytokine responsiveness might be exploited to control IL-9-dependent immune responses.
Signals that induce IL-10 production by peripheral regulatory T cells
Authors: Dean Emma C. , Moseley Carson E. , Hatton Robin D. , Pham Duy , Weaver Casey T.
Published: January 2023

Inflammatory bowel disease (IBD) is an immune-mediated disease of the intestinal tract caused by chronic inflammation that can be relapsing and remitting. This inflammation leads to flares that often require patients to be treated with life-long biologic therapy. Interleukin 10 (IL-10) is an immunosuppressive cytokine produced by effector T cells, especially regulatory T cells (Tregs), to limit inflammatory responses to both foreign and self-antigens. Although IL-10-producing peripheral Tregs (IL-10+ pTregs) are thought to play a mitigating role in murine models of colitis, signals that control transcriptional regulation of the Il10 locus are not well understood. Here, we have examined cytokine and non-cytokine signals that enhance the induction of IL-10+ pTregs in vitro and in vivo. We find that in vitro a mix of inflammatory cytokines acting in concert with IL-2 and on-going TGFβ signaling induce the production of Foxp3+IL-10+ pTregs from Foxp3+IL-10- precursors. Ongoing studies are directed at extending these findings to the in vivo induction of IL-10-producing pTreg cells, and examining the ability of these cells to curb intestinal inflammation driven by aberrant effector T cell responses against the intestinal microbiota. Ultimately, we hope that by identifying signals to amplify our target population, we can perform genomic analyses to better decipher what modulates the transcriptional landscape of the Il10 locus, and identify novel immune therapies for patients with IBD.
Aire is not essential for regulating autoimmune pathology in mice transgenic for human autoimmune-disease associated MHC class II genes HLA-DR2b and HLA-DR4.
Authors: Nalawade Saisha A. , Ji Niannian , Kraig Ellen , Forsthuber Thomas
Published: January 2023

The human MHC class II molecules HLA-DR2b (DRB1*1501) and HLA-DR4 (DRB1*0401) are strongly linked to increased susceptibility to autoimmune diseases, such as multiple sclerosis (MS) and rheumatoid arthritis (RA). However, their contribution to disease pathogenesis is not completely understood. Conceivably, autoimmune disease-associated HLA alleles could shape the repertoire of pathogenic T cells via central or peripheral tolerance. The ectopic expression of tissue-specific antigens promoted by the autoimmune regulator (AIRE) is thought to play a critical role in central tolerance. Aire knockout mice (Aire−/−) develop spontaneous autoimmune pathology characterized by multi-organ lymphocytic infiltrates. Aire deficiency in humans causes multi-organ autoimmune conditions observed in autoimmune polyendocrinopathy syndrome type 1 (APS I). We wanted to investigate whether lack of Aire expression promoted spontaneous MS- or RA-like autoimmune pathology in HLA-DR2b or HLA-DR4 transgenic (tg) mice. We observed that Aire-deficiency modestly enhanced experimental autoimmune encephalomyelitis (EAE) in HLA-DR tg mice, but did not lead to spontaneous neuroinflammation or arthritis. Our results suggest that Aire has a mild and non-essential effect in restraining the autoimmune response in the context of autoimmune disease-associated human HLA alleles.
The effect of Ebola Virus secreted glycoprotein on activated macrophages.
Authors: Bradley Jillian Helena , Shapiro Lindsey , Hitchcock Clara , Kulis Dakota , Needell Lauren , Henry Nathan , Gregg Randal K.
Published: January 2023

During Ebola virus (EBOV) infection, secreted glycoprotein (sGP) and viral protein 40 (VP40) are found in large quantities in the serum of infected animals. Studies suggest that sGP only provides passive immunity by serving as a decoy to anti-EBOV antibodies, and no role for soluble VP40 has been determined. Using an in vitro model incorporating treatment of THP-1 macrophages with recombinant EBOV sGP and recombinant VP40, we examined the impact of the viral proteins upon key macrophage functions.

Treatment with sGP inhibited macrophage production of pro-inflammatory cytokines (TNFα and IL-6) while the yield of anti-inflammatory cytokine (IL-10) remains unaltered. In contrast, VP40 seems to increase the production of pro-inflammatory cytokines. Furthermore, sGP affected only the function of activated macrophages and not the polarization. Phagocytic capacity was unaffected by sGP. Interestingly, the migratory ability of macrophages was diminished by sGP, and cell viability was increased, potentially due to a decrease in expression of CD11b, a vital macrophage integrin. Thus, the results suggests that while EBOV VP40 increases anti-viral immunity, EBOV sGP may operate to diminish functional contributions of non-infected macrophages for the purpose of increasing the potential viral dissemination between macrophages.

Previously macrophages were activated utilizing a TLR4 agonist, LPS. TLR2 agonist zymosan, TLR3 agonist HMW Poly (I:C), TLR7 agonist imiquimod, and TLR9 agonist Class C CpG were examined. Further directions include determining the mechanism behind the inhibition and the specific TLR pathway molecules that are affected by sGP treatment.
A new malaria killer: Fc receptor gamma chain and PLZF identify NK cell subsets that correlate with reduced Plasmodium falciparum parasitemia and increased antibody dependent cellular cytotoxicity against opsonized infected RBCs.
Authors: Hart Geoffrey T. , Tran Tuan , Theorell Jakob , Arora Gunjan , Crompton Peter , Bryceson Yenan , Long Eric O.
Published: January 2023

Plasmodium falciparum (P.f) infection is a major cause of morbidity and mortality world-wide where symptoms and death occur as a result of the blood stage of the P.f. life cycle. To date, there is no effective blood stage malarial vaccine. Natural Killer (NK) cells are key players in the control of hematopoietic cancers and viral infections, however their role in blood stage malaria is unknown. We undertook a comprehensive analysis of NK cell phenotype and function in a cohort of subjects from a malaria clinical study in Mali, Africa. Using an unbiased analysis of different NK cell subsets, we found three major findings: 1. Fc receptor gamma chain (−) or PLZF (−) NK cells correlate with reduced parasitemia 2. Fc receptor gamma chain (−) NK cells correlate with protection from malaria symptoms 3. Fc receptor gamma chain (−) NK cells are enhanced for antibody dependent cellular cytotoxicity (ADCC). Antibodies are known to be protective in malaria, however the mechanism of this protection is still being elucidated. We hypothesized Fc receptor gamma chain (−) NK cells may be clearing opsonized, malaria-infected RBCs in vivo via ADCC. We show that Fc receptor gamma chain (−) NK cells specifically release lytic granules in response to opsonized, infected RBCs. This work indicates the host may increase the number of Fc receptor gamma chain (−) NK cells to aide in clearance of infected RBCs through ADCC. Because the antigens on the surface of infected RBCs are different than the invading parasite, these results could improve the identification of effective targets for blood stage vaccines.
Meningeal inflammation drives long-term engraftment by monocytes that impair CNS immunity
Authors: Rua Rejane , Lee Jane , Silva Alex , Dragan Maric , Johnson Kory , McGavern Dorian B.
Published: January 2023

Tissue-resident macrophages have an embryonic origin and can be replenished by blood monocytes in some tissues during adulthood. Under steady-state conditions, infiltrating monocytes can share phenotypic and genotypic features with their embryonically-derived counterparts. However, little is known about the properties of monocytes/macrophages that establish tissue residency after an inflammatory challenge. The meninges of the central nervous system (CNS) are populated by a dense network of specialized macrophages that serve as immune sentinels. Following infection by lymphocytic choriomeningitis virus (LCMV), these resident macrophages become activated by innate inflammatory cytokines, acquire viral antigens, and are targeted by infiltrating cytotoxic T lymphocytes (CTL), which leads to their depletion. Innate cytokines and chemokines released by CTL also promote a massive recruitment of inflammatory monocytes from the periphery. Surprisingly, these infiltrating monocytes engraft the meningeal niche and remain in situ several months after viral clearance. This leads to significant phenotypic and functional changes in meningeal immunity – a defect that can be partially restored by blocking IFNγ signaling. Importantly, macrophages that establish residency in the meninges after an inflammatory event are deficient in bacterial and immunoregulatory sensors, which impedes their ability to detect pathogens and dampen subsequent meningeal immune responses. Collectively, these data indicate that monocytes can engraft the meninges after an inflammatory challenge and contribute to long-term alterations in CNS immunity.
Modulation of Xanthine metabolism ameliorates inflammation and accelerates diabetic wound healing
Authors: Joshi Amrita , Kimball Andrew , Schaller Matthew , denDekker Aaron , Burant Charles , Kunkel Steven L. , Gallagher Katherine
Published: January 2023

Wound healing in diabetic patients is impaired because of dysregulated innate immune response that results in a chronic inflammatory state. It has been shown by our group and others that metabolites can contribute to a state of chronic inflammation in diabetic tissues. In a murine model of Type-2 diabetes (diet-induced obese mice; DIO), we assessed role of uric acid in driving macrophage-mediated inflammation and delayed wound healing. Using liquid chromatography-mass spectrometry, we show that macrophages from DIO mice have reduced levels of xanthine, a precursor in the uric acid pathway. Xanthine is converted to uric acid via Xanthine oxidase/dehydrogenase (XOR) encoded by the Xdh gene. DIO macrophages express higher levels of Xdh and increased levels of uric acid, suggesting increased XOR activity. Further, exogenous addition of uric acid led to increased production of IL-1β by macrophages. Importantly, pharmacologic inhibition of XOR with Allopurinol resulted in reduced levels of IL-1β expression by macrophages. In order to examine this in vivo, DIO mice were treated with Allopurinol leading to significantly improved wound healing. In summary, our data suggest that the xanthine metabolic pathway can be targeted therapeutically to accelerate wound healing in diabetic patients.
Human NKG2D ligand regulation of Natural Killer cell function and its implications in Cancer and Inflammation
Authors: Dhar Payal , Wu Jennifer
Published: January 2023

NKG2D is an activating lymphocyte receptor which plays an essential role in stimulating immune responses against tumors. The importance of ligand-induced activation of NKG2D in controlling tumor growth has been well established in various animal models. Amongst diverse NKG2D ligands, MHC I chain related molecule MICA/B is expressed most prevalently in human cancers, but absent in rodents. Proteolytically shed-form of MICA/B (sMIC) has been shown to correlate with advanced cancer stages in patients. Previous studies in our lab using two humanized bi-transgenic mice, one expressing native ligand (TRAMP/MICB) and alternatively expressing shedding-resistant mutant (TRAMP/MICB.A2) revealed opposite roles of the two forms of MIC ligand in tumor immunity. Mice with high levels of sMIC exhibited augmented tumor progression, whereas mice expressing MICB.A2 in tumors enjoyed tumor-free survival; raising an intriguing question of why do these similar forms of MIC have different effects on tumor immunity. To address this, we investigated the signaling events and functional outcomes in NK cells upon stimulation by the two forms of ligands. Co-culture studies of NK cells with tumor cells expressing sMICB and MICB.A2 revealed elevated pro-inflammatory cytokine production by NK cells upon stimulation with sMICB. In contrast, NK cells stimulated with MICB.A2 displayed enhanced expression of cytotoxicity mediators and signaling molecules of cytotoxicity pathway. This suggests that sMICB may polarize the NKG2D signaling pathways with preferential activation of inflammatory cytokine pathways. Our data has uncovered a potential mechanism by which sMIC promotes tumor progression and endorses sMIC as a viable target for cancer immunotherapy.
Transient Receptor Potential Ankyrin 1 Mediates the Afferent Arm of the Inflammatory Reflex
Authors: Silverman Harold A. , Addorisio Meghan , Tsaava Tea , Stiegler Andrew , Kressel Adam M. , Chin Christopher A. , Gunasekaran Manojkumar , Pavlov Valentin A. , Chavan Sangeeta S. , Tracey Kevin J.
Published: January 2023

The vagus nerve plays an important role in maintaining an organism’s immune homeostasis through the inflammatory reflex. Inflammatory molecules including LPS, TNF and IL-1β can mediate electrophysiological changes in vagus nerve signaling. Reasoning that these signals are mediated though specific neuronal fibers that can be identified by molecular markers, we administered endotoxin to mice that received optovin, a molecule that interacts with TRPA1 and is light sensitive. Selective stimulation of vagus nerve TRPA1+ fibers significantly reduce serum TNF levels (sham: 920.9 ± 87.9 pg/ml vs. stimulated: 456.8 ± 69.4 pg/ml). This reduction is ablated by proximal vagotomy, indicating that the signals are afferent in nature. Prior work suggests that the afferent vagus nerve is required for IL-1β induced febrile responses and sickness behavior. Here we administered IL-1β to TRPA1 KO mice and observed that body temperature was maintained as compared to wildtype (peak temperature change; wildtype: −2.35 ± 0.15 °C vs. TRPA1 KO:0.18 ± 0.40 °C). When recording electrical activity from the VN, IL-1β induces a significant increase in VN activity in WT mice. However, no significant increase is observed in TRPA1 KOs. Cecal ligation and puncture in TRPA1 KOs caused significantly higher disease severity scores, percent weight loss, and mortality. These results identify TRPA1 as both a marker for fiber specific inflammatory signaling as well as a necessary component for regulating inflammatory homeostasis.
Deficiency in Soluble FasL Terminates Ocular Immune Privilege to Promote Elimination of Intraocular Tumors
Authors: McCullough Peter , Kissinger Emily , Rothstein Ann Marshak , McKenna Kyle C.
Published: January 2023

The eye is a site of immune privilege exemplified by the observation that immunogenic tumor cells, for example P815 mastocytomas, that are rejected when transplanted in the skin of Balb/C J mice, grow progressively forming intraocular tumors when placed in the anterior chamber (a.c.) of the eye. The mechanisms that promote and maintain ocular immune privilege are not completely understood. However, CD95/FasL has been shown to contribute to ocular immune privilege (Griffith TS et al. 1995. Science 270:1189) and is expressed as a membrane associated molecule that can be cleaved to generate a soluble form. To begin to evaluate whether membrane or soluble FasL was required for ocular immune privilege, P815 tumor growth was monitored in mice unable to generate soluble FasL (DCS mice) due to a point mutation introduced in the FasL cleavage site. Twenty-four days after tumor challenge in the a.c. of the eye, tumor burden, measured by weight of challenged eyes, was reduced 5-fold in DCS mice in comparison to wild-type mice (DCS: 99+/−34 mg vs. Balb C/J: 20+/−15mg) and this difference was statistically significant (p<0.0001) and reproducible. The weight of tumors in DCS mice was equivalent to control non tumor challenged eyes suggesting complete tumor elimination. These data suggest that soluble FasL is necessary to maintain ocular immune privilege and promote intraocular tumor growth.
Anti-tumor immunity generated as an artifact of tumor implantation determines the response to immunotherapy in murine models
Authors: Gough Michael J. , Zebertavage Lauren , Bambina Shelly , Kramer Gwen , Friedman David J. , Troesch Victoria , Blair Tiffany , Baird Jason R. , Alice Alejandro , Crittenden Marka R.
Published: January 2023

Cancer cell lines grown in vitro and implanted into mice are a centerpiece of preclinical modeling. However, tumor implantation is an in vivo vaccination event that can impact tumor growth. Checkpoint inhibitors targeting PD1 or CTLA4 interactions are effective at unleashing suppressed pre-existing immunity in murine models, but do not generate new immune responses.

We hypothesized that tumor control by checkpoint inhibitor immunotherapies was dependent on pre-existing immunity generated at tumor implantation.

Using cancer cells engineered to express model antigens, spontaneous tumor models with defined trackable antigens, or unmodified cancer cells, we demonstrate that tumor implantation results in initial T cell mediated immunity. As the tumors progress, these cells become undetectable in the periphery and accumulate in the tumor, but do not prevent tumor progression. While tumors respond to checkpoint inhibitors administered closely following implantation, delayed administration results in a loss of efficacy. This efficacy can be recovered by the addition of radiation therapy to checkpoint inhibitors, permitting cure of established murine tumors that are not controlled by either therapy alone. However, blocking the immune response at tumor implantation using a range of approaches results in a complete loss of efficacy of tumor control by all therapies based around checkpoint inhibitors. We demonstrate an approach to ‘silently’ establish tumors in mice to test therapies that can initiate de novo immunity to tumors.

These data have significant implications for current clinical attempts to use checkpoint inhibitors, which may not be a solution for patients without pre-existing immunity to their tumor.
Differential roles of tumoral and systemic IL-1α in the development of hepatocellular carcinoma
Authors: Liu Haiyan
Published: January 2023

Interleukin-1α (IL-1α) is a pro-inflammatory cytokine that has been shown to be up-regulated in many tumors. However, the role of IL-1α during tumor development is still not clear. IL-1α could be released by necrotic tumor cells passively to the tumor microenvironment or by other cells actively during inflammation. In the current study, we investigated the role of secreted IL-1α released in the tumor microenvironment or systemically in the tumor development using murine hepatocellular carcinoma (HCC) models. We constructed murine HCC hepa1-6 cells stably expressing secreted IL-1α and overexpression of secreted IL-1α had no significant effect on tumor cell proliferation and apoptosis in vitro. Tumor cells expressing secreted IL-1α were orthotopically implanted in the liver. Tumoral IL-1α significantly promoted HCC tumor development. Further studies showed that tumoral IL-1α inhibited T and NK cell activation, and attenuated the cytotoxic T lymphocyte (CTL) cytotoxic activity against tumor cells. Moreover, MDSCs were increased by tumoral IL-1α, and depletion of MDSCs could diminish the effect of the released IL-1α on tumor growth. On the contrary, systemic administration of IL-1α significantly inhibited the tumor development in the same murine HCC models. Systemic administration of IL-1α promoted T cell activation and decreased MDSCs in the tumor microenvironment. Further in vitro experiments showed that recombinant IL-1α could promote T cell activation and enhance CTL cytotoxicity. In conclusion, our studies demonstrated the differential functions of tumoral and systemic IL-1α in HCC development. Thus, our finding provides new insights of IL-1α functions in anti-tumor immunity with implications for tumor immunotherapy.
Inflammation induces GILT expression in human melanoma
Authors: Meador Lydia , Menon Hari , Cui Haiyan , Roe Denise J. , DiCaudo David J. , Hastings Karen Taraszka
Published: January 2023

T cell-mediated immunity can produce durable anti-melanoma responses resulting in improved survival of metastatic melanoma patients. Gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of multiple melanoma antigens to CD4+ T cells and facilitates cross-presentation on MHC class I for activation of CD8+ T cells. Our prior study found that GILT expression is increased in melanocytes of malignant melanoma specimens compared with benign nevi. To determine whether GILT expression is associated with inflammation, expression in halo nevi specimens was compared to nevi without lymphocytic infiltrate by immunohistochemistry. A halo nevus is a benign nevus with a lymphocytic infiltrate which leads to regression of the nevus. GILT, but not MHC class II, expression was increased in melanocytes of halo nevi compared to nevi without lymphocytic infiltrate. Analysis of a publicly available gene expression profiling cohort of 457 cutaneous melanoma specimens revealed that GILT expression was associated with IFN-γ, TNF-α, and IL-1β expression in human melanoma. In vitro exposure of human melanoma cell lines to IFN-γ or inflammatory cytokines, such as TNF-α, induced GILT expression in melanoma cell lines, which lacked GILT expression at baseline. Vemurafenib, a BRAF inhibitor used in the treatment of metastatic melanoma, enhanced IFN-γ-induced GILT and MHC class II expression in a melanoma cell line. Together these data demonstrate that inflammation, alone or in combination with a current therapeutic agent, induces high levels of GILT expression in human melanoma.
Upregulating IL-15 in the tumor microenvironment promotes anti-tumor responses
Authors: Carrero Rosa Maria Santana , Beceren-Braun Figen , Rivas Sarai C. , Anthony Scott M. , Schluns Kimberly S.
Published: January 2023

Previous studies have shown that IL-15 expression within tumors correlates with increased tumor infiltrating T cells, decreased metastasis, and increased patient survival; however, the regulation of IL-15 within the tumor microenvironment (TME) is unclear. Examining IL-15 expression is complicated by difficulties in detecting protein and its unique mechanisms of action involving transpresentation and production of soluble IL-15/IL-15Rα complexes (sIL-15c), which are upregulated by inflammatory signals. Since we have recently shown agonists of the Stimulator of Interferon Genes (STING) pathway are potent inducers of sIL-15c in vivo, we set out to determine if sIL-15c are produced in the TME and are regulated by STING signaling. We find, in analyses of mice implanted with various tumor cell lines, sIL-15c are present at high levels in the interstitial fluid of early tumors and decrease with tumor growth. Depending on tumor type, both the tumor and the stroma are sources of sIL-15c. In analyses of IL-15 reporter mice, IL-15-expressing cells are abundant in the TME with the most predominant cells being CD11b+Ly6Chi. As cell surface IL-15 is negligible, we propose that sIL-15c are the dominant form of IL-15 produced in the TME. Despite the abundance of these IL-15 expressing cells, the relative levels of sIL-15c are low in advanced tumors but can be upregulated by local STING activation. While treatment of tumors with STING agonists leads to tumor regression, optimal STING-mediated immunity and regression of distant secondary tumors required IL-15 expression. These findings reveal the ability of IL-15 expressed within the TME to be upregulated by inflammatory signals and contribute to enhanced anti-tumor responses.
Isolation and analysis of plasma-derived exosomes in patients with glioblastoma
Authors: Garcia Luz Milbeth Cumba , Peterson Timothy E. , Cepeda Mario A. , Leong Hon S. , Johnson Aaron J. , Parney Ian F.
Published: January 2023

Gliomas including glioblastoma (GBM) are the most common malignant brain tumors. Glioma extracellular vesicles (EVs), especially exosomes in plasma have biological effects such as mediating immunosuppression and contain signature tumor-specific cargo, therefore could serve as liquid biopsies and as a non-invasive biomarker. However, isolation of glioma plasma exosomes has not been optimized. The purpose of this study is to develop an optimized method for plasma-derived exosomes isolation and study the immunosuppressive capacity of these particles. Plasma exosomes from glioma patients and normal donors were isolated by brief centrifugation to remove cells/debris followed by one or two-step density gradient ultracentrifugation (DGU). EV size and concentration were determined by NanoSight analysis. Nanoscale Flow Cytometry analysis quantified and characterized the resulting exosome and microvesicle populations isolated by DGU. Protein cargo was screened by Western blot, protein array, and ELISA. One-step DGU efficiently isolated exosomes for NanoSight analysis. Isocitrate dehydrogenase (IDH) WT glioma patients’ plasma exosomes were smaller but higher concentration than normal donors. A second DGU efficiently concentrated exosomes for subsequent cargo analysis. Nanoscale Flow cytometry analysis confirmed that DGU allows isolation and purification of exosomes, not microvesicles. Exosome-specific markers and the immunosuppressive molecule programmed death-ligand 1 (PD-L1), were present in both glioma patient and normal donor exosomes. Screening cytokine and checkpoint molecule arrays suggested reduced pro-inflammatory molecules are found in GBM patients’ plasma exosomes compared to normal donors.