bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of interleukin 4 signaling increases the efficacy of vaccines against opioid use disorders and overdose
Authors: Crouse Bethany , Huseby-Kelcher April , Robinson Christine , Baehr Carly , Pravetoni Marco
Published: January 2023

Opioid use disorder (OUD) affects over 27 million people worldwide and contributes to 118,000 fatal overdoses each year. Vaccines against opioids offer a novel therapeutic strategy for the treatment of OUD, however their clinical efficacy may be limited to a subset of patients. To increase vaccine efficacy and minimize individual variability, our lab has shown previously that blockage of IL-4 increases overall anti-opioid IgG titers and induces IgG class switching, which correlates with greater efficacy in preventing opioid distribution to the brain. The mechanism behind the IL-4-mediated increase in vaccine efficacy is still unknown. We found that the increase in antibody titers is consistent for various anti-opioid vaccine formulation as well as the tetanus, diphtheria, and pertussis (TDaP) vaccine, suggesting this may be a broadly effective strategy to increase vaccine efficacy. This effect was also similar between male and female mice. An increase in the number of germinal centers was seen after IL-4 depletion using both immunofluorescence and flow cytometry. NKT cells, a cell population known to secrete IL-4 during early germinal center formation, were not necessary for vaccine efficacy. Genetic deletions of IL-4, but not necessarily their downstream signaling cascade, increased vaccine efficacy to the same degree as the antibody-based depletion. Finally, RNA-sequencing suggests a possible role of antibody-mediated effector functions in the increase in vaccine efficacy. These pre-clinical data support the use of cytokine-based immunomodulators as molecular adjuvants to increase vaccine efficacy against challenging targets.
Cancer Vaccines in the Pacific Region
Authors: Sika-Paotonu Dianne
Published: January 2023

Pacific communities remain disproportionately affected by some cancers and stand to benefit from research that will contribute to improvements in health outcomes. Significant health inequalities already exist for Pacific Peoples with cancer, where the burden of cancer remains a growing public health concern. Emerging immunotherapeutic based strategies such as therapeutic cancer vaccines being developed to treat cancer, offer potential as one day becoming a frontline treatment for cancer. The purpose of this work was to facilitate appropriate engagement and discussion to explore the potential applicability of therapeutic cancer vaccines within the Pacific setting. As part of wider cancer related discussions and consultation activities, preliminary engagement efforts were made to understand the potential utility of therapeutic cancer vaccines for treating cancer in the Pacific context in Tonga. The need for developing stronger cancer immunotherapies was acknowledged. Within the Pacific setting, therapeutic cancer vaccines would need to be affordable with the appropriate infrastructure required to support their utility in place. Strong support was also expressed for developing technologies to support early cancer detection given the constrained resources in the Pacific Region setting and as cancer presentation in Tonga is usually late and with poor prognostic outcomes. Overall, therapeutic cancer vaccines remain a promising treatment option, however their future applicability within the resource constrained setting, would require further research development and the provision to ensure appropriate resourcing and infrastructure in place for support.
Enhanced functional properties of in vitro expanded lymph node derived human NK cells
Authors: Dogra Pranay , Chin Athena , Kubota Masaru , Matsumoto Rei , Thapa Puspa , Szabo Peter , Poon Maya M.L. , Farber Donna L.
Published: January 2023

Natural killer (NK) cells are innate immune cells with the inherent ability to directly kill tumor and virus infected cells. Due to their ability to kill cancer cells without any prior priming, and their role in preventing metastasis NK cells have since long been the choice for autologous adoptive cell transfer therapy in cancer. However, poor expansion potential of PBMC derived NK cells in vitro is a major roadblock preventing the widespread use of NK cells in immunotherapy. We found that human NK cells isolated from lymph nodes (LNs) express higher levels of genes encoding for stem-like transcription factors (TCF7, LEF1, MYC) compared to NK cells from blood, spleen, bone marrow (BM) and lung. Therefore, we hypothesized that NK cells isolated from LNs will show superior expansion potential in vitro. Flow cytometric analysis shows that LN derived NK cells express high levels of TCF1 protein ex vivo, and show greater proliferation compared to NK cells isolated from blood, spleen and BM following stimulation with IL-2 and IL-15 in vitro. We also observed that a significant frequency of LN NK cells expressed TCF1 even after expansion, suggesting preserved proliferation potential of these cells. Additionally, the expanded NK cells from LN acquired properties of mature, highly functional NK cells such as increased expression of CD16, CD57 and higher Granzyme B expression. Lastly, LN derived NK cells also demonstrate enhanced cytolytic activity in vitro after expansion. Taken together our results suggest that in vitro expanded NK cells from LNs are potentially efficacious anti-tumor agents and could be leveraged for the development of future generation of NK cell directed immunotherapies.
The alarmin cytokine IL-1a drives a feedback-amplification loop between dying tumor cells and inflammatory monocyte-lineage dendritic cells
Authors: Sharma Madhav D , Rodriguez Paulo C , Munn David H
Published: January 2023

Successful immunotherapy must transform the normally suppressive tumor microenvironment into a pro-inflammatory, immunogenic milieu. We show that this critical transformation depends on a previously unrecognized initiating signal delivered by the damage-associated “alarmin” cytokine interleukin-1a (IL-1a). Initially, tumor-intrinsic IL-1a was mobilized from dying tumor cells. This drove rapid local differentiation of a population of monocyte-lineage inflammatory dendritic cells (DCs). We have previously described these inflammation-inducible myeloid DCs in tumors (Sharma et. al, Immunity 48:91–106, 2018), where they serve as key antigen-presenting cells during multiple forms of immunotherapy and chemo-immuno-therapy. Once these inflammatory DCs were elicited by tumor-derived IL-1a, they rapidly re-activated anergic/unresponsive tumor-associated CD8+ T cells. Then DCs and T cells together produced reciprocal host-derived IL-1a, thus completing a potent self-amplifying feedback loop. If this IL-1a-driven feedback-amplification loop was disrupted – either by silencing IL-1a in the tumor cells; or ablating IL-1a in the host cells; or disrupting the IL-1-receptor on the inflammatory dendritic cells – then anti-tumor immune activation was lost across multiple forms of immunotherapy and chemotherapy. To restore the ability to initiate immune activation, the only cells that needed to respond to IL-1a were the specific population of inflammatory monocyte-lineage DCs.
Exploring mechanisms of tumor resistance to CAR T cell therapy in immunocompetent mouse models
Authors: Andrews Alex , Thurston Gavin , Kirshner Jessica R. , DiLillo David J.
Published: January 2023

Chimeric antigen receptor (CAR) T cell therapies have shown striking clinical efficacy in the treatment of hematological malignancies, but limited success so far against solid tumors, suggesting that solid tumors may harbor mechanisms of resistance. To investigate mechanisms of CAR T cell therapy resistance in solid tumors, we developed immunocompetent mouse models to explore immune cell interactions and the effect of an immunosuppressive tumor microenvironment on CAR T cell behavior. We generated murine CAR T cells against a model antigen that contained a CD3z intracellular signaling domain linked to either a CD28 or a 4-1BB signaling domain. We found that murine CAR T cells exhibited a predominantly central memory phenotype together with potent cytotoxic activity and antigen-dependent cytokine release in vitro. Further, we found that murine CAR T cells exhibited strong anti-tumor efficacy against both disseminated (EL4) and subcutaneous (MC38) murine tumors in vivo, whereas non-binding control CAR T cells had no anti-tumor effect. However, in both the disseminated and subcutaneous models, tumors were suppressed but not completely eradicated. Although murine CAR T cells possessing either CD28 or 41BB co-stimulatory domains demonstrated similar in vitro activities, they exhibited strikingly different efficacies and resistance mechanisms in vivo. Collectively, these results highlight the contributions of specific co-stimulatory signaling pathways to CAR T cell therapy in immunocompetent murine models and provide a platform to explore resistance mechanisms to CAR T therapy against solid tumors.
Cytokine Pre-activated γδT cells in Cancer Adoptive Immunotherapy
Authors: Liu Haiyan
Published: January 2023

Adoptive cell therapy harnesses the power of immune system by stimulating and expanding immune cells to combat cancer. The application of tumour-specific αβT cell-based therapy may be hindered by limited tumour antigens. γδT cells are promising candidates for cellular immunotherapy because of their abundant IFN-γ or IL-17 production and MHC-independent cytotoxicity against a broad spectrum of tumors. Numerous attempts to use γδT cells in cancer immunotherapy have been made and resulted in variable efficacy and an overall good safety profile. Better expansion strategies are needed to improve the efficacy of the clinical trials using γδT cell-based immunotherapies. In the current project, we aim to explore the efficacy of cytokine pre-activated γδT cells in cancer immunotherapy. Pre-activated γδT cells by a combination of cytokines were characterised for their surface activation marker expression, cytokine production, and cytotoxicity against tumour cells. We found that IL-12/18, IL-12/15/18, IL-12/18/21 and IL-12/15/18/21 are the most effective cytokine combinations for in vitro expansion of γδT cells in terms of activation and cytokine production. IL-21 upregulated their cytotoxicity against tumour cells in murine systems. All four combinations showed upregulated cytotoxicity of human γδT cells. These findings were extended to in vivo studies using murine hepatocellular carcinoma model.
Human cytomegalovirus preferentially infects early gestation placental macrophages and evades their antiviral immunogenicity through evasion of the type I IFN response
Authors: Johnson Erica L , Swieboda Dominika , Chakraborty Rana
Published: January 2023

Human cytomegalovirus (HCMV) is the leading cause of congenital viral infections. When a pregnant woman develops primary HCMV infection in early gestation, the risk of transplacental infection is ~40%. Studies have documented the presence of HCMV in placental macrophages (Hofbauer cells [HCs]) as a consequence of maternal viremia. However, the interplay between HCMV and placental macrophages (HCs) is poorly defined. With written informed consent, placentae were collected from 12 HIV-1/Hep B/HCMV seronegative women (>18 years). HCs were exposed to human HCMV (TB40/E). qPCR, FACS, ELISA, and Western blot analysis determined the expression of activation markers, cytokines, antiviral genes, and host proteins. Here we found that HCs isolated from early gestation (15–23 weeks) placentae are more susceptible to HCMV infection than HCs isolated from term (>39 weeks) placentae. HCMV infection of early gestation HCs led to a significant upregulation of activation markers, along with elevated secretion of proinflammatory cytokines, compared to HCMV-infected term HCs. In early gestation and term HC, HCMV rapidly induces the type I IFN pathway, leading to secretion of IFNs and strong induction of ISGs. However, HCMV diminishes type I IFN-mediated phosphorylation of STAT2, with early gestation HCs exhibiting the greatest level of inhibition. Our findings show that HCMV can subvert type I IFN signaling to support viral growth at the placenta, particularly during early gestation. Studying mechanisms used by viruses to prevent an immune response is of great importance for the development of new strategies to limit the sequelae of viral infections.
Comparison of Interferons and Other Biomarkers in Influenza and Healthy Donor Samples
Authors: Lavoie Thomas B , Schwartz Barbara , Kisiel Greggory , Panday Alok , Skup Kamil , Clark William
Published: January 2023

Toward an understanding of biomarkers for acute viral diseases we have examined Influenza serum in comparison to healthy donors. We determined the levels of Interferon (IFN) Alpha, Beta, Gamma as well as Neopterin and Beta-2 Microglobulin (B2M) by ELISA in the Serum of 38 Influenza and 65 healthy donors using commercial assays. We measured chemokines and proinflammatory cytokines as biomarkers for IFN Activity by electrochemiluminescence. Assays were performed according to the manufacturer’s protocol. IFN-Omega levels were determined using a newly developed ultra-sensitive single molecule assay sensitive to 0.04 pg/ml. Percentile stratification was by Mann-Whitney non-parametric testing and correlations were determined under Spearman conditions. None of the healthy donors exhibited quantifiable IFN-Alpha or Beta and only two had detectable levels of IFN-Omega. Within the Flu cohort, 68.5% of samples contain quantifiable IFN-Alpha and Omega and 39.5% had IFN-Beta. 79% of the Flu samples displayed IFN-Gamma levels above the 95th percentile determined from the healthy donor distribution. Neopterin, B2M, and CXCL-10 were all significantly elevated in the Flu cohort versus the healthy subclass. Other chemokines either showed no correlation or a mild negative correlation. There was a striking concordance between IFN-Alpha and Omega levels. To our knowledge this is the first study to measure IFN-Omega and a broad panel of other analytes in Flu samples. As such this study may provide a beginning to discriminate between different viral diseases.
B cell-intrinsic expression of Interferon Regulatory Factor 1 supports chronic gammaherpesvirus infection
Authors: Jondle Christopher Ned , Johnson Kaitlin , Uitenbroek Alexis , Sylvester Paul , Tarakanova Vera
Published: January 2023

Gammaherpesviruses are ubiquitous pathogens that are associated with multiple cancers, including B cell lymphomas. These viruses infect naïve B cells and drive a robust germinal center response to amplify the latent viral reservoir and establish life-long infection in memory B cells. It is the increase in mutagenic B cell differentiation caused by the robust gammaherpesvirus-driven germinal center response which is thought to be the primary cause of malignant transformation in viral-driven lymphomagenesis. The host and viral factors that selectively impact gammaherpesvirus-driven germinal center response are poorly defined. We found that global deficiency of the antiviral and tumor suppressive Interferon Regulatory Factor 1 (IRF-1) selectively promotes the gammaherpesvirus-driven germinal center response and expansion of the viral latent reservoir. To establish the degree to which the antiviral effects of IRF-1 are B cell-intrinsic, we generated mice with conditional deficiency of IRF-1. Unexpectedly, IRF-1 B cell-intrinsic function supports the establishment of chronic viral infection and the gammaherpesvirus-driven germinal center response. Indicating that the antiviral function of IRF-1 during gammaherpesvirus infection is mediated though its expression in non-B cell populations.
Conspiring with the enemy: IRF-3 supports latent gammaherpesvirus infection of germinal center B cells
Authors: Johnson Kaitlin E , Sylvester Paul , Aurubin Carlie , Tarakanova Vera
Published: January 2023

Gammaherpesviruses (GHVs) are ubiquitous pathogens that establish lifelong infections in their host. Importantly, these viruses manipulate B cell differentiation to establish long-term latency and are associated with a variety of B cell lymphomas. In a clever feat, GHVs infect naïve B cells and drive the infected B cells along with uninfected bystanders into the germinal center response, where the rapid expansion of B cells results in an exponential increase in the latent viral reservoir. Germinal center B cells are also thought to be the target of malignant transformation.

Type I Interferon (IFN), a major antiviral system of the host, is transcriptionally activated by several host factors, including Interferon regulatory factor 3 (IRF-3). IRF-3 restricts GHV lytic replication during acute infection. Importantly, nothing is known about the role of IRF-3 during chronic GHV infection that is almost exclusively represented by the latent viral life cycle. Additionally, no studies have examined the effect of IRF-3 on the GHV-driven germinal center response.

Unexpectedly and in contrast to the phenotype during acute infection, we discovered that IRF-3 deficiency attenuated the establishment of chronic GHV infection, revealing a a pro-viral role of IRF-3 during viral latency. Further, while there was no effect on germinal center B cell expansion in the absence of IRF-3, the efficiency of germinal center B cell infection by GHV was reduced. Current studies are examining how IRF-3 may be facilitating latent infection of germinal center B cells. This is, to our knowledge, is the first example of IRF-3 functioning in a pro-viral manner in the context of chronic virus infection.
B cell-intrinsic MHCII expression restricts bacterial dissemination from the gut and maintains metabolic tone
Authors: Roland Mary Melissa , Mohammed Ahmed Dawood , Jolly Amy , Kubinak Jason
Published: January 2023

The expression of major histocompatibility complex class II (MHCII) molecules on the surface of B cells is essential for cognate interactions with T cells and ultimately the generation of high affinity serum antibody responses. Serum antibody responses play an important role in limiting the ability of gut microbes to colonize the systemic compartment. This is important because bacterial dissemination from the gut can have severe physiological consequences ranging from life-threatening septicemia to chronic low grade systemic inflammation. Here we use a conditional knockout mouse model (B-MHCII−/− mice) to determine the physiological implications of loss of T cell dependent serum antibody responses due to B cell-intrinsic ablation of MHCII expression. Loss of MHCII expression on B cells in B-MHCII−/− mice results in a significant reduction in the generation of long-lived plasma cells in the bone marrow and reduced circulating levels of IgG1. Reduced levels of circulating IgG1 is associated with increased levels of bacterial dissemination from the gut into the systemic compartment, as B-MHCII−/− mice have significantly increased titers of anaerobic bacterial colony-forming units in their livers. Importantly, increased bacterial dissemination is associated with an inability of B-MHCII−/− mice to gain weight, suggesting that bacterial dissemination in B-MHCII−/− mice drives the development of a metabolic disorder. Collectively, our data imply that beyond MHCII’s role in infectious immunity, MHCII-mediated antibody responses also play a fundamental role in establishing benign symbiosis with resident microbiota to maintain proper metabolic physiology.
Suppression of experimental epidermolysis bullosa acquisita (EBA) by an IgG monoclonal antibody (mAb) to collagen type VII (COL7)
Authors: Samavedam Unni Krishna SRL , Potter Crystal , Angerman Elizabeth , Kasprick Anika , Bieber Katja , Ludwig Ralf , Finkelman Fred D
Published: January 2023

EBA is a chronic inflammatory, mucocutaneous blistering skin diseases caused by autoantibodies against the structural skin protein, COL7. EBA induction requires autoantibody binding as well as complement- and Fcγ receptor (FcγR)-dependent inflammation. Using mouse models of EBA, we evaluated: (1) in IgG1-deficient mice, whether preventing the production of IgG1 anti-COL7 Ab (IgG1 has limited ability to activate complement or FcγRs), influences EBA induced by COL7/adjuvant immunization (active model); and (2) whether a mAb of the pro-inflammatory IgG2c isotype can induce EBA or influence passive EBA induction by a rabbit IgG anti-mouse COL7 or active EBA induction. Disease in the active EBA model develops in half the time and is more severe in genetically susceptible IgG1-deficient mice than in wild-type mice. Blocking IFN-γ, which contributes to IgG2c induction, has little or no effect. 16A1C8, an IgG2c monoclonal anti-COL7 mAb, does not induce EBA, even in hypersusceptible FcγRIIB-deficient mice. Surprisingly, 16A1C8 suppresses EBA induction by rabbit anti-COL7 Ab, and EBA progression in COL7-immunized mice, even though IgG2c is a pro-inflammatory isotype, 16A1C8 does not compete with our rabbit Ab for binding to COL7, and 16A1C8 has little or no effect on neutrophil infiltration or C3 deposition at the dermal-epidermal junction. Thus; (1) autoantibodies that have limited ability to induce pro-inflammatory effector mechanisms can suppress EBA development and severity; and (2) some anti-COL7 mAbs, even those of a pro-inflammatory isotype, may be useful for preventing or even treating EBA.
H5P as an active learning tool builds content-based critical thinking skills in Biological Sciences and student confidence in problem-based learning
Authors: Carr William H , Barry Awa
Published: January 2023

Recent studies have demonstrated that active learning or learning by doing has a more significant positive impact on learning outcomes than reading or watching videos. However the availability of free and open source tools for creating these active learning exercises has been limited by the requirement of computer programing skills. With the development of H5P technology, an open source, on-line tool (www.h5p.org), faculty and even undergraduate students can create interactive exercises that facilitate the learning process and promote formative learning. H5P is a plugin for existing publishing systems that enables the system to create interactive formative assessments, such as multiple-choice quizzes that provide feedback in real-time on responses. Using this technology I created an active learning exercise that was implemented in an undergraduate General Biology I course for Biology majors at Medgar Evers College, CUNY, in Fall 2019. In this exercise students designed interactive multiple choice questions at both low and high levels of Modified Bloom’s Taxonomy. Based on surveys of their subjective impressions, a majority (73%) of the students reported that their confidence increased in explaining course-content (n=44). Content knowledge was also assessed before and after implementing this exercise This exercise was also implemented in an undergraduate Immunology course (n=11) on the topic of the regulation of V(D)J recombination. Here, I present how to introduce and implement this exercise among undergraduate students who are Biology Majors. The outcomes from this work suggest that this platform is an effective and versatile tool for building student confidence in explaining scientific content in the biological sciences.
Elucidating the Role of NEMO and SpiC in DNA Double Strand Break Induced Inhibition of V(D)J Recombination
Authors: Glynn Rebecca , Bassing Craig
Published: January 2023

V(D)J recombination is regulated such that most lymphocytes assemble and express an antigen receptor from only one allele (allelic exclusion). In pre-B cells, RAG-induced DNA double strand breaks (DSBs) at Igκ loci signal via ATM to rapidly repress Rag1/2 transcription, inhibit accessibility of Igk loci, and limit V recombination. We hypothesize that RAG DSB-induced repression of Rag1/2 is critical to transiently limit V recombination, ensure allelic exclusion, and inhibit oncogenic Ig translocations. Fittingly, Atm−/− mice have higher frequencies of developing B cells with RAG DSBs at both Igh and Igk loci and of mature B cells with bi-allelic expression of these genes. Yet, given the multifunctional roles of ATM in the DSB response, Atm−/− phenotypes cannot be directly attributed to Rag1/2 repression. To test our hypothesis, we study mouse models lacking downstream effectors of ATM: i) SpiC, which suppresses Igk accessibility in response to RAG DSBs, and ii) NFκB essential modulator (NEMO), which represses Rag1/2 in response to genotoxic DSBs. I show here that the frequency of surface bi-allelic Igκ expression is normal on SpiC−/− B cells and increased on Nemo−/− B cells. I also show that Nemo−/− pre-B cells exhibit impaired Rag1/2 repression in response to RAG DSBs and concomitant increased RAG cleavage at Igk. Our data support that RAG DSB-induced repression of Rag1/2 transiently inhibits additional Igk rearrangements to enforce Igκ allelic exclusion independent of ATM-mediated DSB repair. I am using Nemo−/− mouse models to determine if DSB-induced Rag1/2 repression requires Nemo in pro-B cells, and if this mechanism is critical to suppress oncogenic Ig translocations independent of defects in DSB repair/checkpoint activation.
E protein support of Zeb2 expression regulates development of multiple hematopoietic lineages
Authors: Huang Xiao , Ferris Stephen , Satpathy Ansuman , Murphy Theresa L , Murphy Kenneth M
Published: January 2023

Zeb2 is an E-box binding transcriptional repressor that were first discovered to be important for epithelial to mesenchymal transition. We recently discovered a genetic circuit involving Zeb2, Nfil3, Id2 that governs dendritic cell (DC) specification in the multipotent common dendritic cell progenitor (CDP). Nfil3 expression is required for the transition from a Zeb2hiand Id2lo stage into a Zeb2lo and Id2hi stage, which has exclusively cDC1 potential. This circuit blocks E protein activity to exclude pDC potential within the progenitor. However how E proteins regulate pDC development is still unclear. Here, we identified an enhancer element 165kb downstream of Zeb2 transcriptional starting site that can be bound by E proteins. Deletion of this enhancer completely abrogates pDC development. Interestingly, the activity of this enhancer is also required for monocytes and B cell development, while tissue resident macrophages used a different enhancer. A circuit involving Zeb2, Id2 and E proteins, where E proteins binding at this cis-element supports Zeb2 expression, is essential for lineage specification at multiple bifurcation point in the hematopoietic tree.
A divergent transcriptional landscape underpins the development and functional branching of MAIT cells
Authors: Koay Hui Fern , Su Shian , Amann-Zalcenstein Daniela , Daley Stephen R , Comerford Iain , Whyte Carly E , Konstantinov Igor E , d’Udekem Yves , Baldwin Tracey , Hickey Peter F , Berzins Stuart P , Mak Jeffrey Y.W. , Kallies Axel , Chen Zhenjun , Nussing Simone , Kedzierska Katherine , Mackay Laura K , McColl Shaun R , Deenick Elissa K , Fairlie David P , McCluskey James , Goodnow Christopher C , Ritchie Matthew E , Belz Gabrielle T , Naik Shalin H , Pellicci Daniel G , Godfrey Dale I
Published: January 2023

MR1-restricted mucosal-associated invariant T (MAIT) cells play a unique role in the immune system. These cells develop intrathymically through a three-stage process, but the events that regulate this are largely unknown. Here, using bulk and single-cell RNA sequencing–based transcriptomic analysis in mice and humans, we studied the changing transcriptional landscape that accompanies transition through each stage. Many transcripts were sharply modulated during MAIT cell development, including SLAM (signaling lymphocytic activation molecule) family members, chemokine receptors, and transcription factors. We also demonstrate that stage 3 “mature” MAIT cells comprise distinct subpopulations including newly arrived transitional stage 3 cells, interferon-γ–producing MAIT1 cells and interleukin-17–producing MAIT17 cells. Moreover, the validity and importance of several transcripts detected in this study are directly demonstrated using specific mutant mice. For example, MAIT cell intrathymic maturation was found to be halted in SLAM-associated protein (SAP)–deficient and CXCR6-deficient mouse models, providing clear evidence for their role in modulating MAIT cell development. These data underpin a model that maps the changing transcriptional landscape and identifies key factors that regulate the process of MAIT cell differentiation, with many parallels between mice and humans.
Activation of AIM2 inflammasome in salivary gland epithelial cells of patients with primary Sjögren’s syndrome
Authors: Hong seungmin , Lee Jaeseon , Jang Se Gwang , Lee Jennifer , Cho Mi-La , Kwok Seung-Ki , Park Sung-Hwan
Published: January 2023

Sjögren’s syndrome (SS) is a chronic and systemic autoimmune disease characterized by lymphocytic infiltration in the exocrine glands. Although an association between inflammasome activation in peripheral blood cells and disease activity of SS has been reported, little is known about inflammasome activation in exocrine glands and its underlying mechanisms. We investigated the levels of AIM2, ASC, caspase-1, IL-1β, and IL-18 in salivary gland tissues and saliva from SS patients. We found that AIM2, ASC, Caspase-1, and IL-18 were increased in the saliva from primary SS patients compared with healthy and sicca controls. Numbers of AIM2-ASC speck also were elevated in the salivary gland epithelial cells of SS patients. The expression of caspase-1 correlated with type I interferon (IFN) signature gene expressions in the minor salivary glands (MSG) of SS patients, and was increased by type I IFN stimulation in salivary gland epithelial cells (SGECs). The AIM2 inflammasome was activated in SGECs after stimulation by the AIM2 ligand, poly(dA:dT). In addition, type I IFN accelerated AIM2 inflammasome activation as determined by the increased expression of caspase-1 in SGECs. In conclusion, the AIM2 inflammasome activation is increased in the SGECs of SS patients, and therefore should be considered an important pathogenic mediator of SS disease.
New classification of CD4+CD8a+ double positive cells in Crohn’s disease patients
Authors: Tanemoto Shun , Sujino Tomohisa , Hagihara Yuya , Takada Yoshiaki , Yoshimatsu Yusuke , Umeda Satoko , Nomura Ena , Ono Keiko , Mikami Yohei , Naganuma Makoto , Kanai Takanori
Published: January 2023

The gastrointestinal tract absorbs nutrition while providing a defense response to foreign antigens including enteric bacteria or food. Once the antigen enter the intestine, inflammatory mononuclear cells are accumulated quickly, however some tissue resident T cells, such as regulatory T cells (Tregs) block the excessive tissue damage. We previously demonstrated that mouse CD4+CD8a+ double expressing (DP)cells and Tregs complementary play an immuno-suppressive role in IE and LP, and a part of IE DP cells is originally developed from Tregs in LP. However, DP cells are not well characterized in human especially in inflammatory bowel disease (IBD) patients.

We obtained human small intestinal tissue from 9 colon cancer patients as normal tissue, and 5 Crohn’s disease(CD) patients, dividing into inflamed and non-inflamed lesion, and analysed by Flow cytometry. We confirmed DP cells are located in both IE and LP in human. First, we demonstrated the ratio of DP cells in normal tissue was as same as in non-inflamed lesion of CD patients. As we expected, the ratio of DP cells in inflamed tissue of CD patients was significantly decreased compared to normal tissue and non-inflamed lesion of CD. Then, we analysed the surface marker of Human DP cells. Surface staining of CD27 and CD45RA showed distinct difference between DP cells and the other CD4 T cells. DP cells expressed more memory T cell marker (CD27−CD45RA−) and less effector T cell marker (CD27+CD45RA−) than the other CD4 subset. Futher more, the more fraction of IE DP cells expressed CD103 than LP DP cells. Conclusively, these data indicated that human DP cell reside both in IE and LP and that DP cells are not a single characterized population, and the DP cells were decreased in inflamed lesion in CD.
Identification of molecules associated with disease progression of autoimmune myocarditis to dilated cardiomyopathy
Authors: Herrera Natalia E , Nalawade Saisha Abhay , Chase Carol Ann , Forsthuber Thomas
Published: January 2023

Myocarditis is an inflammatory disease characterized by immune infiltrates into the myocardium. In many patients, the disease progresses to a chronic phase dilated cardiomyopathy (DCM) in which there is extensive fibrosis leading to ventricular dilation and impaired cardiac function. Less than 50% of these patients survive more than 5 years. Immunosuppressive treatments such as corticosteroids (CSs) are effective in managing the acute phase of the disease, but do not prevent progression to the DCM. Using the experimental autoimmune myocarditis (EAM) animal model, we previously demonstrated that the inflammatory cytokine macrophage migration inhibitory factor (MIF) plays a role in resistance to CS treatment. MIF knockout mice were treated with the corticosteroid Dexamethasone (Dex) and proved to be resistant to both EAM and DCM. We also observed that treatment with MIF inhibitors and Dex decreased the expression of chemokines and adhesion molecules and implicated these molecules in the progression of disease. Within these, osteopontin (OPN), a powerful chemoattractant, was downregulated. We hypothesized that OPN plays a role in the disease process and investigated its role in EAM and DCM using OPN knockout mice. Our results show that OPN knockout mice developed EAM, but that progression to DCM was impaired. Furthermore, we observed a decrease in CD45+CD133+ fibroblast progenitor cells infiltrating the myocardium in the absence of OPN, which could contribute to attenuated disease in KO mice. This study may provide insights into the mechanisms driving progression of EAM to DCM and point to potential therapeutic targets.
Upregulated autophagy in antigen-presenting splenic cells hints at promotion of immune-mediated thrombotic thrombocytopenic purpura (iTTP)
Authors: Schaller Monica , Humbert Magali , Heeb Silvan , Tschan Mario , Hovinga Johanna Kremer
Published: January 2023

Immune-mediated TTP is a life-threatening autoimmune disorder characterized by abundant occlusion of the microcirculation. iTTP is caused by a severe autoantibody-mediated deficiency of the von Willebrand factor cleaving protease, ADAMTS-13. Emerging evidence points to autophagy as key player driving dysregulated autoimmune responses in various diseases. Considering the crucial role of antigen-presenting cells (APCs) in the presentation of autoantigens, we assessed the expression profile of autophagy (ATG)-related genes in iTTP.

We isolated splenic dendritic cells (DC) from one patient carrying the HLA DRB1*11 risk allele and leucocytes from peripheral blood of two healthy donors. Then pan-DCs including plasmacytoid and myeloid DCs were isolated by negative selection followed by FACS sorting. In parallel in vitro differentiated monocyte-derived dendritic cells (Mo-DCs) were cultured from freshly isolated MOs. The expression profile of six essential ATG genes (Beclin-1, Atg5, Atg7, Atg16L1, WIPI1 and LC3B) implicated in autoimmune diseases and the autophagic flux (LC3B expression) were determined by qPCR and detection of LC3B dot formation by immunofluorescence, respectively.

Upregulation of 5 ATG genes (Beclin-1, Atg5, Atg7, WIPI1 and LC3B) was observed in patient’s Mo-DCs compared to controls. Basal autophagy of individual pan-DCs versus Mo-DCs revealed a higher autophagic flux than pan-DCs with the highest autophagic flux displayed in Mo-DCs in the iTTP patient.

The observed activation of autophagy hints at a contribution to the loss of tolerance in iTTP. A more extensive study to investigate if modulators of the respective aberrantly expressed ATG genes might help improve therapy management is ongoing.
Monovalent IgG4 autoantibodies require self-antigen driven affinity maturation to acquire pathogenic capacity
Authors: Fichtner Miriam F L , Vieni Casey , Redler Rachel L. , Jiang Ruoyi , Suarez Pablo , Nowak Richard , Burden Steven J , Bhabha Gira , Ekiert Damian C , O’Connor Kevin C
Published: January 2023

INTRODUCTION

The mechanisms underlying B cell-mediated autoimmune disease and the origin of autoreactive B cells are not well understood. Human monoclonal autoantibodies (mAbs) are valuable tools for investigating both. In this study, we used mAbs derived from patients with the autoimmune disorder, myasthenia gravis (MG). A subset of patients with MG have pathogenic autoantibodies that recognize muscle-specific tyrosine kinase (MuSK). The autoantibodies of MuSK MG are predominantly of the IgG4 subclass and functionally monovalent as a result of Fab-arm exchange.

OBJECTIVE

To gain insight into the origin and development of these unique autoantibodies.

METHODS AND RESULTS

We examined MG patient-derived mAbs, their corresponding germline-encoded unmutated common ancestors (UCA) and monovalent antigen-binding fragments (Fabs) to investigate how antigen-driven affinity maturation contributes to both binding and immunopathology. Mature mAbs, their UCA counterparts and mature monovalent Fabs bound to the MuSK autoantigen and retained their pathogenic capacity. However, monovalent UCA Fabs, which still bound the autoantigen, lost their pathogenic capacity. The mature Fabs were characterized by very high affinity (sub-nanomolar) driven by a rapid on-rate and slow off-rate. However, the UCA affinity was approximately 100-fold less than the mature Fabs, which was driven by a rapid off-rate.

SUMMARY/CONCLUSION

These findings indicate that the autoantigen initiates the autoimmune response in MuSK MG and drives autoimmunity through the accumulation of somatic hypermutations such that monovalent IgG4 Fab-arm exchanged MG autoantibodies reach a high affinity threshold required for pathogenic capacity.
Effects of Morphine on Behavioral and CNS Inflammatory Responses in GP120 Mice Under Immunocompetent vs. Immunodeficient Conditions
Authors: Canonico Dalton , Casale Sadie , Look Tristan , Cao Ling
Published: January 2023

HIV-associated neurocognitive disorder is a common complication of HIV infection, whose development is known to be facilitated by inflammation and exacerbated by morphine. In this study, we infected HIV gp120tg mice with LP-BM5 (a murine retrovirus that can cause systemic immunodeficiency in susceptible mouse strains) to examine morphine’s effect on gp120 in both immunocompetent and immunodeficient conditions. Specifically, we examined the expression of a series of inflammation-related molecules in the brain via qRT-PCR as well as the animals’ learning/memory function using spontaneous alternation T-maze. Morphine treatment (2×25mg pellets) did not significantly affect the development of immunodeficiency induced by LP-BM5 and all brain regions examined (hippocampus, striatum, and frontal lobe) had detectable LP-BM5 viral gag genes. Morphine notably reduced the performance of gp120tg+ mice in the T-maze assay when 2-minute retention was used, regardless of LP-BM5 treatment. Without LP-BM5 treatment, morphine increased hippocampal expression of CCL2, CCL5, CXCL10, IL-12 p40, TNFα, and IFNγ, while it decreased the expression of IL-6, arginase-1, IFNα and IFNβ. For mice who were subjected to LP-BM5 treatment, hippocampal expression of CCL2, CCL5, CXCL10, IL-12p40, and IL-1β were decreased by morphine, while CCL4, TNFα, IFNγ, iNOS, arginase-1 and IFNβ were increased by morphine. Further, expression of the mu opioid receptor was upregulated by morphine without LP-BM5, yet down-regulated by morphine with LP-BM5 treatment. Altogether, the effects of morphine are complex and dependent on the immune status of the host, which will be further elucidated in the future studies. (Supported by NIH/NIDA R21DA044886 (PI, Cao))
Intracellular expression of IFN-λ4 leads to ER stress, enhanced IRF1 signaling and decreased proliferation in hepatic cells that might protect HCV+ patients from liver cirrhosis
Authors: Onabajo Olusegun O , Wang Fang , Lee Mei-Hsuan , Florez-Vargas Oscar , Obajemu Adeola , Castro Mauro A , Vargas Joselin , Liao Shu-Fen , Huang Yu-Han , Shen Chen-Yang , Banday Rouf , O’Brien Thomas , Robertson A. Gordon , Prokunina-Olsson Ludmila
Published: January 2023

Several genetic variants in the human IFNL3/IFNL4 locus have been associated with reduced hepatic fibrosis despite poor clearance of HCV infection. We expanded this analysis to cirrhosis, a more advanced stage of fibrosis. In 2931 individuals with chronic HCV, the IFNL4 genotype that generates IFN-λ4 was associated with protection from cirrhosis (OR=0.65, p=0.012, adjusted for age and sex). The IFNL4 genotype affects the production of IFN-λ4 and, additionally, may affect expression levels of IFN-λ3, making it difficult to delineate the individual contribution of these IFNs. To address this, we established HepG2-based cell models engineered to inducibly express either IFN-λ3 or IFN-λ4. Using RNA-seq based expression profile generated in these hepatic cell lines, we explored the global transcriptome of liver tumors (n=373) from The Cancer Genome Atlas to identify transcription factor networks affected by these IFNs in the liver. Several networks, including IRF1, were upregulated by IFN-λ4 more strongly than by IFN-λ3. Intracellular expression of IFN-λ4 but not of IFN-λ3 also led to potent IRF1-dependent antiproliferative effects. Live cell imaging revealed that IFN-λ4 was poorly secreted, mainly accumulated in lysosomes, and caused apoptosis, suggesting increased ER-stress via the misfolded protein response. Knockdown of DNA damage-inducible transcript 3 (DDIT3), an ER-stress response effector, significantly attenuated the antiproliferative effects of IFN-λ4. This novel interplay of enhanced IRF1 signaling coupled with intracellular accumulation and induction of ER stress by IFN-λ4 may have complex consequences on liver homeostasis during chronic HCV infection but also mediate anti-cirrhotic phenotypes.
The nonreceptor tyrosine kinase SYK induces autoinflammatory osteomyelitis in a mouse model of chronic recurrent multifocal osteomyelitis
Authors: Banoth Balaji , Kanneganti Thirumala-Devi
Published: January 2023

Chronic recurrent multifocal osteomyelitis (CRMO) in humans can be modeled in Pstpip2cmo mice, which carry a missense mutation in the proline-serine-threonine phosphatase-interacting protein 2(Pstpip2) gene. As cmo disease is mediated specifically by IL-1β, not IL-1α, delineating the molecular pathways contributing to the pathogenic IL-1β production is crucial to develop targeted therapies. In particular, our earlier findings support redundant roles for NLRP3 and caspase-1 with caspase-8 in instigating the disease. However, the signaling components upstream of caspase-8 and pro-IL-1β cleavage in Pstpip2cmo mice are not well understood. Therefore, we investigated the signaling pathways in these mice and characterized the central role of the nonreceptor tyrosine kinase SYK in mediating osteomyelitis. We demonstrate that while absent in melanoma 2 (AIM2), receptor-interacting serine/threonine protein kinase 3 (RIPK3), and caspase recruitment domain-containing protein 9 (CARD9) are dispensable for osteomyelitis induction in Pstpip2cmo mice, genetic deletion of Syk completely rescues disease. We further show that SYK centrally mediates signaling upstream of caspase-1 and caspase-8 activation and principally regulates NF-κB and IL-1β signaling in Pstpip2cmo mice to instigate disease. These data provide a rationale for directly targeting SYK and its downstream signaling components in CRMO.
Neutrophil Effector Responses to Cystic Fibrosis Clinical Isolates of Staphylococcus aureus and Pseudomonas aeruginosa
Authors: Fantone Kayla M , Miller Arthur , Fricker Rachel , Yadav Ruchi , Stecenko Arlene , Goldberg Joanna , Rada Balazs
Published: January 2023

Cystic fibrosis (CF) airway disease is characterized by chronic microbial infections and infiltration of polymorphonuclear neutrophil granulocytes (PMNs). In CF, PMNs are unable to clear lung pathogens resulting in lung damage. Neutrophil extracellular traps (NETs) and superoxide production represent defense mechanisms of PMNs to kill extracellular pathogens. NETs and superoxide in the CF lung, however, seem unable to clear Pseudomonas aeruginosa and Staphylococcus aureus, the two main lung pathogens in CF. Our goal of this study was to determine antimicrobial functions of PMNs to S. aureus and P. aeruginosa CF isolates and to observe how the CF airway environment influences them. Human PMNs were isolated from healthy donors. Isolates of S. aureus were obtained from adult CF patients: four strains were methicillin-resistant (MRSA), four were methicillin-sensitive S. aureus (MSSA), and ten CF isolates of P. aeruginosa. PMN effector functions were measured: killing was assessed by bacterial growth assay, NET formation was measured by Sytox fluorescence and superoxide was monitored by Diogenes chemiluminescence. Results were subjected to paired t-test, significant if p<0.05. PMNs from healthy volunteers were able to kill all CF isolates. The extent of killing varied among strains, however, P. aeruginosa was killed more efficiently by blood PMNs than S. aureus clinical isolates. The enhanced killing of P. aeruginosa is correlated with enhanced NET release and superoxide production compared to S. aureus. Killing of all isolates was inhibited by CF sputum pretreatment. Overall, the results show that CF bacterial clinical isolates stimulate PMN effector responses that are impaired by the CF airway environment.
The CF airway environment affects neutrophil antibacterial effector mechanisms: phagocytosis and NET formation
Authors: Tucker Samantha L , Yadav Ruchi , Stecenko Arlene , Rada Balazs
Published: January 2023

Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in the epithelial chloride channel CFTR. Lung disease is the main cause of mortality and morbidity in CF. CF airways are characterized by chronic, mainly neutrophil-mediated inflammation and persistent bacterial infections. Despite their increased presence in the CF lung, airway neutrophils are unable to clear respiratory pathogens like Pseudomonas aeruginosa and Staphylococcus aureus. We hypothesized that the CF airway environment affects two antibacterial effector mechanisms of neutrophils: phagocytosis and neutrophil extracellular trap (NETs) formation. While neutrophils are known to kill P. aeruginosa and S. aureus mainly by phagocytosis-mediated intracellular killing, NET-mediated extracellular killing remains relatively inefficient against these pathogens. To examine this in CF, blood PMNs from healthy donors were incubated in sputum supernatants from CF patients to mimic the CF airway environment. Using this in vitro model, different markers and functions of neutrophils were measured by flow cytometry. Phagocytosis of fluorescently-labelled P. aeruginosa was significantly inhibited by CF sputum treatment. Surface expression of CD33, a myeloid-derived suppressor cell marker known to inhibit phagocytosis, was increased following CF sputum treatment. CF sputum exposure slightly increased both of the NET-specific markers studied: intracellular levels of citrullinated histone H3 and surface expression of gasdermin-D. These results suggest that the CF airway environment contributes to the inability of neutrophils to eliminate bacteria while promoting the release of dangerous, tissue-damaging neutrophil cargo via NET formation.