bluuebunny/crossref_metadata_2025 huggingface dataset:
STK25 acts as a regulator of leukocyte migration
Authors: Rice Matthew R , Matta Bharati , Barnes Betsy J
Published: January 2023

The recruitment of leukocytes to sites of infection is an essential component of the innate immune response to pathogens. Defects in leukocyte migration can render the host susceptible to recurrent infections. In our efforts to identify new mediators of innate immunity, we found that serine/threonine protein kinase 25 (STK25) promotes TLR-induced proinflammatory cytokine production. While STK25 has previously been implicated in the regulation of cell migration, its role in leukocyte trafficking has not been defined. To investigate the involvement of STK25 in leukocyte migration, we evaluated baseline hematological parameters from Stk25-deficient (Stk25−/−) mice via a Hemavet. We found that Stk25−/− mice exhibit reduced circulating lymphocytes, neutrophils, and monocytes at steady-state compared to age- and gender-matched wild-type littermate (WT) mice. Immunophenotyping by flow cytometry confirmed these findings and revealed a significant reduction in the frequency of Ly6Chi inflammatory monocytes in Stk25−/− peripheral blood (PB) relative to WT. In contrast, we observed no apparent change in the frequencies of neutrophils and Ly6Chi inflammatory monocytes in Stk25−/− bone marrow, suggesting a defect in leukocyte migration. Finally, we examined the role of STK25 in neutrophil trafficking via an in vitro chemotaxis assay. We found that PB neutrophils from Stk25−/− mice displayed a defect in migration toward WT serum, suggesting a cell-intrinsic role for STK25 in the control of cell trafficking. Additionally, PB neutrophils from WT mice exhibited reduced migration toward Stk25−/− serum, supporting a cell-extrinsic function for STK25. Thus, our data support a new role for STK25 as a mediator of leukocyte trafficking.
Mechanical stress modulates NLRP3 pathway in macrophages
Authors: Joshi Hemant , Almgren-Bell Alison , Todd Elizabeth M , Morley Sharon Celeste
Published: January 2023

NLRP3 inflammasome activation releases the pro-inflammatory cytokine IL-1b and induces pyroptotic cell death. Dysregulated NLRP3 activation escalates inflammatory diseases such as acute lung injury (ALI), ventilator-induced lung injury (VILI), liver fibrosis, and idiopathic pulmonary fibrosis (IPF). These inflammatory disorders are associated with increased mechanical stress due to changing tissue compliance/stiffness. Resident macrophages sense this mechanical signal via a process known as “mechanotransduction”. However, the underlying mechanism of how mechanotransduction regulates the NLRP3 inflammasome is not fully known.

In this study, we are investigating that how NLRP3 pathway is regulated in lung resident macrophages during mechanical stress. To alter substrate compliance, macrophages are cultured on collagen-coated gels with various physiologically relevant stiffness (elastic moduli; measured in kiloPascal (kPa)) prior to NLRP3 activation. We have found that the NLRP3 inflammasome is sensitive to the substrate stiffness. NLRP3 induced IL-1β and pyroptosis-inducing gasdermin-D production were highest on the softest substrate (0.2 kPa) and decreased on a stiffer substrate (64 kPa). However, NLRC4 inflammasome activation was not mechano-responsive, suggesting ASC adaptor mediated cross signaling in mechanotransduction. Further analysis of gene expression using high-throughput RNA-sequencing, revealed altered expression of inflammatory bio-markers on macrophages when substrate stiffness was varied. Further examination of molecular interactions between adhesion-based mechanotransduction and NLRP3 signaling will help in understanding inflammatory diseases such as lung fibrosis.

AAI career in Immunology Fellowship, R01-AI104732 and R56 AI104732
Incorporating Immunology into the Undergraduate Curriculum to Promote Interdisciplinary Science Education
Authors: Pandey Sumali , Justement Louis B , Taylor Rebekah
Published: January 2023

The national call for undergraduate education reform in the Vision and Change report identified understanding the importance of interdisciplinary science as a key competency; one that is critical for career success and responding to the evolving needs of the STEM workforce. Immunology provides an ideal platform to foster an appreciation of the interrelationships between multiple disciplines in the natural sciences. Interdisciplinary thinking can be fostered by creating educational interventions that relate concepts in immunology with foundational concepts in other sciences and is critical to tackle current societal challenges, such as the effect of global climate change on emerging infectious diseases and the need for new strategies to combat these threats. Although the discipline of immunology integrates easily with other biological fields such as microbiology and physiology, incorporating interdisciplinary thinking into broad educational practice requires intention and collaboration. In this breakout session, we will introduce a conceptual framework that: i) identifies interdisciplinary immunology focused learning outcomes; and ii) demonstrates classroom strategies to address these outcomes. Participants will engage in an active discussion to better understand ways in which Immunology education can be refocused to enhance interdisciplinary learning. The goal of the session is to empower educators by providing a conceptual framework that will enable them to mindfully and intentionally incorporate backward design into their pedagogy to teach interdisciplinary science using immunological topics.

Supported by grant from NSF (#2120806)
Driving social belonging and content retention by using the Flipgrid video discussion app.
Authors: Lukin Kara , Aviszus Katja , Nardi Chelsey
Published: January 2023

Even strong students struggle with the level of detail and interconnectedness required to master immunology. We incorporated Flipgrid assignments to leverage social and emotional learning and cognitive science to enhance students’ engagement with and understanding of content in an undergraduate-level immunology course. The tool is appropriate for elementary through professional school students. Flipgrip is a simple, video recording app used via a cell phone or computer. It can be integrated with most learning management systems. Students created short videos of themselves explaining learning objectives they selected. The videos were evaluated by the educator, updated if needed and shared with the class as tools for review. Peer-evaluation is possible, too. This feedback can be provided quickly in the app.

Through such activities, students increase their sense of social connectedness and belonging which research has shown enhances 1) intrinsic motivation and course engagement, 2) safety in the learning community so students ask peers and educators for support, and 3) overall academic success and retention. The benefits of belonging are especially beneficial for students from historically excluded and underrepresented groups. The approach allows students to become experts in a concept and contribute to discussions in ways they might not feel comfortable doing during class sessions. The videos provide opportunities to see and celebrate diversity among the class in a variety of ways. The creativity students bring to the videos help generate recall clues that allow students to retain information more effectively. Similarly, the collection of videos allows students to use distributive recall to self-assess and solidify content.

Supported by No funding support
Pharmacy Immunology-challenges of teaching a new course in a COVID-affected world of education.
Authors: Fazal Nadeem
Published: January 2023

Immunology education in US schools and colleges of pharmacy do not have a standardized curriculum. Half of 142 colleges of pharmacies have a dedicated course of Immunology with three fourth of them in the first year of pharmacy education. Although it is a part of core curriculum yet more private than public schools allocate about 3 credit hours. Chicago State University College of Pharmacy offered a new 2-credit course of “Pharmacy Immunology” in the fall of 2021 to twenty-four second year pharmacy students for the first time. Ten lectures on basic immunology, i.e., innate and adaptive immunity, prepared the students to understand disorders and diseases associated with the immune system. Four workshops of 3-hours each, on Hypersensitivities, Autoimmunity, Transplantation and Tumor Immunology were added to the syllabus. The workshops employed a Modified Flipped Classroom, Team-Based Learning (TBL), Case-Based Learning (CBL), YouTube videos as a combination of various teaching methods. The course was offered as a standard in-class on-campus module. Student’s performance was assessed by tRAT and iRAT. Data of student performance and perception was collected and analyzed. 58.3% of students agreed that the workshop’s mode of learning helped them. TBL appealed to 29.2%, CBL 45.8%, and PowerPoint lectures 79.2% of the students. Overall, 33.3% of the students disliked workshops as a tool for offering an interactive educational tool. Assessment data confirmed that the pass rate was 100%, with 20.8% passing in iRAT as compared to 79.2% in tRAT. Final assessment and course evaluation data suggest that a hybrid approach including a blended learning technique was the best option, catering for the needs of most students having a diverse learning style.
Open Discovery in an Undergraduate Immunology Course Lab: Testing Anti-Inflammatory Drugs
Authors: Brennan Catherine A , Roshandell Mobina
Published: January 2023

I will present the design and outcomes from a course laboratory module in which undergraduates apply techniques learned earlier in the semester (cell culture, spectrophotometry, standard curves, ELISA) towards the testing of an anti-inflammatory drug of their choice. Students rationalize their dose and assay selections, calculate all dilutions, perform the assays, and analyse and interpret all results. Student-directed course research experiences have been shown to solidify student understanding of advanced scientific concepts. Additionally, I have observed that this exercise, in which students analyse the function of medications used by themselves or loved ones, leads to enthusiastic engagement with and mastery of complex concepts.
Jedi Mindset: Discussing Effective Learning Strategies with Students
Authors: Alard Pascale
Published: January 2023

Students’ ability to retrieve acquired knowledge long-term is critical for every discipline. The research on learning shows undergraduate students use ineffective learning strategies, misjudging passive vs active ways of learning, and prioritizing grade over long-term learning, while they could achieve both by switching to effective learning strategies. Moreover, first generation graduates may be at a disadvantage as they have less opportunity to learn from family member experience. Sixty percent of students who attend medical, dental or graduate school continue to use ineffective strategies, e.g., re-reading.

Our goal was to discuss learning strategies with our incoming graduate students to encourage them to switch to effective ones. We held four consecutive weekly sessions before courses started to discuss the book by Dr. Saundra McGuire entitled, “Teach yourself how to learn”. Incoming graduate students read a few chapters for each session and discussed what they had read with facilitators and senior students, who shared their own experience about the program and provided advice. The final session revisited the various concepts with short podcasts highlighting the different points. A majority of our students were willing to modify their study habits and will be surveyed regularly to assess whether they continue to use effective learning strategies. More importantly, students who switched to effective learning strategies understood concepts better and scored higher on exams, including comprehensive exams. Our study suggests that regularly discussing effective learning strategies with graduate students is easy to implement, and may be beneficial to their understanding of material, performance and long-term retrieval.

Supported by None
Dntt expression reveals developmental hierarchy and lineage specification of hematopoietic progenitors.
Authors: Tussiwand Roxane , Klein Fabian , Roux Julien , Cvijetic Grozdan , Tsapogas Panagiotis
Published: January 2023

Intrinsic and extrinsic cues determine developmental trajectories of hematopoietic stem cells (HSCs) towards erythroid, myeloid and lymphoid lineages. To dissect early lymphoid specification events, we generated two transgenic mouse models reporting and tracing the expression of terminal deoxynucleotidyl transferase (TdT). Surprisingly, transient induction of TdT was detected on a newly identified multipotent progenitor (MPP) subset which lacked self-renewal capacity but maintained multilineage differentiation potential. TdT induction by MPPs reflected a transcriptionally dynamic but uncommitted stage characterized by low expression of lineage-associated genes. Further, analysis using single-cell CITE-Seq indicated that multipotency is associated with expression of Endothelial Cell-Selective Adhesion Molecule (ESAM). Developmental progression along all lineages is defined by downregulation of ESAM and by stable TdT expression within the lymphoid trajectory. Collectively, we propose a new developmental hierarchy of early hematopoietic progenitors for which we provide their transcriptional profile as well as their phenotypic and functional properties at single cell resolution.

Supported by NIH intramural funding
A lineage specific role for PD-1 in agonist selection in the thymus
Authors: Caruso Breanna P , Moran Amy
Published: January 2023

Effective peripheral T cell responses rely upon a diverse T cell repertoire, which is generated during the process of thymic selection. Signaling through the T cell receptor is critical in determining the lineage fate of developing thymocytes; however, it has been established that costimulatory molecules and cytokines can also contribute to fate decisions. Interestingly, coinhibitory molecules, such as programmed cell death protein 1 (PD-1), and their ligands are also expressed in the thymus. Expression of these molecules early in T cell development could regulate lineage fate decisions; however, their potential role in modulating T cell selection is not well-understood. We demonstrate that PD-1 is differentially expressed throughout thymocyte development. Additionally, we show that PD-1’s ligands, PD-L1 and PD-L2, are expressed by thymic epithelial cells. To elucidate the role of PD-1 signaling in regulating T cell selection, we examined mice deficient in PD-1 or PD-L1. Disrupting PD-1 signaling results in an increase in regulatory T cells (Tregs), but not other agonistly selected cell populations. This effect is dose-dependent as mice heterozygous for PD-1 or PD-L1 display an intermediate phenotype. Interestingly, our data suggest that PD-1’s regulation of Treg development is not through its inhibition of T cell receptor signaling, but rather through regulation of IL-2 cytokine production. These data demonstrate that PD-1 signaling restricts Treg development in the thymus, which broadens our understanding of the role of inhibitory proteins to include regulation of T cell development.

Supported by grants from NSF (GRFP AVPRS0015) and NIH (F31 CA261058-01).
Using in vivo barcoding to capture T versus B cell fate commitment in the thymus.
Authors: Abdullah Leena , Emiliani Francesco E , McKenna Aaron , Huang Yina H
Published: January 2023

B cells are a rare subset in the thymus, an environment that supports T cell development and inhibits commitment to other immune cell lineages. Yet thymic B cells play an important role in the negative selection of autoreactive T cells. The origin of thymic B cells from either committed B cells or early thymocyte progenitors remains uncertain. We hypothesize that early thymic progenitor cells are a heterogenous population that can give rise to both T cells and B cells. In our study we combined the CRISPR/Cas9-dependent lineage recorder GESTALT with single cell RNAseq to capture cell lineage and type information to follow progressive cell fate decisions and to identify thymic B cell progenitors and transcriptional regulators permitting B cell differentiation in the thymus. Additionally, to evaluate the functional importance of thymic B cells in shaping the T cell repertoire, we performed TCR-seq on conventional CD4 T cells and Tregs. In the absence of B cells, amino acid usage within either TCR alpha or beta CDR3 regions remained unchanged. However, the dominant TCR alpha CDR3 length from the most abundant thymic Treg clones was reduced compared to wild type. Whether this repertoire shift impacts self-tolerance to B cell or AIRE-dependent antigens, both of which are expressed by thymic B cells, remains to be determined. Together, our data directly assesses lineage relationships leading to thymic T and B cell fate commitment and deepen our understanding of intra-thymic development and function of B cells that are required for central tolerance.

Supported by AI089805 Burroughs-Wellcome Fellowship
The Transcriptional Cofactor Ski in Thymic Epithelial Cells Regulates Peripheral T Cell Responses
Authors: Chiu Honyin , Ziegler Steven F
Published: January 2023

The thymus is an important site for the establishment and maintenance of an appropriate immune response through positive and negative selection of developing T cells. Cortical and medullary thymic epithelial cells (TECs), termed cTECs and mTECs, respectively, interact with developing thymocytes to mediate this selection process. Developing thymocytes also support the maturation and proliferation of mTECs by secreting transforming growth factor-beta (TGF-β) superfamily cytokines. Our lab has previously shown that deletion of TGF-β signaling in TECs enhanced negative selection and functional maturation of thymocytes, and increased the production of regulatory T cells. To investigate whether the proto-oncogene product Ski (Sloan-Kettering Institute), a negative regulator of TGF-β signaling, is associated with thymocyte development in homeostatic condition and inflammatory conditions, we generated mice that deleted Ski specifically in TECs (Foxn1Cre Skifl/fl mice). We found that the mTEC population was decreased as predicted, however we did not find any differences in thymocyte development between the Foxn1Cre Skifl/fl mice and control mice. We also found that CD4 T cells purified from Ski deleted mice showed a defect in proliferation, and Th1 and Th17 differentiation in vitro, while regulatory T cell differentiation was normal. Lastly, we found that these mice were significantly protected from developing experimental autoimmune encephalomyelitis (EAE) disease compared to the control mice. Overall, our findings suggest that Ski signaling in TECs regulates peripheral T cell self-reactivity responses and further study may provide evidence for targeting Ski in T cell driven autoimmune diseases such as multiple sclerosis.

Supported by grants from NIH grant R01-AI136475 (to SZ) and AAI Intersect fellowship (to HC)
Loss of Zfp335 triggers cGAS/STING-dependent apoptosis of post-β selection pre-T cells
Authors: Ratiu Jeremy J , Barclay William , Wang Qun , Shinohara Mari L , Zhuang Yuan
Published: January 2023

Production of a diverse peripheral T cell compartment requires massive expansion of the bone marrow progenitors that seed the thymus. There are two main phases of expansion during T cell development, following T lineage commitment at the DN2 stage and following successful rearrangement and selection for functional TCRβ chains in DN3 thymocytes, which promotes development of DN4 cells to the DP stage. Signals driving expansion of DN2 thymocytes are well studied, however, factors regulating the proliferation and survival of DN4 cells remain poorly understood. Here, we uncover an unexpected link between the transcription factor Zfp335 and control of cGAS/STING-dependent cell death in post-β-selection DN4 thymocytes. Zfp335 controls survival by sustaining expression of Ankle2 and regulating phosphorylation of Baf which functions to promote nuclear envelope integrity and suppress cGAS/STING-dependent cell death. Together, this study identifies Zfp335 as a key transcription factor controlling the survival of proliferating post-β-selection thymocytes and demonstrates a key role for the cGAS/STING pathway driving apoptosis of developing T cells.

Supported by grants from NIH R01-GM059638, P01-AI102853, RO1-AI099100, RO1-NS120417, and RO1-AI160737
The role of skin-derived IFN-kappa in the development of systemic autoimmunity
Authors: Martens Jacob William Shelton , Liu Jianhua , Maz Mitra , Kahlenberg J Michelle
Published: January 2023

Aberrant type I interferon expression and downstream interferon-stimulated gene signatures are central to the immune dysregulation observed in lupus. Recent evidence has suggested that elevated interferon kappa expression in the skin originating from keratinocytes may precede the development of systemic autoimmunity. Using a transgene mouse model overexpressing IFN-κ specifically in keratinocytes, we observe the development of anti-nuclear antibodies and immune complex deposition in the kidneys in the absence of environmental triggers. We also observe immune cell infiltration in the kidneys including activated CD4 T cells, activated cDC2-like dendritic cells, and expansion of IgD-CD27-double negative B cells. A subset of DN B cells has recently been shown to be increased in the blood of lupus patients with active disease and nephritis. This DN B cell subset was shown to be hyperresponsive to TLR7 signaling and showed transcriptional markers indicative of an inclination to differentiate into antibody-secreting plasmablasts. Further studies on the IFN-κ induced DN B cells in our model are needed to confirm phenotype and function of these cells. Future studies also hope to address the role of individual immune cell types in propagating or responding to the skin IFN-κ signal. Elucidating the mechanisms of early immune dysregulation resulting from this heightened IFN signaling would provide promising clinical relevance to treating patients early in their disease course, potentially prior to detrimental systemic manifestations.

Supported by NIAMS(R01 AR071384-05) NIH(T32 AI007413-29)
Association between anti-P. gingivalis antibodies and anti-citrullinated vimentin antibodies in the patients with rheumatoid arthritis.
Authors: Shindo Satoru , Nakamura Shin , Rawas-Qalaji Mohamad , Potempa Jan , Arvikar Sheila , Ouhara Kazuhisa , Shiba Hideki , Kawai Toshihisa
Published: January 2023

Objectives

Anti-citrullinated-protein auto-antibodies (ACPA), especially that react to citrullinated vimentin (CV), are implicated in the pathogenesis of Rheumatoid arthritis (RA). P. gingivalis (Pg), a key pathogen of periodontitis, can citrullinate the host proteins by production of Pg-derived peptidylarginine deiminase (PPAD). This study evaluated the relationship among antibodies (Abs) that react cyclic citrullinated protein (CCP), CV and periodontal pathogens in the serum of RA patients or control subjects.

Methods

Blood serum of healthy subjects (n=10) and RA patients (n=26) were obtained from Precision for Medicine. Six distinctive peptide sequences of Vimentin (#1–#6) and corresponding CV (#1’–#6’) peptides were synthesized. ELISA was employed to detect IgG Ab titers for CCP, intact vimentin and CV peptides. Fixed periodontal pathogens, Pg 33277, F. nucleatum, P. intermedia, A. actinomycetemcomitans (Aa) Y4, S. mitis, S. gordonii, A. odontolyticus, and C. gingivalis were also used as IgG ELISA antigens.

Results

The levels of anti-CCP Ab were higher in RA patients with anti-Pg Ab than those without anti-Pg Ab. Since elevated anti-Pg Ab is hallmark of periodontitis, it is conceivable that RA patients with anti-Pg Ab would have periodontitis. However, there was no association between anti-CCP Ab and the Abs reactive to the other bacteria. Very interestingly, RA patients with anti-Pg Ab, but not those without anti-Pg Ab, showed significantly elevated Ab response to #6’ CV peptide compared to intact #6 vimentin peptide, among six peptide sequences tested.

Conclusions

Our findings indicated that periodontitis may be associated with the induction of ACPA via PPAD-mediated citrullination of host proteins.

Supported by NIH NIDCR grants, DE-027851, DE-028715 and DE-331851
IgA and FcαR are critical components of plasmacytoid DC response to autoantibody-containing immune complexes in SLE.
Authors: Waterman Hayley R , Duffort Matthew , Posso Sylvia , Buckner Jane H , Hamerman Jessica A
Published: January 2023

Systemic lupus erythematosus (SLE) is a heterogenous autoimmune disease characterized by the presence of circulating autoreactive anti-nuclear antibodies (ANAs). ANAs form immune complexes (ICs) upon binding to nuclear antigens and these ANA-ICs promote a feedforward loop by enhancing immune responses in cells that recognize ANA-ICs via Fc receptors (FcRs). Plasmacytoid dendritic cells (pDCs) are FcR expressing cells that promote SLE pathology. ANA-ICs deliver nucleic acids to endosomal TLR7 and TLR9. These TLRs induce type I IFN secretion and immune activation upon nucleic acid recognition. Type I IFNs, including IFNα, and interferon stimulated genes are correlated with SLE disease activity. IgG isotype facilitated ANA-IC internalization via FcgRIIA has been the most studied antibody-mediated route for IFNα production in pDCs. The importance of IgA isotype ANAs remains under investigated despite these autoantibodies being present in half of SLE patients. We show here the novel result that human pDCs express the IgA-specific FcαR (CD89) by flow cytometry, that pDCs from SLE patients express increased FcαR compared to pDCs from healthy controls, and that increased pDC FcαR expression in SLE correlates with hallmark IFNα gene set. Additionally, we found that IgA autoantibodies were a critical component of pDC ANA-IC activation when these ANA-IC were generated with serum from IgA autoantibody positive SLE donors complexed with smRNP nuclear antigen. Therefore, our study shows that IgA-containing immune complexes and FαRI are important contributors to pDC type I IFN production in SLE.

Supported by LRA Lupus Mechanisms and Targets Award (JAH), R21AI154841 (JAH), and ITHS TL1 training grant (HRW).
Small molecule inhibition of spleen tyrosine kinase mitigates gut-liver axis inflammation and histopathology in a novel model of IBD-associated liver disease.
Authors: Jewell Mark L , Xue Yue , Huang Hsin-I , Lamagna Chrystelle , Hammer Gianna E
Published: January 2023

In light of recent studies identifying gain-of-function variants of spleen tyrosine kinase (Syk) as a cause of multi-organ inflammatory disease, Syk inhibition has become an attractive pharmacological target. Therapeutic potential of Syk inhibition may extend beyond that of gain-of-function mutations, since Syk activity initiates downstream responses to several pattern-recognition receptors, which are collectively thought to trigger pathology in several inflammatory diseases, including inflammatory bowel disease (IBD). We previously identified the IBD-linked gene Tnfaip3 (A20) as a suppressor of Syk-dependent inflammatory signals in dendritic cells (DCs) and report here that small molecule inhibition of Syk with FDA-approved drug Fostamatinib mitigates the small intestinal inflammation that develops spontaneously in mice with DC-specific loss of A20 (A20cko mice). In our investigation of IBD in these mice we surprisingly found that A20cko mice also had striking inflammation of the liver that shared many biochemical and histological features characteristic of IBD-associated liver disease, including extensive bridging fibrosis. Importantly, Syk inhibition normalized liver biochemistries and significantly reduced intrahepatic inflammation and fibrosis. Mechanistically, plasma proteomics and liver immunophenotyping pinpointed the key impact of Syk inhibition on restricting chemokine expression linked to recruitment of pro-inflammatory monocytes into in the liver. Collectively this work identifies the interplay of Syk and A20 signaling as drivers of inflammation along the gut-liver axis and suggests Syk is a valid target for pharmacological intervention for IBD and IBD-associated liver disease.

Supported by R01AI145930-01A1
CB2 engagement enhances group 2 innate lymphoid cell expansion and induction of airway hyperreactivity
Authors: Hurrell Benjamin P , Helou Doumet Goerges , Shafiei-Jahani Pedram , Howard Emily Diane , Painter Jacob Dean , Quach Christine , Akbari Omid
Published: January 2023

Cannabinoids modulate the activation of immune cells and physiological processes in the lungs. Group 2 innate lymphoid cells (ILC2)s are central players in type-2 asthma, but how cannabinoids modulate ILC2 activation remains to be elucidated. Using a combination of cannabinoid receptor (CB)2 KO mice, a CB2 antagonist and agonist, we here provide evidence that CB2 signaling in ILC2s is important for the development of ILC2-driven airway inflammation in both mice and human. We show that both naïve and activated murine pulmonary ILC2s express CB2. CB2 signaling did not affect ILC2 homeostasis at steady state, but strikingly stimulated ILC2 proliferation and function upon activation using various models of airway inflammation including IL-33, IL-25 and Alternaria alternata. As a result, ILC2s lacking CB2 induced lower lung inflammation, as we made similar observations using a CB2 antagonist. Conversely, CB2 agonism remarkably exacerbated ILC2-driven airway hyperreactivity and lung inflammation. Mechanistically, transcriptomic and protein analysis revealed that CB2 signaling induced CREB phosphorylation in ILC2s. Human ILC2s expressed CB2, as CB2 antagonism and agonism showed opposing effects on ILC2 effector function and development of airway hyperreactivity in humanized mice. Collectively, our results define CB2 signaling in ILC2s as an important modulator of airway inflammation. Furthermore, our findings highlight the stimulatory capacity of cannabinoids on ILC2s and offer new therapeutic avenues, including the use of substances or pathways able to modulate CB2 and capable of alleviating lung function in patients with lung inflammation.
The role of Cul5 in Group 2 innate lymphoid cells (ILC2s)
Authors: Guha Ipsita , Dar Asif Amin , Kumar Binod , Porter Nadia , Oliver Paula M
Published: January 2023

Over the past few years we have witnessed a change in the study of allergic inflammation from a view that holds T helper type 2 cells at the centre of immune orchestration to one that appreciates the significant contribution made by innate lymphoid type 2 cells (ILC2s). ILC2 cells are considered to be the innate counterpart of Th2 cells and these two evolutionarily related cells cooperate to protect the host against helminth infection and, when dysregulated, in the pathogenesis of allergic diseases. We recently determined that the E3 ubiquitin ligase Cul5 limits the differentiation and pathogenicity of Th2 cells and protects against allergic airway remodelling in mice. Here we show that Cul5 also regulates ILC2 cells. Specifically, following IL-33 treatment, mice lacking Cul5 in all hematopoietic cells contained increased numbers of ILC2 cells in their lungs and this corelated with significant Type 2 inflammation. Additionally, in the absence of Cul5, ILC2 cells were more likely to produce IL-9. Similar results were also observed in mice lacking Cul5 specifically in ILC2 cells (Cul5fl/fl-R5/+), supporting that Cul5 restricts the numbers and function of ILC2 cells. Ex vivo and following in vitro culture, Cul5-deficient ILC2 cells exhibited distinct features that would likely impact their responsiveness to environmental cues such as cytokines. Here we will detail how Cul5 impacts the differentiation and function of ILC2 cells and how this may alter the outcome of type 2 immune responses.
TSLP and IL-33 distinctively modulate the allergic response by differentially modulating Th2 cells and ILC2s
Authors: Gurram Rama Krishna , Zhu Jinfang (Jeff) , Leonard Warren J
Published: January 2023

Alarmins are endogenous chemotactic or immune-activating proteins released after cell injury or immune activation, and these include thymic stromal lymphopoietin (TSLP) and IL-33. Mice lacking these cytokines exhibit defective type 2 responses, but there is controversy regarding the roles of these cytokines in allergic responses in vivo. Here, we demonstrate that the nature of the allergic response is dictated by the alarmin. Mice lacking IL-33 were resistant to pulmonary inflammation induced by papain, but they exhibited normal OVA-induced allergic inflammation. In contrast, mice lacking either TSLP or TSLPR had significantly lower pulmonary inflammation in response to OVA challenge but responded normally to papain. Moreover, while intranasal papain challenge rapidly induced the release of IL-33, OVA challenge predominantly released TSLP. Steady-state pulmonary ILC2s were activated in response to IL-33, and correspondingly, Il33 KO mice showed a similar defect in effector Th2 generation to that observed in animals lacking ILC2s. In contrast to IL-33, TSLP did not activate steady-state ILC2s. Instead, conditional deletion of TSLPR in T cells hampered Th2 cell generation in the OVA-induced asthma model, but these mice responded normally to papain. These data demonstrate that Th2 cells rely on IL-33-stimulated ILC2s, whereas in the OVA-induced asthma model, TSLP acts on Th2 cells to promote type 2 responses. Thus, TSLP and IL-33 are both alarmins, but they have distinctive effects on allergic responses based on their different release kinetics and on their differential actions on Th2 cells versus ILC2s. These findings indicate approaches to selectively target different allergic responses, with potential translational impact.

This research was supported by the Intramural Research Programs of NIAID and NHLBI, NIH.
LMAN1, a Negative Regulator of the Response to House Dust Mite
Authors: Tigno-Aranjuez Justine , Miller Madelyn , Swaby Lindsay , LaFratta Maggie , Zhang Bin
Published: January 2023

We have utilized an unbiased receptor capture approach (CaptiRec) to screen for receptors on dendritic cells which directly bind HDM. Using this technique, we have identified LMAN1 (Lectin, Mannose Binding 1) as a candidate receptor for HDM. LMAN1 is a mannose binding lectin which is primarily known to act as a cargo transporter between the ER, ERGIC, and Golgi compartments. The role played by LMAN1 in the recognition of and response to HDM, is currently unknown. We verify the ability of LMAN1 to directly bind HDM proteins biochemically and in a cellular context. Furthermore, we present evidence to demonstrate that LMAN1 downregulates NF-κB responses in response to inflammatory cytokines and to HDM. Loss of LMAN1 on DCs used for allergic sensitization leads to exacerbated airway inflammation in a HDM model of allergic asthma. Lastly, peripheral DCs of asthmatics show a significant reduction in expression of LMAN1 compared to healthy controls. These findings have potential implications for the development of novel therapeutic interventions for allergy and allergic asthma.

Supported by American Lung Association Innovation Award IA-695632
Sexual dimorphism in the antigen-specific T cell response in S. Typhimurium infections within extra-lymphoid tissues
Authors: D’Souza Shaina J , Immethun Vicki , Godwin Matthew , McLachlan James B
Published: January 2023

Differences in susceptibility to and severity of infectious diseases between the sexes are well established. Females often generate a beneficially stronger immune response when combatting infection but show an increased risk of developing autoimmunity. While the sex-related hormone estradiol is known to affect CD4 T cell cytokine release in vitro, less is known about T cells behavior in vivo. To address this, we intravenously infected male and female mice with attenuated Salmonella Typhimurium (S. Tm). By day 15 post infection (p.i.), male mice showed significantly lower rates of survival. Males also generated a higher number of S. Tm-specific CD4 T cells in the liver but, conversely, displayed higher bacterial burdens. To isolate the role of sex hormones, mice were gonadectomized before reaching sexual maturity and then infected. Ovariectomized mice lost significantly more weight than ovary-intact females by day 15 p.i. Like normal males, ovariectomized females demonstrated a significantly higher number of S. Tm-specific CD4 T cells but also higher liver bacterial burdens. To investigate the role of extra-lymphoid tissues such as the liver, we infected lymphotoxin-alpha (LTa) mice that genetically lack lymphoid tissues. Surprisingly, we found the stark difference in survival and CD4 T cell expansion between the sexes was lost. These differences in T cell expansion, and the commensurate effect on survival, in the presence or absence of endogenous sex hormones or lymphoid tissues indicate that females may have evolved a separate ability to regulate immune responses outside of traditional lymphoid organs. These studies will provide insights into sex-based differences seen in cellular immunity against bacterial pathogens in vivo.

Supported by a grant from the Keck Foundation
Mycobacterium tuberculosis reduces global histone acetylation in human macrophages which is restored by Sirtuin inhibitors
Authors: Jagannath Chinnaswamy , Singh Vipul , Mishra Abhishek , Khan Arshad , Zhang Kangling
Published: January 2023

Mycobacterium tuberculosis (Mtb) infects and grows in human macrophages unless they are activated earlier using IFN-γ. IFN-γ polarized M1-Macrophages (MΦs) and IL-4 driven M2-MΦs respectively kill Mtb or are permissive. Mtb infected M1-MΦs showed an increased expression of iNOS and autophagy-regulating ATGs compared to M2-MΦs. Despite increased bactericidal function, M1-MΦs were unable to eradicate Mtb.

Hypothesis

Because M1- and M2-MΦs differentially expressed Sirtuin1, 2, 3 and Sirt5, we hypothesized that, Sirtuin-dependent histone deacetylation would affect gene expression and bactericidal function

Methods

M0 (naive), M1 or M2-MΦs were treated with inhibitors of Sirtuin1, Sirt2, Sirt3 and Sirt5 followed by Mtb infection, and evaluation of histone modifications using a novel triomics approach. Viability of Mtb was measured before and after Sirtuin blockade and correlated to mRNA expression for iNOS and ATG5 using qPCR.

Results

a) Relative to uninfected M0-MΦs, M1-MΦs showed higher levels of histone acetylation followed by M2-MΦs. b) Mtb infection reduced global histone acetylation in all three MΦ phenotypes, with an concurrent increase in histone methylation. c) Blockade of Sirt1, 2, 3 but not Sirt5 led to a dramatic restoration of histone acetylation in all three phenotypes, although M2-MΦs were still resistant to Sirt3 inhibitors. d) M0, M1- and M2-MΦs killed Mtb more effectively after Sirt2 blockade compared to Sirt1, 3 and 5 inhibition, which was associated with an increase in mRNA for iNOS and ATG5.

Conclusion

We conclude that Mtb decreases histone acetylome of human MΦs and Sirtuin inhibitors provide a new epigenetic mechanism to restore macrophage bactericidal function against tuberculosis.

Supported by NIH NIAID RO1 AI-122070 and AI-161015.
Core fucosylation of N-linked glycans: a novel player in memory CD8+ T cell differentiation following acute viral infection
Authors: Abdelbary Mahmoud , Harbour Jake , Fancher Samantha , Nappi Taylen , Nolz Jeffrey C
Published: January 2023

Core fucosylation (CF) of N-linked glycans is indispensable for normal physiology, but whether CF regulates any aspect of T cell activation, differentiation, or function remains unknown. Fucosyltransferase 8 (Fut8) is the only enzyme in mammals that catalyzes CF and following activation, both expression of Fut8 and CF synthesis increases on CD8+ T cells. To test the hypothesis that CF of N-glycans regulates memory T cell differentiation following acute viral infection, we generated mice with a floxed Fut8 gene. WT (CD4-Cre−) and Fut8−/− (CD4-Cre+) mice were infected with LCMV Armstrong (LCMV-Arm) and the differentiation of LCMV-specific CD8+ T cells was analyzed by measuring the surface expression of killer-cell lectin like receptor G1 (KLRG1) and the α chain of interleukin 7 receptor (CD127) to identify short-lived effector and memory precursor T cells, respectively. LCMV-specific CD8+ T cells from both WT and Fut8−/− followed the same pattern of expansion and contraction without any significant difference in the overall frequency of LCMV-specific CD8+ T cells. However, Fut8−/− retained a higher percentage of CD127+ KLRG1− and a lower percentage of KLRG1+ CD127− CD8 T cells throughout the course of infection suggesting CF influences the progression to long-term memory. At day 42 post infection, mice were infected with virulent Listeria monocytogenes expressing the LCMV epitope GP33–41. Both WT and Fut8−/− GP33-specific CD8+ T cells expanded equally. However, Fut8−/− retained a higher frequency of GP33-specific CD8+ T cells and did not undergo contraction until >3 weeks post-2ry challenge. Taken together, our findings suggest that CF of N-linked glycans regulates memory CD8+ T cell differentiation following acute viral infection.

Supported by grants from NIH (R21 AI159401)
Tim-3 deletion on Treg increases virus-specific T cell response and reduces viral burden in chronic LCMV infection
Authors: Nieves-Rosado Hector M , Banerjee Hridesh , Kane Lawrence P
Published: January 2023

Tim-3, a surface protein, is upregulated on Treg during chronic viral infections. During HCV infection in humans there is an increase in frequency of Tim-3+ Treg, which have increased proliferation, survival, and expression of immunosuppressive cytokines like IL-10. Similarly, it’s been recently reported that there is an increase in Tim-3+ Treg in people with HIV (PWH). However, the role of regulatory T cells (Treg) during chronic viral infection has not been fully defined. We hypothesize that Tim-3 promotes an effector phenotype on Treg during chronic viral infection, which limits the virus-specific T cell response and impairs viral clearance. Using an inducible Treg-specific Tim-3 loss-of-function (Tim-3 Treg KO) model, we found a significant decrease in morbidity during LCMV chronic infection. Tim-3 Treg KO mice mounted a higher virus-specific T cell response and had lower viral burden in blood, liver, and kidney at 30dpi. Lack of Tim-3 expression on Treg limited their ability to upregulate IL-10, TGFb, CD39, PD-1, CD25, CTLA-4 and CD44. Similarly, in PWH Tim-3+ Treg have increased expression of IL-10 compared to HIV(−). In addition, Tim-3+ Treg from PWH show significant upregulation of Ki67+, CD73+, Gzmb+ and TGFb+ compared to their Tim-3− Treg counterpart. Our findings demonstrate that modulation of a surface protein in Treg can lead to a reduction in viral burden and enhance LCMV-specific T cell response. We found that Tim-3+ Treg have an increased suppressive phenotype, compared with Tim-3− Treg in PWH, regardless of viral suppression. Elucidation of the mechanism of Tim-3 function in Treg may provide insights into the biology of chronic infections and for developing novel potential strategies to achieve HIV remission off ART.

Supported by grants from NIH (T32GM008208, R01AI138504, U01AI131285)
Upregulation of CD9 identifies cytokine-producing T follicular helper (Tfh) cells during viral infection
Authors: Antao Olivia Quinn , Sanchez Gina M , Mayer Daniel , Weinstein Jason S
Published: January 2023

T follicular helper (Tfh) cells, a subset of CD4+ T cells, critically regulate the adaptive immune response by promoting germinal center (GC) B cell maturation into memory B cells and antibody secreting plasma cells. Tfh cells promote the GC response through the production of canonical cytokines IL-21 and IFNg, or IL-4 and CD40L signaling. Tfh cells progressively differentiate to regulate the germinal center response, but other than previous studies using cytokine reporter mice, the stages of this differentiation remain unclear. We identified that CD9, a tetraspanin protein, is temporally upregulated on a subset of Tfh cells following acute LCMV infection. To assess the function of the CD9 expressing Tfh cells, we compared migration to GC honing chemokines CXCL12 and CXCL13 and cytokine production between CD9hi and CD9lo Tfh cells at 8 days post LCMV infection. We found that CD9hi Tfh cells exhibited enhanced migration to CXCL12 and CXCL13 compared to CD9lo cells. Ex vivo stimulation of Tfh cells revealed that the CD9hi subsets were the primary producers of IL-21 and IFNg. Transcriptional analysis demonstrated that that CD9hi and CD9lo Tfh cells are distinct subsets characterized by enhanced expression of cell cycling genes in the CD9hi fraction. Thus, not only does CD9 identify the cytokine producing population of Tfh cells but marks a functionally and transcriptionally distinct subset promoting GC responses during viral infections.
Ena/VASP protein-mediated actin polymerization contributes to naive CD8+ T cell activation and expansion by promoting T cell-APC interactions in vivo
Authors: Waldman Monique M , Rahkola Jeremy T , Willett Benjamin A S , Chung Jeffrey W , Sigler Ashton L , Friedman Rachel S , Kedl Ross M , Jacobelli Jordan
Published: January 2023

Naive T cell activation in secondary lymphoid organs such as lymph nodes (LNs) occurs upon recognition of cognate antigens presented by antigen presenting cells (APCs). T cell activation requires cytoskeleton rearrangement and sustained interactions with APCs. Ena/VASP proteins are a family of cytoskeletal effector proteins responsible for actin polymerization and are frequently found at the leading edge of motile cells. Ena/VASP proteins have been implicated in motility and adhesion in various cell types, but their role in primary T cell activation has not been explored. Our goal was to determine the contribution of Ena/VASP proteins to T cell activation and expansion in vivo. Our results showed that naïve T cells from Ena/VASP-deficient mice have a significant reduction in antigen-specific T cell accumulation following Listeria monocytogenes infection. The kinetics of antigen-specific T cell impairment were further confirmed in Ena/VASP-deficient T cells stimulated via dendritic cell immunization. To investigate the cause of this T cell expansion defect, we analyzed T cell-APC interactions in vivo by 2-photon microscopy and observed fewer Ena/VASP-deficient naïve T cells interacting with APCs in LNs during priming. We also found that Ena/VASP-deficient T cells formed conjugates with significantly less actin polymerization at the T cell-APC synapse, and that these conjugates were less stable than their WT counterparts. Thus, we conclude that Ena/VASP proteins contribute to T cell actin remodeling downstream of T-APC interactions required for the initiation of stable T cell conjugates during APC scanning and for efficient activation and expansion of T cells in vivo.

Supported by NIH (R01AI125553; T32AI007405)