bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional and non-transcriptional functions of IRF3 in host defense
Authors: Chattopadhyay Saurabh , Subramanian Gayatri , Zhang Ying , Veleeparambil Manoj , Sen Ganes C
Published: January 2023

Innate immune response is the first line of host defense against microbial infection. Interferon Regulatory Factor 3 (IRF3), a critical transcription factor, is rapidly activated during virus infection to trigger numerous antiviral genes, including the interferons. Our studies have revealed that in addition to triggering these genes, IRF3 activates direct apoptosis of virus-infected cells by a newly discovered antiviral apoptotic pathway, RIPA (Chattopadhyay et al, Immunity 2016, EMBO J, 2010). In RIPA, IRF3 is differentially modified by linear polyubiquitination of two lysine residues. Moreover, a knock-in mouse strain, without the transcriptional activity of IRF3, can still mount antiviral response by its RIPA branch. Importantly, the use of pathway-specific mutants of IRF3 revealed that both transcriptional, and RIPA, branches contribute to the overall antiviral functions of IRF3. To investigate the contribution of the transcriptional branch of IRF3, we screened a shRNA library of the human ISGs. Our screen identified a small subset of novel IRF3-dependent genes, which exhibit antiviral functions in human and mouse cells. These newly-identified ISGs are protective against a wide range of clinically relevant human viruses, e.g. RSV, HPIV3, HSV-1, HCMV. In-depth investigation revealed that these ISGs regulate cellular autophagy pathway to control virus replication. The presentation will highlight how both pathways of IRF3 mount an optimum host response against viral as well as non-viral pathogenesis.
Distinct innate signaling and activation of dendritic cells during the neonatal period determines their functional response to respiratory syncytial virus infection
Authors: Lau-Kilby Annie W , Kehl Margaret , Malloy Allison MW
Published: January 2023

Dendritic cells (DCs) are among the first to respond to infection, providing the critical link between innate and adaptive immunity. Neonates often have weak innate immune responses that can limit adaptive immunity and contribute to increased susceptibility and more severe infection with intracellular pathogens, such as respiratory syncytial virus (RSV). We have previously shown differential migration and defective activation of neonatal murine respiratory CD103+ DCs, which is associated with diminished RSV-specific CD8 T cell responses in neonatal mice. In order to define the age-dependent regulation of respiratory DC function, we have developed a neonatal mouse model of RSV to track antigen presentation via infection with an ovalbumin-expressing RSV and an antibody against H2kb-OVA257–264. We found adequate transfer of RSV-derived OVA257–264 by neonatal CD103+ DCs to the dLN, suggesting poor CD8 T cell responses are not due to limited antigen presentation. However, neonatal CD103+ DCs expressing H2kb-OVA257–264 failed to upregulate CD80/86, which is critical to T cell stimulation. RNA-seq analysis of H2kb-OVA257–264+ CD103+ DCs implicated defective type I IFN signaling in neonates that may influence reduced activation. In support of this, while we find type I IFN to be diminished in the neonatal lung, treatment of neonates with IFN-α failed to restore the activation of neonatal H2kb-OVA257–264+ CD103+DCs.

Our data demonstrate a distinct developmental regulation of innate immunity in early life and identify type I IFN signaling as contributing to defective DC activation during RSV infection in neonates. Defining age-dependent mechanisms controlling immunity may lead to intelligently designed vaccines for this age group.
Aging of B-1a Cell Derived Natural Antibodies
Authors: Holodick Nichol E , Rothstein Thomas L.
Published: January 2023

Streptococcus pneumoniae is the most common cause of pneumonia, which claims the lives of people over the age of 65 seven times more frequently than those aged 5–49. B-1a cells provide immediate and essential protection from S. pneumoniae through production of natural immunoglobulin, which is germline-like due to minimal insertion of N-region additions. We have previously demonstrated B-1a derived phosphorylcholine (PC)- specific and total IgM moves away from the germline with age as a result of selection. Consequently, the protective capacity of natural serum IgM against S. pneumoniae infection is significantly decreased in aged mice. Anti-phosphatidylcholine (PtC) antibodies, like anti-PC antibodies, contain few N-additions, and have been shown to be essential in protection from bacterial sepsis. Herein, we demonstrate the number of N-additions in PtC-specific B-1a cell antibodies do not change with age. Furthermore, the average percent of B-1a cells binding PtC-liposomes (13–15%) did not change in aged mice. Therefore, in aged mice the majority of natural IgM derives from non-PtC binding B-1a cells producing antibody that has moved away from the germline. In direct contrast, the PtC-binding B-1a cell population preserves its germline status. These results suggest a difference in selection pressures and/or self-renewal upon B cells expressing different specificities. Elucidating the role of different B-1a cell progenitors and cues influencing B-1a cell circulation will be interesting and necessary lines of investigation to further understand how diversification and selection affect the maintenance of B-1a cells and the efficacy of the natural IgM they produce with increasing age.
A Mouse Model of Acute Ethanol and Burkholderia Infection
Authors: Monroy Fernando Porfirio , Jimenez Victor , Settles Erik , Currie Bart , Keim Paul S
Published: January 2023

Alcohol consumption both, acute and chronic has well-documented effects on the immune system. Melioidosis is an infection caused by Burkholderia pseudomallei of public health importance in Southeast Asia and Northern Australia. Alcohol consumption has been described as risk factor associated with B. pseudomallei infections in humans. Evidence supports the importance of alcohol consumption as a risk factor for development of meliodosis, but few experimental studies have focused on ethanol effects on Burkholderia spp. infection. In these studies, we propose the use of the closely related B. thailandensis E264 as a useful BSL-1 model to study the effects of alcohol on host’s immune responses. Female C57BL/6 were administered ethanol (4 g/kg) intraperitoneally 30 min before intra-nasal infection with 1 × 105 B. thailandensis CFU. Animals were euthanized at 24 and 72 hr post-infection (PI), blood and tissues were collected to determine bacterial dissemination. Our results at 24 hr showed bacterial dissemination to the lung, liver, and spleen in the alcohol group. Spleen had the largest number of bacteria present. At 72 hr PI, bacteria were found only in the lungs and the brains of mice administered alcohol. Mice in the alcohol group had significantly larger spleens at both time points. Furthermore, serum and tissue cytokine concentrations (TNF-α, GM-CSF, IL-10) were decreased by alcohol. These results provide insight into the effects of a culturally prevalent meliodosis risk factor, acute alcohol consumption as a predisposing factor for melioidosis. Future work will determine mechanisms of alcohol on bacterial dissemination and how this translates to B. pseudomallei and melioidosis rates of infection.
Antibiotic treatment causes a reduction in antigen-specific T cell memory and increased susceptibility to secondary infection
Authors: Benoun Joseph M , Labuda Jasmine C , Fogassy Zachary N , Pham Oanh , Pham Quynh-Mai , Puddington Lynn , McSorley Stephen J
Published: January 2023

Salmonella enterica serovar Typhi causes recurrent and relapsing infection in antibiotic-treated individuals, suggesting that rapid bacterial clearance hinders the development of adaptive immunity. We have developed an antibiotic-treatment model in mice to examine this issue. This model demonstrates poor secondary protection after antibiotic treatment and allows the use of antigen-specific reagents to examine this issue in detail. Infection with Salmonella Typhimurium (BRD509-2W1S) caused expansion of Salmonella-specific CD4 T cells that were detected in peripheral blood, lymphoid tissues, and various non-lymphoid tissues. In contrast, antibiotic-treated mice had lower CD4 clonal expansion that persisted for months after infection. This reduced clonal frequency of Salmonella-specific CD4 T cells correlated with diminished protective immunity to secondary infection. In order to examine the protective contribution of circulating and non-circulating CD4 T cells, parabiosis experiments were performed whereby mice previously infected with BRD509-2W1S were paired with naïve mice for 30 days. Previously infected mice were more capable of controlling secondary infection compared to naïve parabionts or naïve control mice, however naïve parabionts were partially protected, indicating the requirement of both tissue resident and circulating cellular populations. Current experiments are underway to restore full protection during antibiotic intervention. Greater understanding of how antibiotics hinder CD4 memory development may allow for therapeutics to boost protective immunity to secondary or relapsing Salmonella infections. (Funding: NIH 5P01AI056172-09)
Type-1 interferon induced cross-regulation of IL-10 function: a novel mechanism in type 1 diabetes development
Authors: Lozano Marcos Iglesias , Arun Anirudh , Lam Brandon , Lee W.P. Andrew , Brandacher Gerald , Raimondi Giorgio
Published: January 2023

Type 1 Diabetes (T1D) is caused by T cell-mediated destruction of insulin-producing beta cells of the pancreas. The “fertile field” hypothesis of T1D proposes that in a genetically predisposed individual, an inflamed environment prompts the organ towards autoimmunity. In this study, we tested whether chronic and localized accumulation of type I interferons (TI-IFNs) creates a “fertile field” that releases diabetogenic T cells from immuneregulation. In vitro incubation of mouse T cells with IFN-beta renders memory and regulatory T cells with a dramatic defect in the production of phospho-STAT3 in response to the regulatory cytokine IL-10 (but not to IL-6). This resulted in prevention of upregulation of IL-10 induced genes. Altered signaling was NOT caused by reduction of IL-10 receptor expression, nor induction of Suppressor of Cytokine Signaling (SOCS) 1 or 3. Instead, microarray analysis suggested a novel role for the transcription factor STAT1 in dampening IL-10 signaling. IFNbeta induces STAT1 expression causing a reversal of the intracellular STAT1/STAT3 protein ratio that favors a competitive role of STAT1. In agreement, STAT1-KO T cells exposed to IFN-beta did not show any defect in IL-10 signaling. In diabetes-prone NOD mice we discovered a persistent reduction of IL-10 signaling in Tmem and Treg restricted to pancreatic and mesenteric lymph nodes (correlating with the reported local accumulation of TI-IFNs). Treatment of these mice with an IFNRA blocking antibody (that delays the onset and reduces the incidence of T1D) showed a clear restoration of IL-10 signaling.

Overall, these data reveal a novel immune defect that, if properly targeted could enhance the efficacy of immunomodulatory strategies to control T1D.
Critical role for OX40 signaling in Thymic and Peripheral Treg Expansion
Authors: Kumar Prabhakaran , Bhattacharya Palash , Epstein Alan L , Prabhakar Bellur S
Published: January 2023

Regulatory T-cells (Treg) play a pivotal role in suppressing self-reactive effector T-cells (Teff) and help maintain the critical balance between self-tolerance and autoimmunity. Restoration of homeostatic balance between Teff/Treg cells has been shown to ameliorate many autoimmune diseases including type-1 diabetes. Recently, we have reported a novel strategy for the selective expansion of Tregs using OX40L (a TNF-superfamily ligand) and Jagged (JAG)-1 (a Notch family ligand) treatment independent of canonical TCR-signaling, but mediated through specific interaction between OX40L-OX40 and JAG1-Notch3 preferentially expressed on Tregs. OX40L-JAG1 treatment of NOD mice with soluble OX40L and JAG1 increased proliferation of peripheral Tregs and delayed the onset of diabetes. In the present study, we show that OX40L treatment significantly increased Tregs in the thymus by inducing proliferation of thymic Tregs independent of TCR-stimulation upon addition of exogenous IL-2. OX40−/− mice had reduced numbers of thymic Tregs compared to wild type (WT) controls. The reduction in thymic Treg numbers likely due to impaired ability of proliferation in response to OX40L. However, we did not observe any change in IL-2 sensitivity of thymocytes in OX40−/− mice. Mechanistic studies suggested that OX40L-IL-2 induced thymic Treg proliferation was mediated through NF-kB and AKT-mTOR-S6 signaling. Further, phenotypic characterization studies revealed a significant decrease in TIGIT+Treg population, often associated with Treg activation, in both thymus and spleen of OX40−/− mice compared to WT controls. Taken together, these results suggest a critical role for OX40L/OX40 signaling in the development as well as function of Tregs.
Treg mediated immune system homeostasis requires NKAP association with HDAC3
Authors: Dash Barsha , Arocha Sinibaldo Romero , Chung Ji Young , Shapiro Virginia S.
Published: January 2023

NKAP is a transcriptional regulator essential for conventional T cell maturation. It does not have any previously defined DNA binding domains and is thought to interact with HDAC3 to exert its functions. The deletion of NKAP at the double positive stage (DP) in T cell development using the CD4-cre NKAP conditional knockout (cKO) mouse model renders thymocytes unable to incorporate α2,8-linked sialic acids and upregulate the complement inhibitory protein CD55, leading to rapid elimination of RTEs by complement. Here, we interrogate the importance of NKAP in Tregs. To address this, we created the Foxp3-YFP-cre NKAP cKO mouse model to delete NKAP only in the Treg lineage. Loss of NKAP in Tregs results in their disappearance, culminating in systemic autoimmunity, similar to the “scurfy” phenotype of Foxp3 mutant mice. Mice with Tregs lacking NKAP have increased conventional T cell proliferation, lymphocytic infiltration into various organs, and crusty skin on tail and ears, which necessitate euthanasia by three weeks. Preliminary results indicate that the disappearance of Tregs is a result of increased complement deposition. Transgenic expression of wildtype NKAP in a cre-inducible manner in cKO mice ameliorates the lymphoproliferative disease by restoring normal Treg numbers, and the mice are healthy. Interestingly, transgenic expression of a mutant version of NKAP that has lost its ability to bind HDAC3 is unable to prevent autoimmunity. Previous studies have demonstrated that HDAC3 is important for the generation and function of Tregs. Our results indicate that NKAP’s binding with HDAC3 is important for its role in Treg mediated immune system homeostasis.
Indole-3-carbinol ameliorates murine colitis symptoms through alterations in gut microbial composition and metabolomic pathways, particularly through decreasing disease-associated Bacteroides acidifaciens species.
Authors: Busbee Philip Brandon , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

Colitis is an autoimmune disease characterized by acute or chronic inflammation of the large intestine. Currently there is no cure for patients suffering from colitis, and most treatments involve the use of immunosuppressive drugs that can have adverse side-effects or increased toxicity. In the current study, we show that indole-3-carbinol (I3C), a naturally-occurring plant product found in a number of cruciferous vegetables, was able to ameliorates symptoms in two well-established murine colitis models. In particular, I3C was able to prevent weight loss, reverse colon shortening, as well as reduce accumulation of disease-associated infiltrating immune cells and inflammatory biomarkers, such as serum amyloid A. We performed 16S rRNA metagenomic sequencing to investigate alterations in the gut microbiome after induction of colitis by TNBS and treatment with I3C. 16S analysis of cecal flushes and validation by PCR revealed that TNBS-induced colitis leads to a significant increase in the species Bacteroides acidifaciens, whereas colitis mice treated with I3C had decreased levels of this species, comparative to naïve controls. In addition, I3C was able to modulate gut microbial metabolites by way of altering short chain fatty acid (SFCA) production during disease induction. Lastly, transfer of disease and treated fecal material in antibiotic-treated disease mice confirmed I3C-mediated alterations in the gut microbial environment contributed to the ability of this natural compound to alleviate deleterious effects attributed to colitis. Collectively, these data suggest that I3C is able to ameliorate colitis by preventing pathogenic gut microbial dysbiosis and restoring gut microbiome composition to a more homeostatic state.
Persistence of gut microbiota may require repeated colonizations
Authors: Basu Srijani , Rao Aashima , Oswal Neelam , Bal Vineeta , Rath Satyajit , George Anna
Published: January 2023

Reports indicate that, variously, 10–50% of bacteria in the gut are coated with IgA and that bacterial diversity correlates with IgA amounts. In support of these observations, it was recently shown that there is an overall reduction in bacterial diversity in Rag1−/− mice. In extension to these findings, we report that taxa that are severely depleted in Rag1−/− mice include Proteobacteria and SFB, which are IgA-coated in wild-type (WT) mice. We reasoned that IgA might be required for these taxa to take up residence in the gut. Further, since germ-free SCID mice can be stably colonized with SFB, we explored the possibility that retention of SFBs may require continuous exposure to the organisms. We tested this by co-housing Rag1−/− mice with WT mice and found that by day 5, Rag1−/− mice had acquired both Proteobacteria and SFB. In addition, when F2 littermates obtained from WT x Rag1−/− crosses were compared for microbial diversity, both were present in the Rag1−/− pups. These data raise the possibility that mice are being colonized continuously. We also report that members of Proteobacteria separate equally into IgA-bound and IgA-unbound fraction, raising the possibility that the IgA-unbound fraction may represent new colonizers. Finally, in agreement with earlier reports, which showed that transfer of IgA-coated bacteria increases the susceptibility to DSS-colitis in germ-free mice, we find that Rag1−/− mice, which lack coated bacteria, are more resistant to DSS-colitis.
Evaluating the Therapeutic Potential of Transplantation of Neural Precursor Cells for Treating the Autoimmune Disease Multiple Sclerosis
Authors: McIntyre Laura Lynn , Plaisted Warren , Coleman Ronald , Loring Jeanne , Lane Thomas , Walsh Craig M
Published: January 2023

Multiple Sclerosis is a chronic, autoimmune disease for which there is currently no cure. Previous studies suggest transplantation of neural precursor cells (NPCs) is a promising therapeutic strategy to treat neurological disorders. Using two mouse models of MS, experimental autoimmune encephalomyelitis (EAE) and murine hepatitis virus (MHV), we have observed remyelination and decreased neuroinflammation when mice received an intra-spinal transplant of syngeneic mouse GFP-NPCs. This recovery was due to replacement of damaged cells and had little effect upon immune responses. In contrast, transplantation of xenogeneic human embryonic stem cell (hESC) derived NPCs (hNPCs) or induced pluripotent stem cell (iPSC) derived NPCs (hiNPCs) modulates the host immune response. Mice receiving intra-spinal transplants of human NPCs displayed less demyelination, decreased neuroinflammation, and an increase in T regulatory cells (Tregs). Recovery was not a result of cell replacement by hNPCs because transplanted cells underwent rapid xenograft rejection. Importantly, ablation of Tregs abrogated clinical and histopathological improvement mediated by hNPC transplantation. Furthermore, hNPCs promote the induction of naïve T cells into Tregs in co-cultures in vitro. However, the mechanisms and factors that propagate Treg induction and function resulting in subsequent endogenous remyelination remain undiscovered. We hypothesize rejection of NPCs plays a role in generation of Tregs in vitro and in vivo. Future experiments will focus on elucidating the factors from hNPCs that are responsible for Treg differentiation and evaluate the ability of syngeneic, allogeneic, and xenogeneic NPCs to generate Tregs.
CGEN-15001: a Novel B7-like regulator of immune homeostasis and inducer of Ag-specific tolerance
Authors: Hecht Iris , Rotman Galit , Chiang Ming-Yi , Neria Eyal , Miller Stephen D , Podojil Joseph R
Published: January 2023

B7 proteins play critical immunomodulatory roles and provide attractive targets for development of novel therapies for cancer and autoimmunity, both of which involve improper immune tolerance. A major medical need in autoimmunity is restoration of immune tolerance to self-antigens.

CGEN-15001, an Fc fusion protein of a novel B7-like protein that we identified, regulates immune homeostasis by inhibiting Th1/Th17 responses while enhancing Th2 and anti-inflammatory cytokines such as IL-10, and promoting iTreg differentiation. This was observed in vitro using murine cells and in PBMCs of healthy donors and MS patients, as well as in vivo in R-EAE model and in HY transplantation model. Short-term administration of CGEN-15001 in R-EAE model of MS and in NOD model of T1D resulted in durable therapeutic effects which lasted long after cessation of treatment, suggestive of restoration of immune tolerance. Treg blockade with anti-IL-10 or anti-TGFβ, and transient Treg neutralization with anti-CD25 abolished these therapeutic effects. Importantly, transferring CD4+ T cells from CGEN-15001-treated mice to naïve recipients protected them from R-EAE induction in Ag-specific manner, demonstrating induction of Ag-specific tolerance. In contrast, tolerance transfer was not achieved following CTLA4-Ig treatment in spite of the remission it induced in the donor mice.

In summary, CGEN-15001 has a unique mode of action, combining immunomodulation and regulation of immune homeostasis, as well as re-establishment of Ag-specific tolerance via enhancing Treg differentiation, leading to durable disease amelioration. These findings support the potential of CGEN-15001 to provide a promising therapeutic approach across autoimmune diseases.
Human gut-derived commensal Prevotella histicola suppress experimental autoimmune encephalomyelitis in humanized mice
Authors: Shahi Shailesh K , Freedman Samantha , Luckey David , Karau Melissa , Marietta Eric , Choung Rok-Seon , Gibson-Corley Katherine , Patel Robin , Rodriguez Moses , David Chella S , Taneja Veena , Murray Joseph A , Mangalam Ashutosh K
Published: January 2023

Our recent multiple sclerosis (MS) microbiome study indicates an important role of gut microbiota in pathogenesis of MS, an autoimmune disease of central nervous system. Gut microbiota help in maintaining immune-homeostasis by regulating balance between CD4+CD25+FoxP3+ regulatory T cells (Tregs) and pro-inflammatory Th1/Th17 cells. A shift of balance towards Th1/Th17 cells leading to inflammation and demyelination in central nervous system (CNS), is suggested to be responsible of disease initiation and/or relapses in MS. Therefore, gut commensals capable of restoring the microbiome to a healthy state could provide novel therapeutic options for treating autoimmune diseases including MS. Here, we report identification of human gut-derived commensal bacteria, Prevotella histicola (P. histicola), which can suppress an autoimmune disease in HLA class-II transgenic model of experimental autoimmune encephalomyelitis (EAE), an experimental model of MS. P. histicola suppresses disease through modulation of systemic immune response. P. histicola challenge led to a decrease in pro-inflammatory Th1 (IFNγ) and Th17 (IL17) cytokines, and increase in the frequencies of CD4+FoxP3+ Tregs, tolerogenic dendritic cells and suppressive macrophages. We also observed that mice with EAE had a altered gut microbiota compared to naïve mice and treatment with P. histicola restored gut flora to a normal state which further support an important role of gut microbiota in EAE/MS. Our study provides compelling evidence that administration of gut commensals may regulate a systemic immune response and have a possible role in the treatment of autoimmune diseases.
A unique tolerizing dendritic cell phenotype induced by the synthetic triterpenoid CDDO-DFPA is protective against EAE
Authors: Wei Hsi-Ju , Pareek Tej K , Liu Qi , Letterio John J.
Published: January 2023

Dendritic cells (DCs) have emerged as relevant targets for the treatment of multiple sclerosis (MS) and other autoimmune disorders. Strategies that exploit their tolerogenic potential are now under clinical investigation. However, the essential pathways and optimal methods for the induction of tolerogenic DCs (TolDCs) remain intense areas of research. Triterpenoids are a class of small molecules with potent immunomodulatory activity linked to activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) target genes, and that suppress the manifestations of experimental autoimmune encephalomyelitis (EAE). Here we reveal DCs as a principal target of the immune modulating activity of triterpenoids in the context of EAE. Exposure of bone marrow-derived murine DCs to the triterpenoid 2-cyano-3, 12-dioxooleana-1, 9(11)-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA) results in the induction of heme oxygenase-1 (HO-1), transforming growth factor-beta (TGF-β) and interleukin-10 (IL-10), as well as the repression of NF-kB, endothelin-1 (EDN-1) and of the pro-inflammatory cytokines IL-6, IL-12 and TNF-α. CDDO-DFPA exposed DCs retained expression of cell surface ligands and their capacity for antigen uptake but were impaired in their induction of Th1 and Th17 cells in vitro and in vivo. TGF-β was identified as the factor mediating suppression of T cell proliferation by CDDO-DFPA pretreated DCs in vitro, and MOG (35–55) pulsed CDDO-DFPA-pretreated DCs failed to passively induce EAE in vivo. These findings demonstrate the potential therapeutic utility of CDDO-DFPA in the treatment and prevention of autoimmune disorders, and more broadly the capacity of triterpenoids to induce tolerance via modulation of the DC phenotype.
CpG type-A induction of an early protective environment in experimental multiple sclerosis
Authors: Manni Giorgia , Gargaro Marco , Crooks James , Pirro Matteo , Scalisi Giulia , Turco Antonella , Romani Rita , Matino Davide , Rostami Abdolmohamad , Puccetti Paolo , Gran Bruno , Fallarino Francesca
Published: January 2023

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory, demyelinating disease of the CNS that mimics human multiple sclerosis (MS), and it is thought to be driven by Th1 and Th17 myelin-reactive cells. Although adaptive immununity is clearly pivotal in the pathogenesis of EAE – with an essential role of CD4+ T cells – little is known of early, innate responses in this experimental setting. CpG-rich oligodeoxynucleotides (ODNs), typically found in microbial genomes, are potent activators of TLR9 in plasmacytoid dendritic cells (pDCs). In this study, we compared the effects of two types of CpG, namely, type A and type B, on EAE. We found that treatment with CpG type-A ODN (CpG-A) – known to induce high amounts of IFN-α in pDCs – significantly reduced disease severity in EAE, relative to controls (12.63 ± 1.86 vs. 23.49 ± 1.46, respectively; p = 0.001). Treatment also delayed onset of neurological deficits and reduced spinal cord demyelination, while increasing the percentage of splenic regulatory (Foxp3+CD4+) T cells. CpG-A likewise reduced the levels of IL-17 and IFN-γ in the CNS. Mechanistic insight into those events showed that CpG-A promoted a regulatory phenotype in pDCs. Moreover, adoptive transfer of pDCs isolated from CpG-A–treated mice inhibited CNS inflammation and induced disease remission in acute-phase EAE. Our data thus identify a link between TLR9 activation by specific ligands and the induction of tolerance via innate immunity mechanisms.
Selective inhibition of matrix metalloproteinase-9 in CD4+ T-cells reduces clinical severity in a murine model of Multiple Sclerosis.
Authors: Onwuha-Ekpete Lillian C. , Tokmina-Roszyk Dorota , Fields Gregg B.
Published: January 2023

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that mediate the degradation of extracellular matrix components. Their functions are essential for normal physiological processes such as leukocyte trafficking, but their dysregulation is associated with various pathologies including Multiple Sclerosis (MS). During MS, CD4+ T-cells activated in the periphery penetrate the blood-brain barrier (BBB), recruit immune cells, initiate destruction of the myelin sheath, and cause axonal loss. Experimental Autoimmune Encephalomyelitis (EAE) is a well-established murine model of MS. In EAE, MMP-9 is required for penetration of the BBB and generation of auto-antigens. Recent studies have demonstrated that MMP-9 contributes to normal intracellular function of various cell types including antigen activated T-cells; however, the intracellular role of MMP-9 in immune cell activation during EAE pathogenesis is not known. In our studies, we demonstrate that the MMP-9 is important for homeostatic maintenance as well as a robust antigenic stimulation. Upon activation, CD4+ T-cells treated with an MMP-9 inhibitor demonstrate a reduced ability to enter cell cycle, proliferate, and produce cytokines. In addition, RNA-seq analysis highlight key proteins associated with the T-cell receptor signaling pathway that are impacted. Furthermore, treatment of EAE mice resulted in reduced clinical severity. This reduction is associated with a treatment regimen that starts on Day 7 as opposed to Day 0, suggesting that CD4+ T cells are more sensitive to the impact of MMP-9 inhibition compared to other immune cells. These results emphasize the importance of MMP-9 as a therapeutic target in CD4+ T-cell mediated autoimmune diseases.
A dynamic approach for treating autoimmune disease.
Authors: Keeler Geoffrey D. , Silverberg Emily L , Hoffman Brad E
Published: January 2023

Cell-based immunotherapies have the potential to reverse autoimmune disease by inducing or restoring immune-tolerance. Extensive preclinical studies have demonstrated that regulatory T cell (Tregs) play a key role in both induction and maintenance of tolerance. Several strategies aimed at harvesting and expanding autologous Tregs and then re-infusing them into patients have been investigated. Unfortunately, development of a large scale in vitro method has not been achieved, especially for human cells. On the other hand, a clinically relevant in vivo approach for inducing Ag-specific tolerance has successfully been demonstrated using ectopic expression of an antigen in the liver. Here, we have developed a long-lasting immunotherapy that induces functionally suppressive, Ag-specific Tregs in the murine model of multiple sclerosis using a liver directed adeno-associated virus (AAV) vector expressing myelin oligodendrocyte glycoprotein (MOG). Modulation of EAE disease in C57BL/6 is typically limited to a single immunodominant epitope. By engineering the AAV vector to express full-length protein (MOG1-247), we have created an immunotherapy that is capable of dynamically inducing Ag-specific tolerance against multiple immunogenic epitopes (e.g MOG35-55, MOG119-132). Mice received a single peripheral injection of AAV.MOG vector, or control. EAE was induced between 2 weeks and 8 months later using various MOG epitopes emulsified in adjuvant. Within 14-days, control mice developed significant neurological deficits, whereas none of the treated mice exhibited any symptoms. This work completely abrogates the deficiencies associated with ex vivo expanded Tregs providing a new paradigm for treating autoimmune diseases.
IL-23 induces regulatory T cell plasticity with implications for inflammatory skin diseases.
Authors: Kannan Arun Kumar , Su Zhi , Gauvin Donna M , Lippert Stephanie E , Marie Honore P , McGaraughty Steve P , Kort Michael E , Scott Victoria E , Gauld Stephen B
Published: January 2023

Foxp3+ regulatory T cells (Tregs) are a major fraction of T cells in the skin. At steady state, Treg cells make little to no inflammatory cytokines and control against inflammation. However, under certain autoimmune inflammatory settings (including psoriasis) Treg cells have been shown to become dysfunctional, making pro-inflammatory cytokines and contributing to disease pathogenesis. To better understand the role of Treg cells in psoriasis we analyzed the homeostasis and function of these cells in a mouse model of psoriasiform dermatitis (IL-23 mediated skin inflammation). We find that IL-23 induced inflammation drove both proliferation and increased accumulation of Foxp3+T cells in the skin. Furthermore, IL-23 induced a population of Foxp3+T cells that expressed RORgt and made IL17A. The induction of this putative pathogenic population was completely abrogated by a RORgt inverse agonist that inhibits RORgt signaling in vivo. IL-23 was also sufficient to drive the generation of Foxp3+RORgt+IL17A+ cells in vitro, suggesting a cell intrinsic role for IL-23. Interestingly, IL-23 did not induce production of other pro-inflammatory cytokines like IFN-g by Treg cells. This is in line with recent studies where both increased accumulation and the ability of Foxp3+ T cells to make IL17A was reported in lesional skin of psoriasis patients. Current efforts are focused on better defining the characteristics of Foxp3+RORgt+IL17A+ cells in lesional skin of psoriatic patients and mice treated with IL-23. Collectively, our data suggests that IL-23 drives Treg cell plasticity by inducing a population of Foxp3+RORgt+IL17A+ cells that is regulated by RORgt signaling and could be relevant to disease pathogenesis.
Families of neurotransmitter receptors are differentially regulated across T cell subsets
Authors: Rosenberg Kenneth M , Singh Nevil J
Published: January 2023

Although primarily expressed in neurons, a surprising array of neurotransmitter receptors (NR) has been found to be expressed in immune cells, and in particular T cells. This suggests that T cell responses may be amenable to direct regulation by the nervous system. However, the absence of adequate information about the subset-specific and activation-dependent variations in NR expression in T cells makes it challenging to decipher how and when such regulation might impact T cell immunity. In this study, we examined the levels of several NR components in different mouse T cell subsets. RNA from FACS-purified CD8 or CD4 (naïve, memory, or regulatory) T cells were analyzed by RT-PCR for 84 known NR transcripts. Preliminary data show that 19 of these are expressed in at least one of the T cell subsets studied. Of these, 3 subunits were stably expressed across all subsets with the remaining 16 transcripts showing at least a 2-fold expression change between groups. 32% of genes showed the greatest expression in naïve CD4 T cells while a further 32% were upregulated most highly among memory CD4 T cells. Intriguingly, the glutamate receptor Gria3 is uniquely expressed by CD8 T cells, while the vasopressin receptor Avpr1a is specific to regulatory CD4 T cells. We also compared our profiles with previously reported metadata including the ImmGen expression database. This study now allows us to develop a functional model for where specific neuronal outputs may impinge on different T cell subsets under physiological contexts.
Elucidating the Role of CD27 in Treg Function during Th17 Responses
Authors: Remedios Kelly Anne , Zirak Bahar , Rosenblum Michael D
Published: January 2023

Tregs and Th17 cells have a very close and complex relationship. Tregs and Th17 cells share a dependence on TGFb for their differentiation and it has recently been shown that Tregs require the Th17-driving transcription factor STAT3 to regulate Th17 responses. Despite sharing a dependence on STAT3, Tregs within healthy tissue tend to produce very low amounts of IL-17. In contrast, in multiple Th17-driven diseases (including Multiple Sclerosis, Psoriasis and Crohn’s) Tregs within the diseased tissue produce increased levels of the IL-17. The costimulatory receptor CD27 and its ligand, CD70, have been shown to play a role in suppressing Th17 responses through the epigenetic suppression of IL-17 and CCR6. We have found that CD27 is highly expressed on Tregs in both human and murine skin, but whether CD27 plays a role in promoting Treg function (and suppressing acquisition of Th17-like traits) remains unknown. Utilizing CD27-deficient mice (CD27KO), we found that Tregs in the skin of CD27KO mice in the steady state produced more IL-17 compared to their wild-type (WT) counterparts. To test the role of CD27 specifically on Tregs during Th17-driven inflammation in vivo, we have developed an adoptive transfer model of cutaneous candida albicans infection where Tregs play an essential role in the development of productive Th17 responses. Using this model, we found that CD27 suppressed Treg expression of IL-17 and CCR6 in a cell-intrinsic manner. We also found that IL-6 could induce the production of IL-17 from Tregs in vitro which was suppressed when co-cultured with CD70 expressing BMDCs. Our studies suggest that the CD27-CD70 pathway promotes Treg function by limiting production of Th17 effector molecules.
Age difference in the immune response to endotoxin (LPS) shapes Th2-mediated airway inflammation and development of asthma.
Authors: Ruiz Beatriz León , Tato Andre Ballesteros
Published: January 2023

In addition to genetic factors, environmental exposures is also of primary importance for the development of asthma/allergy disorders in children, as demonstrated by epidemiological data showing children growing up in traditional farms seem to develop asthma less often than children growing up in urban areas. According to the hygiene hypothesis, this is due to increased exposure to endotoxin (LPS) and other farm-related microorganism-derived compounds. However little is known about how early-life contact to microbial compounds influence the development of asthma. Here we show that house dust mite (HDM) allergen sensitization in adult mice (8 weeks-old) induced T-helper type 2 (Th2)- driven airway inflammation, i.e., influx of activated Th2 cells into the airways, airway eosinophilic inflammation and increased IgE-producing plasmablasts. In contrast, HDM allergen sensitization with low-dose LPS (10 μg) abrogated HDM-driven Th2-mediated allergic airway inflammation without increasing antigen-specific Th1 cell trafficking into the airways. Importantly, unlike adults, infant mice (3 weeks-old) exposed to HDM with low-dose LPS (10 μg) developed Th2 responses allergic airway inflammation and required allergen sensitization with high-dose LPS (100 μg) to effectively suppress allergen-specific Th2 responses and allergic inflammation. These results show that airway exposure levels of endotoxin provide different protection against asthma and allergies in adults and infants and suggest that Infants are more vulnerable to prime type 2 cell dominant responses to inhaled allergens in the presence of low dose endotoxin, which will shape future effector Th2 responses and Th2-mediated airway inflammation throughout adult life.
Adipose type one innate lymphoid cells regulate macrophage homeostasis through targeted cytotoxicity
Authors: Boulenouar Selma , Lynch Lydia
Published: January 2023

Adipose tissue has a dynamic immune system that adapts to changes in diet and maintains homeostatic tissue remodeling. Recently, adipose type 1 innate lymphoid cells (AT1-ILCs) were reported to promote proinflammatory M1 macrophages in obesity. However, their role at steady state remains unknown. Here we reveal heterogeneous populations of AT1-ILCs in humans and mice. Using parabiotic mice fed HFD, we found that AT1- ILCs had differential trafficking and proliferation features. AT1-ILCs displayed fluctuations in response to short and long term HFD and could revert to regular levels upon diet restriction. Unexpectedly, all AT1-ILCs were able to kill macrophages at steady state, especially tissue repair-associated M2 macrophages. T1-ILCs-depleted and perforin-deficient mice had increased M2:M1 ratios. Diet-induced obesity impaired AT1-ILCs killing ability. Adoptive transfer of AT-1ILCs from lean to obese mice reduced total macrophages but increased M1:M2 ratio. Collectively, we show that AT1-ILCs are essential for regulating macrophage homeostasis through targeted cytotoxicity.
Mechanisms of Glucocorticoid Resistance in Hypereosinophlic Syndromes
Authors: Stokes Kindra N , Yoon Pryscilla , Makiya Michelle , Gebreegziabher Meheret , Holland-Thomas Nicole , Haugen Brandon , Pazdrak Konrad , Klion Amy D. , Khoury Paneez
Published: January 2023

Glucocorticoids (GC) are anti-inflammatory agents commonly used to treat patients with hypereosinophilic syndrome (HES). GC promote eosinophil apoptosis and downregulation of Th2 cytokine production. Although abnormalities of GC receptor (GR) function and elevations in β-GR isoforms have been implicated in GC resistance in asthma, little is known about the mechanisms of GC resistance in HES. To explore the etiology of GC-resistance in HES, samples were analyzed from 19 subjects with HES enrolled on a prospective study of GC-responsiveness and 33 subjects with HES enrolled on a natural history study of eosinophilia for whom response to GC was known. GC responders were defined by a reduction in absolute eosinophil count (AEC) to <1000/mL after 0.5–1mg/kg of GC for ≥1 week. In the prospective trial, GM AEC was significantly decreased in the GC responders (n=14; GM 2430 to 245/mL, p<0.001) but not in GC non-responders (n=5; GM 12800 vs GM 9927, NS). In contrast, an expected rise in absolute neutrophil count was seen in 12/14 GC responders (GM 3.39 to 6.07, p<0.01) and 5/5 non-responders (GM 2.51 vs. 5.27, p<0.01), suggesting that GC resistance in HES is not a global phenomenon. Expression of GR isoforms (α, β, γ and P) was assessed by RT-PCR in RNA from purified eosinophils and showed no differences between responders (n=23) and non-responders (n=12). A panel of serum cytokine levels were measured in 37 subjects. Only IL-5 levels were significantly different between the groups (GM 86.2 pg/mL in non-responders vs. 15.3 in non-responders, p=0.019). IL-5, at levels comparable to those seen in GC non-responders, inhibited GC-induced eosinophil apoptosis in vitro (n=7). These data suggest that increased IL-5 may play a major role in GC resistance in HES.
Dysregulation of Inhibitory Receptor Expression in Systemic Sclerosis
Authors: Fleury Michelle , Belkina Anna C , Gupta Ashna , Zammitti Christopher , Simms Robert , Snyder-Cappione Jennifer , Lafyatis Robert , Dooms Hans
Published: January 2023

Systemic sclerosis (SSc) is an autoimmune connective tissue disorder associated with aberrant fibrosis of the skin and internal organs that can lead to organ failure and death in patients. With few treatment options many patients succumb to fibrosis of the lungs or kidneys, or due to vascular damage culminating in pulmonary disease; the 50 percent survival rate is only about 10 years. We collected PBMCs from 36 SSc patients that were not on immune-suppressants, along with 25 age-matched healthy controls. Using a 15 color flow cytometry panel our lab has observed increases in the inhibitory receptors PD-1, TIGIT, and Tim-3 on PBMCs from SSc patients. This increase in expression is cell type specific with PD-1 and TIGIT shown to be increased on defined T cell types including CD4+ T cells, both Treg and non-Treg subsets, as well as CD8+ T cells. Tim-3 was only statistically significantly increased on a mature subset of CD16+ CD56med natural killer cells. This increase was not seen for all inhibitory receptors. The expression of LAG-3 remained low across all cell types observed. This increase in inhibitory receptor expression was enhanced in patients with a high percent of Tregs within the CD4+ gate. In vitro blockade of these inhibitory receptors provided differential cytokine secretion in patients versus healthy subjects, suggesting a possible role in disease pathogenesis. The observation of increased inhibitory receptors in SSc as well as the ability to modulate cytokines by blocking the interaction of these receptors with their ligands could provide insights into possible immune modulation in patients.
The major roles of monocytes/macrophages and their products in skin inflammation induced by skin deposited lupus IgG
Authors: Deng Guo-Min , Xu Guangqiong , Guo Xuanxuan , Fei Xibin , Li Xiaoyan
Published: January 2023

Skin injury is second common in patients with systemic lupus erythematosus (SLE), but pathogenesis of skin injury remains unclear. Based on skin deposition of IgG in SLE, the role of skin deposited IgG in the pathogenesis of skin injury in SLE was studied. We established skin deposited lupus IgG by intradermal injection of lupus IgG in mice and investigated the role of skin deposited lupus IgG in pathogenesis of skin injury in SLE. We found that skin deposited lupus IgG made by intradermal injection of lupus serum IgG induced skin inflammation, which appeared at 3 h and peaked at 3 d after intradermal injection. This skin inflammation was related to the dose of injected IgG but not to systemic disease activity. This skin inflammation was significantly decreased in mice depleted of monocytes but not lymphocytes. This skin inflammation was markedly decreased in mice with TNF-a deficiency. Lupus IgG promoted monocyte differentiation into dendritic cells (DCs) and enhanced the expression of TNF-α. TNF-α played a major role in the proliferation of keratinocytes. The results indicate that the deposition of IgG in skin exerts an important role in the pathogenesis of lupus skin injury. IgG signaling pathway is a therapeutic target in skin injury of SLE.
Expression of Immune Checkpoint Receptors in Multiple Myeloma Patients
Authors: Kwon Minsuk , Choi Yoon Seok , Park Junsik , Shin Eui-Cheol
Published: January 2023

Multiple myeloma is a malignancy of plasma cells and the second most common hematologic cancer. High-dose chemotherapy plus autologous stem cell transplantation has been performed to achieve remission, but almost all the patients will finally relapse. Alternative therapeutic options are necessary for the multiple myeloma patients. Immune checkpoint inhibitors, such as anti-PD-1 have shown practice-changing therapeutic effectiveness for advanced non-small cell lung cancer, as well as Hodgkin’s lymphoma. To investigate T cell exhaustion in multiple myeloma patients, we assessed the expression of various immune checkpoint receptors, such as PD-1, Tim-3, LAG-3 and TIGIT, in the paired peripheral blood mononuclear cells (PBMC) and bone marrow mononuclear cells (BMMC), by multicolor flow cytometry. In BMMC, PD-1+, and PD-1+TIGIT+ were increased in CD8+ T cells, compared to the paired PBMC. Only a few patients showed Tim-3+ or LAG-3+ T cell population, and the most identified Tim-3+ or LAG-3+ T cell populations were observed in PD-1hiCD8+ T cells in the BMMC. There was significantly increased PD-1+, and PD-1+TIGIT+ expression in CD69+CCR7−CD8+ population compared to CD69− CCR7−CD8+ in BMMC. On direct ex vivo MHC multimer analysis of BMMC, tumor-associated-antigen specific CD8+ T cell showed increased PD-1+, TIGIT+, PD-1+TIGIT+ and CD69+ expression. PD-L1 was also expressed on malignant plasma cell, and the level of expression was correlated with the disease burden. Collectively, our data show that the immune checkpoint receptors-expressing T cells are enriched in bone marrow, where malignant plasma cells reside. Functional restoration of these T cells by immune checkpoint blockers will be investigated in further study.