bluuebunny/crossref_metadata_2025 huggingface dataset:
A speaking-intensive model for case studies in undergraduate immunology
Authors: Redmond Sarah , Mohamed Attia
Published: January 2023

Effective communication is consistently identified as a key attribute of successful graduates seeking employment or additional training. While most undergraduate programs place a focus on written communication, many interactions students have leading up to and during these new experiences require dialogue with peers, overseers, or the public. To prepare students for those interactions we use a group-presentation model in which students are responsible for three 50-minute presentations in their undergraduate immunology course. Student-led class sessions present challenges – real-time assessment, reliance on students to accurately and thoroughly convey information, and class engagement throughout the presentation. We use case studies as the basis for each presentation, with a set structure that requires presenters to address background, clinical details, scientific literature, and case-specific questions. Basic information is given to non-presenters and presenters receive additional information allowing for a preparatory meeting. Using a hybrid model for discussions, non-presenters can attempt to “solve” the case and ask questions before the class meets. In addition to the in-class assessment, students are graded on their preparation for the preparatory meeting and reflect on each other’s performance. Students were sampled at the beginning and end of the course using the CURE assessment (5 point scale), and reported a 0.74 point increase in critical thinking skills and a 0.44 point increase in speaking skills, as compared to a 0.09 point increase in other academic skills. We will share instructor, teaching assistant, and student perspectives.
Characterization of AIM2 expression in human macrophages during M. tuberculosis infection
Authors: Ontiveros Carlos Oscar , Arnett Eusondia , Schlesinger Larry S.
Published: January 2023

Tuberculosis (TB) is a leading contributor to global mortality and is a well-known threat to global health. The disease-causing bacterium, Mycobacterium tuberculosis (M.tb), has demonstrated resistance to current treatments, thus necessitating novel methods to fight the disease. The investigation of host-bacterium interactions may provide insight for development of effective host-directed therapies, thus circumventing antibiotic resistance. The body’s defense system against airborne M.tb includes alveolar macrophages (AMs) responsible for elimination of foreign agents. However, they do not effectively eliminate M.tb, leading to bacterial propagation. It was previously found that the NOD-like receptor, absent in melanoma 2 (AIM2), plays key roles in M.tb infection in murine macrophages. However, the expression of AIM2 in primary human cells during M.tb infection remains unknown. To this end, we characterized AIM2 expression in human monocyte-derived macrophages (MDMs) and AMs during M.tb infection via quantitative real-time PCR (qRT-PCR). We found that M.tb induces AIM2 transcripts in human MDMs and AMs, in a time-dependent manner. We also investigated the contribution of peroxisome proliferator-activated receptor (PPAR)γ to AIM2 expression due to its known regulation of several genes involved in the human macrophage response to M.tb. We found that PPARγ mediates AIM2 expression. In conclusion, the increased AIM2 gene expression observed in MDMs and AMs during M.tb infection necessitates further investigation into its function and its potential use as a target for M.tb host-directed therapy.
Myeloid derived suppressor cells (MDSC) from HIV-infected individuals are defective in innate immunity to Mycobacterium tuberculosis thus increasing the risk of tuberculosis
Authors: Garg Ankita J , Spector Stephen A
Published: January 2023

Tuberculosis (TB) during HIV-infection can occur at any disease stage, even when CD4+ T cell number are reconstituted and virus replication suppressed. We previously showed that compared to HIV-uninfected, immune suppressive CD14+ MDSC are higher in HIV-infected individuals on combined antiretroviral therapy (cART) and with virus suppression. Here we examined if MDSC isolated from HIV-infected individuals are responsible for defective immunity to M tuberculosis (Mtb). PBMCs from HIV-infected individuals on cART and with viral suppression were isolated and CD14+HLA DRhi (DRhi) and HLA DR−/lo (MDSC) cell subsets were sorted by flow cytometry (FACS) and analyzed for the expression of TLR-2, -4 and IL-27R. FACS analysis showed that MDSC and DRhi subsets had comparable expression of surface TLR-2 (p=0.21) and TLR-4 (p=0.13); expression of IL-27R was high on MDSC (p=0.002). MDSC and DRhi subsets were infected with GFP expressing Mtb Erdman strain at MOI 5 in the presence of neutralizing IL-27 or isotype match control antibody. FACS analysis at 2-hrs post infection showed a higher expression of intracellular ROS in DRhi as compared to MDSC subsets (p= 0.05). Neutralization of IL-27 increased ROS expression of DRhi (p=0.005) but not MDSC subset (p=0.07). Furthermore, MDSC as compared to DRhi subset exhibited a higher intracellular replication of Mtb at day-5 post infection (p=0.01). Lastly, gene expression of intracellular MyD88 in MDSC as compared to DRhi subset was lower in response to Mtb (p=0.05). These findings suggest that MDSC in HIV-infected individuals inhibit protective immunological response to Mtb and increase the risk of TB disease in these individuals.
Involvement of Sphingosine Kinase (SK1) during autophagosome formation plays a critical role in controlling the growth of Mycobacterium tuberculosis within Human Mesenchymal Stem Cells
Authors: Khan Arshad , Singh Vipul Kumar , Mann Lovepreet , Papanna Ramesha , Canaday David , David Sunil A , Tsetkov Andrey S , Jagannath Chinnaswamy
Published: January 2023

Mycobacterium tuberculosis (Mtb) evades host defense mechanisms by infecting and persisting in Mesenchymal Stem Cells (MSCs). Thus, MSCs contain M. tuberculosis DNA and harbor viable bacilli, and our recent study showed that autophagy plays a critical role in restricting the growth of Mtb in human MSCs. Activation of human MSCs using rapamycin enhanced the bactericidal function of MSCs. Here, we examined the autophagy pathway associated with the control of Mtb growth within MSCs. When compared to human macrophages, MSCs had increased expression of Sphingosine Kinase 1 (SK1) during Mtb infection, which was also found to be associated with overall increase in autophagic flux and LC3B positive puncta. SK1 also more often co-localized with gfpMtb containing autophagosomes within MSCs when compared to human macrophages. Percentage of SK1 and LC3B double positive M. tuberculosis containing autophagosomes were also significantly higher in MSCs compared to macrophages suggesting that Mtb induces SK1 mediated autophagy in MSCs during infection. To validate the role of SK1 mediated autophagy in inhibition of Mtb growth within MSCs, we knocked down SK1 using siRNA and scrambled control and examined the survival of M. tuberculosis. SK1-siRNA treated MSCs showed an enhanced survival of Mtb, and replication compared to control siRNA treated cells over 21 days of incubation. These results together indicate that SK1 mediates autophagy, autophagosome formation and autophagy dependent inhibition of Mtb growth within MSCs.
Deletion of MCP-1 enhances susceptibility to carbapenem-resistant Klebsiella pneumoniae via modulating neutrophil and macrophage function
Authors: Jin Liliang , Ghimire Laxman , Paudel Sagar , Cai Shanshan , Jeyaseelan Samithamby
Published: January 2023

Monocyte chemoattractant protein-1 (MCP-1) is important for monocyte recruitment to the lungs in response to bacterial infection. MCP-1 is also essential for neutrophil-dependent host defense during Escherichia coli infection. Carbapenem-Resistant Klebsiella pneumoniae (CRKP) infection has been rapidly growing as a life-threatening nosocomial disease in the U.S. and world. However, the role of chemokines and cellular responses in protective immunity to pulmonary infection with CRKP is largely unknown. Here, we investigated the role of MCP-1 in pulmonary host defense against CRKP infection using MCP-1 knockout (KO) mice. We determined survival, bacterial burden, and neutrophil influx, cytokine/chemokine expression as well as cellular functions, such as formation of neutrophil extracellular traps (NETs) and macrophage extracellular traps (METs). MCP-1 is essential for survival and restricting bacterial outgrowth organs. Compared to the C57Bl/6 (control) mice, KO mice showed impaired influx of neutrophils in the airways as assessed by nuclear cell morphology in BALF and by myeloperoxidase activity (MPO) in lung parenchyma. Moreover, the NETs and METs induced by CRKP were reduced in KO mice in both in vitro or in vivo. Intriguingly, subsequent to CRKP infection, KO mice demonstrated increased pyroptosis through enhanced activation of caspase-1, higher expression of IL-1β and cleaved gasdermin-D in the lungs. These findings enhance our understanding of the critical role of MCP-1 in modulating neutrophil function, such as NETosis, METosis and pyroptosis and suggest that therapies targeting modulation of NETs, METs and pyroptosis during CRKP infection can be explored through clinical trials.
Type III interferons are expressed in tuberculosis granulomas and may influence signaling in epithelioid macrophages
Authors: Talukdar Priyanka , Mattila Joshua T.
Published: January 2023

Granulomas are the pathologic hallmark of Mycobacterium tuberculosis (Mtb) infection. Balanced pro- and anti-inflammatory cytokines regulate inflammation in tuberculosis (TB) and determine outcomes at the lesional and organismal level. Understanding what constitutes protective and detrimental inflammation in TB is important for the improvement of current TB therapeutics. The type III interferon family is composed of four isoforms and includes IL-29, IL-28A, IL-28B, and the recently classified IFNλ4. These cytokines may be important regulators of inflammation in TB yet little is known about their relationship with TB or whether they have different properties in granulomas than type I interferons (IFNα/β), a family of cytokines associated with severe TB. To better understand the relationship between IFNλ, IFNα, and TB we characterized IFNλ expression in formalin-fixed paraffin-embedded granulomas from cynomolgus macaques and investigated cytokine expression and STAT phosphorylation by macaque peripheral blood, lung, and lymph node cells. We found that IL-29 and IL-28B are expressed by epithelioid macrophages and neutrophils in granulomas and IFNλ4 had a unique expression in the transcriptionally active region of epithelioid macrophage nuclei suggesting a possible regulatory role for IFNλ4 in these cells. Experiments were also performed to compare IFNα and IFNλ’s effect on antimycobacterial responses in monocyte-derived macrophages, and ability to induce STAT phosphorylation and pro- and anti-inflammatory cytokine expression by T cells, macrophages, monocytes, and neutrophils. Our findings suggest that IFNλ may have unappreciated roles in regulating inflammation at the site of disease in TB.
Phenotypic analysis of alveolar macrophage populations in old mice and their response to Mycobacterium tuberculosis infection
Authors: Lafuse William P , Rajaram Murugesan VS , Wu Qian , Moliva Juan Ignacio , Torrelles Jordi B , Turner Joanne , Schlesinger Larry S.
Published: January 2023

The elderly are more susceptible to pulmonary infections including tuberculosis. Here, we examined the impact of aging on the phenotype and response of mouse alveolar macrophages (AMs) to Mycobacterium tuberculosis (M.tb). AMs were isolated from bronchoalveolar lavage fluid (BALF) of young (3 month) and old (18 month) C57BL/6 mice. AMs from old mice expressed higher mRNA levels of CCL2, IFN-β, IL-10, IL-12p40, TNFα and MIF than young mice and old mice contained higher levels of CCL2, IFN-β, IL-1β and MIF in their BALF. Flow cytometry analysis of AMs showed two distinct AM populations, a major CD11c+ CD11b− and a minor CD11c+CD11b+ population, the latter was significantly increased in old mice. Expression of CD206, TLR2, CD16/CD32, MHC class II and CD86 was higher in the CD11c+CD11b+ AMs and these AMs expressed monocytic markers Ly6C, C3CR1, and CD115, suggesting they are of monocytic origin. Sorted CD11c+CD11b+ AMs expressed higher mRNA levels of CCL2, IL-1β, and IL-6, whereas CD11c+ CD11b− AMs expressed higher mRNA levels of immune-regulatory cytokines IFN-β and IL-10. CD11c+CD11b+ AMs phagocytosed more M.tb, and M.tb in these AMs expressed higher RNA levels of genes required its survival. Our studies identify two distinct AM populations in old mice, specifically a novel CD11c+CD11b+ AM population which is hyper-inflammatory and phagocytoses M.tb more efficiently, yet is more permissive for M.tb survival than the CD11c+CD11b− population which is more immuno-regulatory in nature. Our results also indicate that the lung cytokine environment of old mice contains an unusual array of both pro-inflammatory and immune-regulatory cytokines.
Alveolar macrophage responses to Mycobacterium tuberculosis reveal human-to-human variation in important immunobiology pathways gleaned from functional genomics study
Authors: Azad Abul , Papp Audrey , Zhou Xiaofei , Pietrzak Maciej , Wang Min , Arnett Eusondia , Montoya Maria , Handelman Samuel , Rempala Grzegorz , Lin Shili , Sadee Wolfgang , Schlesinger Larry
Published: January 2023

Human alveolar macrophages (AMs) are the primary target of Mycobacterium tuberculosis (M.tb) in the lung, where M.tb establishes intracellular residence and growth. This study characterizes M.tb-AM interactions and processes driving inter-subject variability. AMs were obtained by bronchoalveolar lavage from 28 healthy donors and cultured in the presence and absence of virulent M.tb strain H37Rv engineered to produce bioluminescence. Luminescence intensity (RLU) was measured at 2, 24, 48, and 72h post infection. At 2, 24, and 72h, we also measured 27 secreted proteins and AM RNA transcriptomes using AmpliSeq, targeting 20,804 genes, including 2,228 non-coding RNAs. RLUs reveal large differences in M.tb uptake at 2h, varying rates of intracellular adaptation between 2 and 24h, and a range of growth rates from 48 to 72h, resulting in discrete donor groups for each type of interaction. We identify 5039 significant differentially expressed RNAs (DE genes), revealing immune-related pathways, gene networks, and hub genes. A sub-group of three AMs with rapid growth rates (72/48h RLU ratios) displays the most significant DE RNA profiles, including TREM1 networks, IL8 and IL1B, CXCL10, and most prominently IDO1 at 72h, recently identified as a key protein characteristic of active TB, in a cluster of 73 tightly co-expressed genes. Large inter-individual differences in secreted proteins are mostly consistent with the RNA results. Our results identify known and novel key genes in the early phase of M.tb infection in a subset of HAMs, potentially relevant to in vivo infection and TB risk. These key genes can guide a search for genetic variants that contribute to TB risk, and potential targets for diagnostics, treatment, and prevention strategies.
Non-canonical activation of Caspase 8 mediates pyroptosis
Authors: Poltorak Alexander
Published: January 2023

Cell death and inflammation are intimately linked during homeostasis, host defense, and tumorigenesis. Pathogenic Yersinia inhibits the MAP kinase TGFβ-activated kinase 1 (TAK1) via the effector YopJ, thereby silencing cytokine expression while activating caspase-8–mediated cell death. Here, using Yersinia pseudotuberculosis in corroboration with costimulation of lipopolysaccharide and (5Z)-7-Oxozeaenol, a small-molecule inhibitor of TAK1, we show that caspase-8 activation during TAK1 inhibition results in cleavage of both gasdermin D (GSDMD) and gasdermin E (GSDME) in murine macrophages, resulting in pyroptosis. The ensuing cell death is rapid and morphologically is similar to pyroptosis. Loss of GsdmD delays membrane rupture, reverting the cell-death morphology to apoptosis. The IL1 response arises from asynchrony of macrophage death during bulk infections in which two cellular populations provide signal 1 for transcriptional upregulation and signal 2 for the inflammasome assembly. Furthermore, we found that human macrophages are resistant to YopJ-mediated pyroptosis, with dampened IL-1β production. Our results uncover a form of caspase-8–mediated pyroptosis and suggest a hypothesis for the increased sensitivity of humans to Yersinia infection compared with the rodent reservoir. Altogether, this knowledge will be fundamental for our understanding of the mechanism of regulation of cell death and the discovery of new drug targets that can span areas from infections to tissue injury both in and ex vivo. This work was supported by the NIH grant AI056234 and Russian Science Foundation Grant 15-15-00100 (to A.P.)
Calpain drives pyroptotic vimentin cleavage, intermediate filament loss, and cell rupture that mediates immunostimulation
Authors: Davis Michael Alan , Fairgrieve Marian R. , den Hartigh Andreas , Yakovenko Olga , Duvvuri Bhargavi , Lood Christian , Fink susan , Thomas Wendy E. , Gale Michael J
Published: January 2023

Pyroptosis is an inflammatory form of programmed cell death following cellular damage or infection. It is a lytic process driven by gasdermin D-mediated cellular permeabilization and presumed osmotic forces thought to induce swelling and rupture. We found that pyroptotic cells do not spontaneously rupture in culture but lose mechanical resilience. As a result cells were susceptible to rupture by extrinsic forces such as shear stress or compression. Cell analyses revealed that all major cytoskeleton components were disrupted during pyroptosis and that sensitivity to rupture was calpain-dependent and linked with cleavage of vimentin and loss of intermediate filaments. Moreover, while release of LDH, HMGB1, and IL-1β occurred without rupture, rupture was required for release of large inflammatory stimuli—ASC-specks, mitochondria, nuclei, and bacteria. Importantly, supernatants from ruptured cells were more immunostimulatory than those from non-ruptured cells. These observations reveal undiscovered cellular events occurring during pyroptosis, define the mechanisms driving pyroptotic rupture, and highlight the immunologic importance of this event.
Nucleosomal double-stranded DNA triggers APOL1 expression through the STINGTBK1-IRF3 pathway
Authors: Davis Shamara E , Khatua Atanu , Popik Waldemar
Published: January 2023

African Americans are four times more likely to develop kidney disease. Racial disparities in the frequency of nondiabetic kidney disease has led to the discovery of the apolipoprotein L1 (APOL1) gene variants, G1 and G2, which exist exclusively in individuals of recent African ancestry. The presence of these kidney risk alleles strongly correlates with several forms of nondiabetic kidney disease including lupus nephritis (LN). However, the mechanism by which APOL1 variants lead to a faster progression to LN in systemic lupus erythematosus (SLE) patients carrying APOL1 risk alleles is unclear. Increased levels of type I interferons (IFNs) and nucleosomal dsDNA fragments (nsDNA) in the blood is the hallmark of lupus erythematosus. We hypothesize that accumulation of dsDNA in the cytosol of podocytes that play a key role in maintenance of the filtration barrier in the kidney activates the DNA sensing pathways leading to expression of type I IFNs and proinflammatory cytokines damaging podocytes. Although type I IFNs stimulate APOL1 expression, it is unknown whether dsDNA induces APOL1 accumulation independently of IFN. Here we show that nsDNA triggers the expression of APOL1 through the activation of the cGAS/IFI16-STING-TBK1-IRF3 pathway. We have also shown that inhibition of type I IFN only partly inhibited APOL1 accumulation in podocytes, demonstrating that nsDNA directly activates expression of the APOL1 gene. We conclude that blocking type I IFN signaling and targeting elements of the nsDNA-induced pathway may represent a novel therapeutic approach to inhibit LN progression in African Americans SLE patients carrying APOL1 kidney risk alleles.
Proteomic profiling of autoinflammatory diseases, NOMID and CANDLE before and after treatment with interleukin-1 or with interferon blockade
Authors: Alehashemi Sara , Sawhney Megha , Sellers Brian , de Jesus Adriana , Cheung Foo , Biancotto Angélique , Goldbach-Mansky Raphaela
Published: January 2023

Systemic Autoinflammatory diseases (SAIDs) present with chronic sterile inflammation. NLRP3 mutation cause the IL-1 mediated SAID Neonatal-Onset Multisystem Inflammatory Disease (NOMID), that presents early in life with fever, rash, neutrophilic meningitis, hearing loss and eye inflammation. Chronic Atypical Neutrophilic Dermatosis, Lipodystrophy and Elevated Temperature (CANDLE) caused by additive loss-of-function mutations in proteasome genes including PSMB8, leads to chronic type 1 interferon signaling, characterized by early-onset fever, neutrophilic panniculitis, lipodystrophy, cytopenias, myositis and lymphocytic meningitis. IL-1 blocking treatments are approved for NOMID, and JAK-inhibitors show efficacy in CANDLE treatment.

We used proteomic analysis to compare differentially-regulated proteins in active NOMID and CANDLE compared to healthy children before and after treatment to identify inflammatory pathways that distinguish NOMID and CANDLE and are uniquely responsive to either IL-1 or IFN signaling blockade.

Serum samples from active NOMID (n=12) and CANDLE (n=7) before and after treatment and healthy controls (n=7) were subjected to proteomic profiling using SomaLogic platform (n=1129 proteins). Enriched pathways were identified in REACTOME and KEGG. Cytoscape was used for network extraction and visualization.

Differentially expressed proteins in NOMID vs CANDLE suggested endothelial cell function, coagulation and complement cascades and inflammation related pathways dysregulation. Distinct protein markers (i.e. cytokine and chemokines) are consisted the different immune dysregulation in NOMID and CANDLE; and uniquely respond to either IL-1 or IFN signaling blockade.
Regulation of GLI3 expression by TLR4 signaling
Authors: Matissek Stephan Josef , Elsawa Sherine F
Published: January 2023

While acute inflammation is an important host response to infections, chronic inflammation has emerged as a mechanism driving many diseases including autoimmune and malignant diseases. Monocytes are a subset of immune cells that secrete inflammatory cytokines in response to infections through toll-like receptor (TLR) signaling. The cross-talk between TLR signaling and other pathways has been shown to be important for downstream effector functions. We identify a novel role for the transcription factor GLI3, a negative regulator of Hedgehog signaling, in mediating TLR4-induced inflammation. Stimulation of monocytes with LPS increases GLI3 mRNA and protein expression. This occurred in a dose- and time-dependent manner. Investigation of the signaling mechanism suggests that LPS induced GLI3 occurs through the adaptor protein TRIF (but not MyD88). Targeting of transcription factors NFkb, AP-1 and IRF3 by pharmaceutical inhibitors suggests that TLR4-mediated GLI3 is regulated by IRF3. Transfection of our monocyte cell lines with shRNA targeting GLI3 suggests a regulatory role of transcription factor GLI3 in mRNA expression of essential cytokines involved in inflammation including IL-6 and CCL2. We generated conditional GLI3 knockout in mice myeloid cells using LysM-cre mediated recombination. IFA-elicited macrophages from mice with conditional GLI3 knockout driven by LysM-cre stimulated with LPS show reduced IL-6 and CCL2 secretion compared to macrophages from wild-type littermates. Taken together, these data suggest that GLI3 plays a role in innate immunity by regulating TLR4-induced inflammation.
Ligand-induced IFNγ down regulation calibrates myeloid cell IFNγ responsiveness
Authors: Crisler William J , Eshleman Emily , Lenz Laurel L
Published: January 2023

Inflammation triggers production of type II IFN (IFNγ), which induces expression of gamma-activated genes (GAGs) that enhance antigen presentation and antimicrobial activity associated with M1-type macrophage activation. Negative regulation of these responses is important to prevent excessive macrophage activity and inflammation. We report here that stimulation with IFNγ transiently reduces myeloid cell IFNGR to raise the threshold for STAT1 activation in response to subsequent IFNγ. IFNγ production contributed to reduced monocyte surface IFNGR during the first day after systemic Listeria monocytogenes (Lm) infection. IFNγ treatment also reduced IFNGR staining on cultured bone marrow derived macrophages (BMDM) and dendritic cells (BMDCs), and human monocytes. These reductions in IFNGR were independent of type I IFNs, the primary trigger for IFNGR reductions later after Lm infection. Reductions in IFNGR correlated with reduced macrophage ifngr1 transcript abundance but this was independent of effects on the proximal Ifngr1 promoter. IFNGR down regulation following a brief exposure to IFNγ was further observed to transiently reduce myeloid cell sensitivity to subsequent IFNγ, measured as induction of phosphorylated (p)STAT1. Prolonged or elevated IFNγ could overcome the suppression of pSTAT1 activation. These data suggest transient IFNγ production reduces IFNGR to calibrate M1-type myeloid cell activation.
IRF3 signaling in stromal cells contributes to sepsis pathogenesis
Authors: Walker Wendy , Harper Jourdan , Lopez Charlie , Nellikappallil Jose , Heipertz Erica L
Published: January 2023

Interferon regulatory factor 3 is a transcription factor that induces type 1 interferons and related inflammatory genes following activation of pattern recognition receptors. Our prior report demonstrates that IRF3-KO mice are substantially protected from sepsis in a lethal mouse model of cecal ligation and puncture (CLP). Here, we demonstrate that IRF3 remains relevant in the context of a CLP model incorporating clinical treatments (lactated ringer’s solution and antibiotics). 92.3% of IRF3-KO mice survived sepsis in this model, compared to 61.5% of wild-type mice. Septic IRF3-KO mice also had an improved disease score and lower levels of serum cytokines (IL-6 and IL-12), relative to wild-type counterparts. To examine if IRF3 exerts its effect within leukocytes, we generated bone marrow chimeras. We found that chimeras with IRF3-KO leukocytes did not exhibit a significant survival advantage over chimeras with wild-type leukocytes. However, chimeras with IRF3-KO stroma exhibited substantially improved survival relative to chimeras with wild-type stroma, paired with lower disease score and serum cytokines (IL-6 and IL-12). In vitro experiments with thioglycollate-elicited macrophages from IRF3-KO and wild-type mice demonstrate that IRF3 does not directly reduce the amount of IL-6 produced in response to cecal bacteria. However, wild-type monocytes that are adoptively transferred into septic hosts produce less IL-6 when the host is IRF3-KO vs wild-type. These experiments reveal the surprising finding that IRF3 exerts its major effect on sepsis pathogenesis through its presence in stromal cells, with indirect effects on leukocytes.
Assessment of the global chromatin landscape and transcriptome of peripheral iNKT cell subsets
Authors: Paynich Mallory L , Engel Isaac , Seumois Gregory , Liang Shu , de la Mata Sara Herrera , Sethi Ashu , Premlal Ashmitaa Logandha Ramamoorthy , Greenbaum Jason A , Vijayanand Pandurangan , Rao Anjana , Scott-Browne James , Kronenberg Mitchell
Published: January 2023

Vα14i invariant Natural Killer T (iNKT) cells are important modulators of immune responses. They differentiate into three effector cell subsets in the thymus, NKT1, NKT2, and NKT17, which resemble Th1, Th2, and Th17 CD4 T cells. After exiting the thymus, iNKT cells localize to tissues and do not recirculate, but the impact of tissue localization on the epigenetic landscape and transcriptome of each subset is not known. We assessed global chromatin accessibility and transcriptional activity using ATACseq and RNAseq in iNKT cell subsets isolated from the thymus, spleen, liver, and lung. By comparing iNKT cell subsets within each tissue, we found over 5,000 differentially accessible regions of chromatin. This is consistent with previous analyses showing great differences in the transcriptomes of the thymic iNKT cell subsets. Interestingly, relatively limited differences in the accessible chromatin or transcriptome were observed when comparing the same subset from different sites, e.g., NKT1 cells from different tissues. An exception is the unique set of accessible chromatin regions and transcripts identified in all iNKT cell subsets in the lung. Lung iNKT cells exhibited features of increased AP-1 transcription factor activity, which has been correlated with both activation and tissue residency, found in other lung lymphocyte populations. After antigen challenge, an iNKT cell subset with features similar to T follicular helper cells is abundant; these cells are highly different from the other subsets. Taken together, these data indicate that the epigenome and transcriptome of iNKT cells is highly impacted by subset decision, and much less by tissue localization, although dynamic changes occur after antigen exposure.
Sustained E protein activity selectively inhibits ILC2 development
Authors: Berrett Hannah R , Alberola-Ila Jose
Published: January 2023

Innate lymphoid cells (ILCs) are among the first cells to respond to invading pathogens. They coordinate the immune response through the use of effector programs, primed cell responses that cause release of specific cytokines. There are three ILC subsets, ILC1s, ILC2s, ILC3s. All three share a developmental requirement for Id2, a dominant negative inhibitor of the E protein family of transcription factors. To examine how E protein activity affects ILC subset development, we expressed ET-2 during ILC development in mice. ET-2 is a chimeric molecule which restores some E protein activity by competing with Id2 for E protein binding. We examined ILCs in their site of development, the bone marrow, and in a site of mature cells, the lungs. While we did not see changes in overall populations of ILC subsets between control and ET-2 mice, we did see a selective decrease in the percentage of ET-2-expressing ILC2s in ET-2 mice in both bone marrow and lungs. However, these ET-2-expressing ILC2s were still capable of secreting their effector program cytokines of IL-5 and IL-13. These results indicate that partially sustaining E protein activity negatively affects ILC2 development, but not that of ILC1s or ILC3s.
Interleukin-37 improves aging-associated declines in adaptive immunity leading to enhanced suppression of Ph+ leukemia
Authors: Henry Curtis J , Eisenmesser Elan , Dinarello Charles A , DeGregori James
Published: January 2023

Currently over 600 million people worldwide are over the age of 65. This number is predicted to reach 1.6 billion by 2050, representing a 17 percent increase in the world’s aged population. This amazing achievement also presents with concerns, notably our inability to effectively treat older patients with diseases of varying etiologies. Chronic inflammation, which commonly manifests in aged populations, has been shown to promote oncogenesis and compromise immunity. However, the relationship between these aging-associated manifestations are still unclear.

In preliminary studies we found that aging-associated inflammation promotes the expansion of inhibitory immune cells in the spleen, increases the surface expression of the inhibitory mediators PD1/PD-L1 on immune cells, and attenuates interferon-gamma (IFN-γ) production in both CD4+ and CD8+ T-cells. Surprisingly, we have discovered that the anti-inflammatory cytokine interleukin-37 (IL-37) significantly reduces inflammation in aged mice and restores IFN-γ production in both T-lymphocyte populations to almost youthful levels. Importantly, recombinant IL-37 (rIL-37) significantly extends the survival of aged mice with Ph+ leukemia. These observations suggest that chronic inflammation attenuates immunity in aged mice which can be rejuvenated by IL-37 exposure. Since attenuated immunity and increased disease prevalence are hallmarks of aging, our studies highlight the potential use of rIL-37 as a treatment strategy to improve immunity in aged populations to combat disease.
Keap1-Nrf2 system controls cellular metabolism to govern NKT cell homeostasis and effector functions
Authors: Pyaram Kalyani , Kumar Ajay , Chang Cheong-Hee
Published: January 2023

Kelch-like ECH-associated protein (Keap1) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) proteins work in concert as anti-oxidant system to regulate reactive oxygen species (ROS) levels. Additionally, Keap1-Nrf2 system has been shown to play a role in T cell-mediated inflammation but its role for maintenance and function of innate T cells is unclear. Using mice with T-cell specific deletion of Keap1 (KO) leading to higher Nrf2 activity, we recently reported that Keap1-Nrf2 system plays an essential role in the development and homeostasis of invariant natural killer T (NKT) cells. At steady state, KO NKT cells show increased rates of proliferation as well as apoptosis together with altered cell metabolism involving higher glucose uptake, glucose transporter-1 expression, mitochondrial mass and mitochondrial potential but lower ROS than WT NKT cells. Interestingly, upon activation with specific ligand a-GalCer, KO NKT cells proliferated lesser than WT NKT cells. Gene expression data indicated higher glycolysis and lower glutaminolysis in activated KO NKT than WT NKT cells. Further, unstimulated WT NKT cells were found to have elevated levels of glutamine and glutamate. NKT cells stimulated in the absence of glutamine survived and proliferated poorly but had no effect on cytokine production suggesting the importance of glutamine metabolism and keeping a check on glycolysis for the homeostasis and proliferation of NKT cells. Overall, our findings indicate that, via Nrf2, Keap1 positively regulates glutaminolysis and negatively regulates glycolysis to maintain NKT cell homeostasis and promote activation-driven proliferation. Here, we report a yet unknown role of Keap1-Nrf2 system in the cell metabolism of T cells.
Liver macrophages controls Mycobacterium tuberculosis growth by enhancing autophagy
Authors: Thandi Ramya Sivangala , Tripathi Deepak , Radhakrishnan Rajesh Kumar , Paidipally Padmaja , Vankayalapati Ramakrishna
Published: January 2023

Alveolar macrophages are the first cells to come in contact with TB pathogen in lung, but unable to eliminate Mycobacterium tuberculosis (M. tb) completely and serves as niche. In the current study, we compared M. tb growth and cytokine production by murine alveolar and liver macrophages. We found three-fold higher CFU in alveolar macrophages compared to liver macrophages (5 ± 1.7 × 106 vs. 1.776 ± 0.5 ×106 CFU) indicating that liver macrophages are more efficient in inhibiting M. tb growth. In contrast, M. tb H37Rv infected alveolar and liver macrophages produced equal amounts of TNF-α, IL-6, IL-10 & IL-1β. There is no significant difference in the apoptosis and M1/M2 paradigm of M. tb infected alveolar and liver macrophages. Flow cytometry analysis revealed the percentage of LC-3B+ M. tb infected liver macrophages were two-fold higher compared to percentage of LC-3B+ M. tb infected alveolar macrophage. This was confirmed by real time PCR. We also noted distinctly enhanced LC-3B puncta formation in infected liver macrophages as examined by confocal microscopy. We found significantly enhanced M. tb CFU in liver macrophages transfected with LC-3B, ATG-5, ATG-7 and Beclin-1 siRNA compared to liver macrophages transfected with control siRNA (p<0.01; 0.006; 0.01&0.002) respectively. Our results suggest that autophagy is involved in better restriction of M. tb growth by liver macrophages. Studies are underway to compare metabolic changes of control and M. tb H37Rv infected alveolar and liver macrophages and in vivo relevance to our current findings using mouse model of M. tb infection.
Unrelated respiratory infections compromise established cellular immunity by promoting apoptosis of pre-existing lung-resident memory CD8 T cells
Authors: Hayward Sarah L , Li ZR Tiger , Lobby Jenna L , Eggert Joel O.P. , Kohlmeier Jacob E
Published: January 2023

Lung-resident memory T cells (lung TRM) are critical for protective heterosubtypic immunity against influenza viruses. However, the efficacy of cellular immunity against respiratory pathogens such as influenza wanes over time due to the gradual loss of flu-specific lung TRM. One possible mechanism for this decline is frequent exposure to environmental and biological insults resulting in localized inflammation that promotes the death of established lung TRM. We investigated whether unrelated infections could exacerbate the loss of pre-existing, flu-specific lung TRM and reduce the efficacy of cellular immunity to subsequent influenza challenge. Infection of influenza-immune mice with Sendai virus, an unrelated murine parainfluenza virus, resulted in significantly higher viral titers and greater morbidity following influenza challenge compared to PBS-treated controls. This loss of protective cellular immunity corresponded to a significant decrease in the number of pre-existing flu-specific lung CD8 TRM compared to PBS controls due to increased apoptosis. This loss of pre-existing lung TRM following Sendai infection was not due to competition for limited resources or tissue niches between flu- and Sendai-specific lung TRM. Sendai infection had no impact on the number of systemic flu-specific memory CD8 T cells in the spleen. The loss of lung TRM required a respiratory infection, as LCMV infection and intranasal delivery of TLR agonists did not significantly reduce the number of pre-existing flu-specific lung CD8 TRM. Together, these data suggest that tissue damage induced by unrelated respiratory infections can promote the loss of pre-existing lung TRM and compromise cellular immunity against respiratory pathogens.
Prior Bacillus Calmette-Guérin (BCG) vaccination ameliorates the pathogenesis of Type 2 diabetes mellitus mice infected with Mycobacterium tuberculosis
Authors: Radhakrishnan Rajesh Kumar , Tripathi Deepak , Thandi Ramya Sivangala , Paidipally Padmaja , Vankayalapati Ramakrishna
Published: January 2023

Previously, we found experimentally induced type 2 diabetes mellitus (T2DM) mice are highly susceptible to Mycobacterium tuberculosis (Mtb) infection. In the present study, we determined whether prior BCG vaccination has any effect on the susceptibility of T2DM mice infected with Mtb. C57BL/6 mice were vaccinated with BCG or treated with PBS, three months later some mice were induced with T2DM and after another month fifty percent of all groups of mice were challenged with Mtb. All Mtb infected T2DM mice and 40% of uninfected T2DM mice died within 10 months. In contrast, only 40% of Mtb infected BCG vaccinated T2DM mice and 20% of uninfected BCG vaccinated T2DM mice died. Lung bacterial burden was significantly less in Mtb infected BCG vaccinated T2DM mice compared to PBS treated Mtb infected mice. Pro and anti-inflammatory cytokine levels were significantly high in the lungs of Mtb infected BCG vaccinated T2DM mice compared to all other groups of vaccinated and PBS treated mice infected with Mtb. Our findings suggest that prior BCG vaccination protects Mtb infected mice from T2DM induced pathogenesis. Studies are underway to determine the BCG induced mechanism/s those protect T2DM mice challenged with Mtb.
Acetylcholine and Cholinergic Lymphocytes in the Immune Response to Influenza
Authors: Phillips Joy A , Horkowitz Alex , Feuer Ralph
Published: January 2023

Acetylcholine acts to control inflammation in part via binding to α7 nicotinic acetylcholine, receptors on inflammatory macrophages, inhibiting NF-κB nuclear translocation and down-regulating ongoing inflammatory cytokine synthesis. This pathway is presumed to function in the lungs, but the source of local acetylcholine in response to pulmonary inflammation is as yet unknown. We have found evidence that the airway acetylcholine concentration increases over the course of influenza infection, peaking 10 days after infection before rapidly decreasing. The kinetics of acetylcholine increase and loss is mirrored by the appearance and disappearance of cholinergic CD4 T cells in the lungs and airways during influenza illness and resolution. These CD4 T cells bind to influenza-specific tetramers, indicating antigen specificity. This is the first evidence for cholinergic lymphocyte participation in the response to respiratory viral infection. The role for acetylcholine during infleunza recovery is as yet unknown. Decreasing the airway acetylcholine concentration at the later stages of influenza infection leads to extended inflammation marked by increased expression of Allograft-inhibitory factor 1 (Aif-1, IBA1). Together, the results indicate that acetylcholine produced by cholinergic lymphocytes act to regulate pulmonary inflammation during viral infection.
CD4+ resident memory T cells dominate immunosurveillance and orchestrate local recall responses
Authors: Beura Lalit K , Fares-Frederickson Nancy J , Steinert Elizabeth Marie , Scott Milcah C , Thompson Emily A , Fraser Kathryn A , Schenkel Jason M , Vezys Vaiva , Masopust David
Published: January 2023

Migratory dynamics of T cells influence outcomes of infections and cancer. This study addresses how CD4+ T cell immunosurveillance strategies compare to CD8+ T cells by analyzing migration, transcriptome, phenotype, and function. After acute viral infection, memory CD4+ T cells predominantly utilized residence to survey nonlymphoid tissues, albeit not as stringently as observed for CD8+ T cells. In contrast, memory CD4+ T cells were more likely to be resident within lymphoid organs than CD8+ T cells. Migration properties of memory-phenotype CD4+ T cells in non-SPF parabionts were similar, generalizing these results to diverse infections and conditions. CD4+ and CD8+ resident memory T cells (TRM) shared overlapping transcriptional signatures and location-specific features, such as granzyme B expression in the small intestine, revealing tissue-specific and migration property-specific, in addition to lineage-specific, differentiation programs. Functionally, mucosal CD4+ TRM reactivation locally triggered both chemokine expression and broad immune cell activation. Thus, residence provides a dominant mechanism for regionalizing CD4+ T cell immunity, and location enforces shared transcriptional, phenotypic, and functional properties with CD8+ T cells.
Single-cell mRNA analysis of colon phagocyte heterogeneity identifies two major macrophage developmental pathways
Authors: Kang Byunghyun , Alvarado Luigi J. , Lehman Michael , Kim Bong-Hyun , Li Peng , Cho Hyeseon , He Jianping , Larochelle Andre , Kelsall Brian L
Published: January 2023

Tissue macrophages (MPs) develop distinctive functions due to unique transcriptional programs driven by local environmental ques. In the intestine, it is known that MPs are heterogeneous cells that differentiate from blood monocytes and embryonic precursors, and are essential for homeostasis with the vast microbiome through their ability to kill invading microbes, clear apoptotic cells, and produce regulatory cytokines. However, the full extent of MP heterogeneity, their developmental relationships, and how the intestinal microbiota affects MP generation is not known. Here, we performed single cell RNA sequencing of colonic myeloid cells from specific pathogen free (SPF) and germ free (GF) C57BL/6 mice and found extensive heterogeneity of colon MP, with at least six different identifiable populations, with less heterogeneity of dendritic cells (DCs). Unsupervised modeling of developmental pathways predicted two major trajectories from common CCR2+precursors resulting in CD11c+CD9+MMRintCD121b+and CD11c−CD9intMMRhiCD121b−MP populations. The generation of both mature populations of colon MPs was impaired in germ-free mice, which coincided with the appearance of a dominant unique population of MPs, whereas the generation of DC populations in GF mice were unaffected or increased. Furthermore, the two major MP populations were clearly distinct from other populations regarding their gene expression profile, localization within the lamina propria, and ability to phagocytose macromolecules from the blood. These data uncover the diversity of intestinal myeloid cell populations, highlight the importance of microbiota on the unique developmental as well as anatomical and functional fates of colon MPs.
Examining the Immune Composition of the Intestinal Mucosa by Mass Cytometry
Authors: Tyler Christopher J , Lundborg Luke , Yeasmin Shaila , Rivera-Nieves Jesus
Published: January 2023

BACKGROUND

The inflammatory bowel diseases (IBD) can affect various segments of the gastrointestinal tract, and the biology behind regional differences in disease remains poorly understood, in part due to a lack of understanding of regional variation of the particular cell subsets present. By utilizing mass cytometry, our objective was to identify regional differences in the immune composition of the digestive tract in healthy individuals.

METHOD

Intestinal pinch biopsies were obtained from the following sites of healthy individuals (n=10); duodenum, terminal ileum, ascending colon, transverse colon, descending colon, and rectum. Immune cells were isolated from biopsies and stained with a panel of 37 markers for analysis using mass cytometry.

RESULTS

Marked variations in immune cell composition were observed along the digestive tract, in particular T and B lymphocytes. Increased abundance of T lymphocytes (>30% of total CD45+ cells) was identified in the duodenum and terminal ileum, whereas B lymphocytes became more abundant (>30% of total CD45+ cells) towards the distal end of the tract. In addition, intestinal homing molecules displayed variation at each site; integrin α4β7, a target for IBD therapeutic intervention, was increasingly expressed in the terminal ileum and ascending colon, compared with the descending colon (Mean 40% vs 25% abundance, n=10, p<0.01).

CONCLUSIONS

Mass cytometry has the ability to identify and characterize numerous distinct immune subsets throughout the intestinal mucosa, allowing a deep understanding of regional differences in immune composition. This understanding may shed light on the pathogenesis of IBD and allow standardization of intestinal sampling during the evaluation of therapeutics.