bluuebunny/crossref_metadata_2025 huggingface dataset:
Migrating Type 2 Dendritic Cells Prime Mucosal Th17 Cells Specific to Small Intestinal Commensal Bacteria
Authors: Ngoi Soo , Yang Yi , Iwanowycz Stephen , Gutierrez Jennifer , Li Yingqi , Williams Christina , Hill Megan , Chung Dongjun , Allen Carter , Liu Bei
Published: January 2023

Dendritic cells (DCs) are professional APCs equipped with MHC-restricted Ags, costimulations, and cytokines that effectively prime and differentiate naive T cells into distinct functional subsets. The immune signals that DCs carry reflect the route of Ag uptake and the innate stimuli they received. In the mucosal tissues, owing to the great variety of foreign Ags and inflammatory cues, DCs are predominantly activated and migratory. In the small intestine, CD4 Th17 cells are abundant and have been shown to be regulated by DCs and macrophages. Using a mouse commensal bacteria experimental model, we identified that the early priming step of commensal-driven Th17 cells is controlled by bona fide Zbtb46-expressing DCs. CCR7-dependent migration of type 2 DCs (DC2s) from the small intestine to the mesenteric lymph nodes (MLNs) is essential for the activation of naive CD4 T cells. The migratory DC2 population in the MLNs is almost exclusively Esam+ cells. Single-cell RNA sequencing highlighted the abundance of costimulatory markers (CD40 and OX40) and chemokines (Ccl22 and Cxcl16) on MLN migratory DCs. Further resolution of MLN migratory DC2s revealed that the Th17-polarizing cytokine IL-6 colocalizes with DC2s expressing CD40, Ccl17, and Ccl22. Thus, early Th17 cell differentiation is initiated by a small subset of migratory DC2s in the gut-draining lymph nodes.
Single-Cell Transcriptomic Analysis Reveals Neutrophil as Orchestrator during β-Glucan–Induced Trained Immunity in a Teleost Fish
Authors: Mu Di , Yang Jin , Jiang Yu , Wang Zhuang , Chen Weijie , Huang Jianchang , Zhang Yuanxing , Liu Qin , Yang Dahai
Published: January 2023

Trained immunity defines long-term memory of innate immunity based on transcriptional, epigenetic, and metabolic modifications of myeloid cells, which are characterized by elevated proinflammatory responses toward homologous or heterologous secondary stimuli in mammals. However, the evidence of trained immunity-associated immune cells and its molecular mechanism in teleost fish remains largely unknown. In this study, we established a trained immunity activation model in turbot (Scophthalmus maximus) and found that administration with β-glucan induces protection against a bacterial infection. Through single-cell RNA sequencing to annotate 14 clusters of innate and adaptive immune cells, as well as two clusters of blood cells, from head kidney and spleen, respectively, we characterized that neutrophil displays cardinal features of trained immunity by analyzing the expression abundance of trained immunity database–related genes at the single-cell level. Subsequently, through establishing an in vivo training and in vitro neutrophil challenge model, we found that the trained neutrophils exhibit a significant elevation of the IL-1R signaling pathway after Edwardsiella piscicida infection. Furthermore, inhibition of neutrophil’s IL-1R signaling pathway through anakinra treatment impaired the heightened production of reactive oxygen, nitrogen species, lactate, as well as the neutrophil extracellular traps formation and bacterial killing ability. Taken together, these findings characterized neutrophil as the orchestrator to express features of trained immunity, and revealed that the IL-1R signaling pathway plays a critical role in induction of trained immunity for bacterial clearance in teleost fish.
Heterotypic Influenza Infections Mitigate Susceptibility to Secondary Bacterial Infection
Authors: Cipolla Ellyse M , Yue Molin , Nickolich Kara L , Huckestein Brydie R , Antos Danielle , Chen Wei , Alcorn John F
Published: January 2023

Influenza-associated bacterial superinfections have devastating impacts on the lung and can result in increased risk of mortality. New strains of influenza circulate throughout the population yearly, promoting the establishment of immune memory. Nearly all individuals have some degree of influenza memory before adulthood. Due to this, we sought to understand the role of immune memory during bacterial superinfections. An influenza heterotypic immunity model was established using influenza A/Puerto Rico/8/34 and influenza A/X31. We report in this article that influenza-experienced mice are more resistant to secondary bacterial infection with methicillin-resistant Staphylococcus aureus as determined by wasting, bacterial burden, pulmonary inflammation, and lung leak, despite significant ongoing lung remodeling. Multidimensional flow cytometry and lung transcriptomics revealed significant alterations in the lung environment in influenza-experienced mice compared with naive animals. These include changes in the lung monocyte and T cell compartments, characterized by increased expansion of influenza tetramer-specific CD8+ T cells. The protection that was seen in the memory-experienced mouse model is associated with the reduction in inflammatory mechanisms, making the lung less susceptible to damage and subsequent bacterial colonization. These findings provide insight into how influenza heterotypic immunity reshapes the lung environment and the immune response to a rechallenge event, which is highly relevant to the context of human infection.
Conserved Role of mTORC1 Signaling in B Cell Immunity in Teleost Fish
Authors: Cao Jia-feng , Ding Li-guo , Wang Qing-chao , Han Guang-kun , Qin Da-cheng , Cheng Gao-feng , Dong Zhao-ran , Mu Qing-jiang , Kong Wei-guang , Liu Xia , Yu Yong-yao , Xu Zhen
Published: January 2023

Mammalian studies have demonstrated that B cell immune responses are regulated by mechanistic target of rapamycin complex 1 (mTORC1) signaling. Teleost fish represent the oldest living bony vertebrates that contain bona fide B cells. So far, whether the regulatory mechanism of mTORC1 signaling in B cells occurred in teleost fish is still unknown. In this study, we developed a fish model by using rapamycin (RAPA) treatment to inhibit mTORC1 signaling and demonstrated the role of mTORC1 signaling in teleost B cells. In support, we found inhibition of mTORC1 signaling by RAPA decreased the phagocytic capacity, proliferation, and Ig production of B cells. Critically, Flavobacterium columnare induced specific IgM binding in serum, and these titers were significantly inhibited by RAPA treatment, thus decreasing Ab-mediated agglutination of F. columnare and significantly increasing the susceptibility of fish upon F. columnare reinfection. Collectively, our findings elucidated that the mTORC1 pathway is evolutionarily conserved in regulating B cell responses, thus providing a new point for understanding the B cells functions in teleost fish.
Helminth Infection–Induced Increase in Virtual Memory CD8 T Cells Is Transient, Driven by IL-15, and Absent in Aged Mice
Authors: Hussain Tabinda , Nguyen Angela , Daunt Carmel , Thiele Daniel , Pang Ee Shan , Li Jasmine , Zaini Aidil , O’Keeffe Meredith , Zaph Colby , Harris Nicola L , Quinn Kylie M , La Gruta Nicole L
Published: January 2023

CD8 virtual memory T (TVM) cells are Ag-naive CD8 T cells that have undergone partial differentiation in response to common γ-chain cytokines, particularly IL-15 and IL-4. TVM cells from young individuals are highly proliferative in response to TCR and cytokine stimulation but, with age, they lose TCR-mediated proliferative capacity and exhibit hallmarks of senescence. Helminth infection can drive an increase in TVM cells, which is associated with improved pathogen clearance during subsequent infectious challenge in young mice. Given the cytokine-dependent profile of TVM cells and their age-associated dysfunction, we traced proliferative and functional changes in TVM cells, compared with true naive CD8 T cells, after helminth infection of young and aged C57BL/6 mice. We show that IL-15 is essential for the helminth-induced increase in TVM cells, which is driven only by proliferation of existing TVM cells, with negligible contribution from true naive cell differentiation. Additionally, TVM cells showed the greatest proliferation in response to helminth infection and IL-15 compared with other CD8 T cells. Furthermore, TVM cells from aged mice did not undergo expansion after helminth infection due to both TVM cell–intrinsic and –extrinsic changes associated with aging.
Cutting Edge: Promoting T Cell Factor 1+ T Cell Self-Renewal to Improve Programmed Cell Death Protein 1 Blockade
Authors: Wang Peter H , Washburn Robert , Maniar Rohan , Mu Michael , Ringham Olivia , Kratchmarov Radomir , Henick Brian S , Reiner Steven L
Published: January 2023

Immune checkpoint blockade is limited by resistance to treatment, with many patients not achieving durable antitumor responses. Self-renewing (T cell factor 1+ [TCF1+]) CD8+ T cells have recently been implicated in efficacy of anti–programmed cell death protein 1 (anti–PD-1). Mice challenged with syngeneic tumors were treated with anti–PD-1 and/or a reversible inhibitor of PI3K δ, designed to promote T cell self-renewal. Growth of tumors in untreated mice was characterized by waning proportions of TCF1+ T cells, suggesting self-renewing T cells become limiting for successful immunotherapy. Higher proportions of TCF1+ T cells in tumor and blood correlated with better control of tumor growth. Combining anti–PD-1 and inhibitor of PI3K δ conferred superior protection compared with either monotherapy and was associated with higher frequency of TCF1+ T cells in tumor and blood compared with anti–PD-1 alone. These findings reveal predictive importance of self-renewing T cells in anti-tumor immunity and suggest that resistance-directed strategies to enhance T cell self-renewal could potentiate the efficacy of PD-1 blockade.
γδ TCRs Function as Innate-like Receptors in the Bovine γδ T Cell Response against Leptospira
Authors: Gillespie Alexandria E , Loonie Kathleen , Lefevre Lucas , Hope Jayne C , Baldwin Cynthia L , Connelley Timothy K
Published: January 2023

Leptospira serovar Hardjo are bacterial pathogens of cattle that also cause zoonotic disease in humans. Vaccine-mediated protection against Leptospira serovar Hardjo in cattle is associated with a workshop cluster 1 (WC1)+ γδ T cell response that can be recalled in vitro from PBMC by antigenic stimulation. This provides a model system in which to examine protective vaccine-induced γδ T cell responses in a γδ T cell high species. Only a small proportion (5–10%) of WC1+ γδ T cells from immunized cattle are Leptospira responders, implying that Ag specificity is determined by clonally distributed receptors. Both WC1 and TCR are known to be required for Leptospira-specific responses by bovine WC1+ γδ T cells. Through variegated expression patterns and V(D)J recombination, respectively, they have the capacity to confer Ag specificity. In this study, we develop and use a high-throughput TCR-sequencing approach to study the TCRγ and TCRδ repertoires of naive ex vivo PBMC, Leptospira-responding, and Leptospira nonresponding WC1+ γδ T cells to examine the potential role of γδ TCR in determining Ag specificity. Our results provide novel insights into the PBMC γδ TCR repertoires in cattle, demonstrating the TCRγ repertoire to be clonally stratified and essentially public, whereas the TCRδ repertoire shows much higher levels of clonal diversity and is essentially private. TCR repertoire analysis of Leptospira-responding WC1+ γδ T cells identifies no signature of TCR-mediated selection, suggesting that TCR functions largely as an innate-like receptor and does not act as a primary determinant of Ag specificity in the response to this pathogen.
IL-10 Negatively Controls the Primary T Cell Response of Tilapia by Triggering the JAK1/STAT3/SOCS3 Axis That Suppresses NF-κB and MAPK/ERK Signaling
Authors: Li Kang , Li Jiaqi , Wei Xiumei , Wang Junya , Geng Ming , Ai Kete , Liang Wei , Zhang Jiansong , Li Kunming , Gao Haiyou , Yang Jialong
Published: January 2023

The braking mechanisms to protect the host from tissue damage and inflammatory disease caused by an overexuberant immune response are common in many T cell subsets. However, the negative regulation of T cell responses and detailed mechanisms are not well understood in early vertebrates. In the current study, using a Nile tilapia (Oreochromis niloticus) model, we investigated the suppression of T cell immunity by IL-10. Tilapia encodes an evolutionarily conserved IL-10, whose expression in lymphocytes is markedly induced during the primary adaptive immune response against Aeromonas hydrophila infection. Activated T cells of tilapia produce IL-10, which in turn inhibits proinflammatory cytokine expression and suppresses PHA-induced T cell activation. Moreover, administration of IL-10 impairs the proliferation of tilapia T cells, reduces their potential to differentiate into Th subsets, and cripples the cytotoxic function, rendering the animals more vulnerable to pathogen attack. After binding to its receptor IL-10Ra, IL-10 activates the JAK1/STAT3 axis by phosphorylation and enhances the expression of the suppressor of cytokine signaling 3 (SOCS3), which in turn attenuates the activation of the NF-κB and MAPK/ERK signaling pathways, thus suppressing the T cell response of tilapia. Our findings elucidate a negative regulatory mechanism of T cell immunity in a fish species and support the notion that the braking mechanism of T cells executed through IL-10 existed prior to the divergence of the tetrapod lineage from teleosts. Therefore, this study, to our knowledge, provides a novel perspective on the evolution of the adaptive immune system.
CTLs Get SMAD When Pathogens Tell Them Where to Go
Authors: Suarez-Ramirez Jenny E , Cauley Linda S , Chandiran Karthik
Published: January 2023

Vaccines protect against infections by eliciting both Ab and T cell responses. Because the immunity wanes as protective epitopes get modified by accruing mutations, developing strategies for immunization against new variants is a major priority for vaccine development. CTLs eliminate cells that support viral replication and provide protection against new variants by targeting epitopes from internal viral proteins. This form of protection has received limited attention during vaccine development, partly because reliable methods for directing pathogen-specific memory CD8 T cells to vulnerable tissues are currently unavailable. In this review we examine how recent studies expand our knowledge of mechanisms that contribute to the functional diversity of CTLs as they respond to infection. We discuss the role of TGF-β and the SMAD signaling cascade during genetic programming of pathogen-specific CTLs and the pathways that promote formation of a newly identified subset of terminally differentiated memory CD8 T cells that localize in the vasculature.
Ring Finger Protein 215 Negatively Regulates Type I IFN Production via Blocking NF-κB p65 Activation
Authors: Wu Yun , Chen Delin , Hu Yiwen , Zhang Shuqing , Dong Xinhuai , Liang Hao , Liang Minqi , Zhu Yujia , Tan Chahui , An Shu , Zhu Xun , Yuan Jie , Li Mengfeng , He Zhenjian
Published: January 2023

Germline-encoded pattern recognition receptors (PRRs) recognize molecules frequently found in pathogens (pathogen-associated molecular patterns [PAMPs]) during viral infection. This process induces production of IFNs, leading to expression of IFN-stimulated genes to establish a cellular antiviral state against viral infection. However, aberrant activation of the IFN system may cause immunopathological damage and systemic autoimmune diseases such as systemic lupus erythematosus. Stringent control of IFN signaling activation is critical for maintaining homoeostasis of the immune system; yet, the mechanisms responsible for its precise regulation remain to be elucidated. In this study, we identified that ring finger protein 215 (RNF215), a zinc finger protein, was upregulated by viral infection in human macrophages. In addition, we demonstrated that RNF215 inhibited the production of type I IFNs at least in part via interacting with p65, a subunit of NF-κB, and repressed the accumulation of NF-κB in the promoter region of IFNB1. Moreover, we found that the expression of RNF215 negatively correlated with type I IFNs in patients with systemic lupus erythematosus, indicating that RNF215 plays an important role in the pathogenesis of autoimmune diseases. Collectively, our data identified RNF215 as a key negative regulator of type I IFNs and suggested RNF215 as a potential target for intervention in diseases with aberrant IFN production.
A Multi-Omics Approach Reveals Features That Permit Robust and Widespread Regulation of IFN-Inducible Antiviral Effectors
Authors: Göczi Loránd , Csumita Mária , Horváth Attila , Nagy Gergely , Póliska Szilárd , Pigni Matteo , Thelemann Christoph , Dániel Bence , Mianesaz Hamidreza , Varga Tamás , Sen Kaushik , Raghav Sunil K , Schoggins John W , Nagy Laszlo , Acha-Orbea Hans , Meissner Felix , Reith Walter , Széles Lajos
Published: January 2023

The antiviral state, an initial line of defense against viral infection, is established by a set of IFN-stimulated genes (ISGs) encoding antiviral effector proteins. The effector ISGs are transcriptionally regulated by type I IFNs mainly via activation of IFN-stimulated gene factor 3 (ISGF3). In this study, the regulatory elements of effector ISGs were characterized to determine the (epi)genetic features that enable their robust induction by type I IFNs in multiple cell types. We determined the location of regulatory elements, the DNA motifs, the occupancy of ISGF3 subunits (IRF9, STAT1, and STAT2) and other transcription factors, and the chromatin accessibility of 37 effector ISGs in murine dendritic cells. The IFN-stimulated response element (ISRE) and its tripartite version occurred most frequently in the regulatory elements of effector ISGs than in any other tested ISG subsets. Chromatin accessibility at their promoter regions was similar to most other ISGs but higher than at the promoters of inflammation-related cytokines, which were used as a reference gene set. Most effector ISGs (81.1%) had at least one ISGF3 binding region proximal to the transcription start site (TSS), and only a subset of effector ISGs (24.3%) was associated with three or more ISGF3 binding regions. The IRF9 signals were typically higher, and ISRE motifs were “stronger” (more similar to the canonical sequence) in TSS-proximal versus TSS-distal regulatory regions. Moreover, most TSS-proximal regulatory regions were accessible before stimulation in multiple cell types. Our results indicate that “strong” ISRE motifs and universally accessible promoter regions that permit robust, widespread induction are characteristic features of effector ISGs.
Monocyte/Macrophage Heterogeneity during Skin Wound Healing in Mice
Authors: Pang Jingbo , Maienschein-Cline Mark , Koh Timothy J
Published: January 2023

Monocytes (Mos)/macrophages (Mϕs) orchestrate biological processes critical for efficient skin wound healing. However, current understanding of skin wound Mo/Mϕ heterogeneity is limited by traditional experimental approaches such as flow cytometry and immunohistochemistry. Therefore, we sought to more fully explore Mo/Mϕ heterogeneity and associated state transitions during the course of excisional skin wound healing in mice using single-cell RNA sequencing. The live CD45+CD11b+Ly6G− cells were isolated from skin wounds of C57BL/6 mice on days 3, 6, and 10 postinjury and captured using the 10x Genomics Chromium platform. A total of 2813 high-quality cells were embedded into a uniform manifold approximation and projection space, and eight clusters of distinctive cell populations were identified. Cluster dissimilarity and differentially expressed gene analysis categorized those clusters into three groups: early-stage/proinflammatory, late-stage/prohealing, and Ag-presenting phenotypes. Signature gene and Gene Ontology analysis of each cluster provided clues about the different functions of the Mo/Mϕ subsets, including inflammation, chemotaxis, biosynthesis, angiogenesis, proliferation, and cell death. Quantitative PCR assays validated characteristics of early- versus late-stage Mos/Mϕs inferred from our single-cell RNA sequencing dataset. Additionally, cell trajectory analysis by pseudotime and RNA velocity and adoptive transfer experiments indicated state transitions between early- and late-state Mos/Mϕs as healing progressed. Finally, we show that the chemokine Ccl7, which was a signature gene for early-stage Mos/Mϕs, preferentially induced the accumulation of proinflammatory Ly6C+F4/80lo/− Mos/Mϕs in mouse skin wounds. In summary, our data demonstrate the complexity of Mo/Mϕ phenotypes, their dynamic behavior, and diverse functions during normal skin wound healing.
Double-Stranded RNA Induces Mortality in an MDA5-Mediated Type I Interferonopathy Model
Authors: Emralino Francine Lianne , Satoh Saya , Sakai Nobuhiro , Takami Masamichi , Takeuchi Fumihiko , Yan Nan , Rutsch Frank , Fujita Takashi , Kato Hiroki
Published: January 2023

Gain-of-function mutations in the viral dsRNA sensor melanoma differentiation–associated protein 5 (MDA5) lead to autoimmune IFNopathies, including Singleton–Merten syndrome (SMS) and Aicardi–Goutières syndrome. However, much remains unclear regarding the mechanism of disease progression and how external factors such as infection or immune stimulation with vaccination can affect the immune response. With this aim, we generated mice with human MDA5 bearing the SMS-associated mutation R822Q (hM-R822Q). hM-R822Q transgenic (Tg) mice developed SMS-like heart fibrosis, aortic valve enlargement, and aortic calcification with a systemic IFN-stimulated gene signature resulting in the activation of the adaptive immune response. Although administration of the viral dsRNA mimic polyinosinic-polycytidylic acid [poly(I:C)] did not have remarkable effects on the cardiac phenotype, dramatic inflammation was observed in the intestines where IFN production was most elevated. Poly(I:C)-injected hM-R822Q Tg mice also developed lethal hypercytokinemia marked by massive IL-6 levels in the serum. Interrupting the IFN signaling through mitochondrial antiviral signaling protein or IFN-α/β receptor alleviated hM-R822Q–induced inflammation. Furthermore, inhibition of JAK signaling with tofacitinib reduced cytokine production and ameliorated mucosal damage, enabling the survival of poly(I:C)-injected hM-R822Q Tg mice. These findings demonstrate that the MDA5 R822Q mutant introduces a critical risk factor for uncontrollable inflammation on viral infection or vaccination.
Cytomegalovirus Seropositivity in Older Adults Changes the T Cell Repertoire but Does Not Prevent Antibody or Cellular Responses to SARS-CoV-2 Vaccination
Authors:   COVID-in-LTC Investigator Group, Breznik Jessica A , Huynh Angela , Zhang Ali , Bilaver Lucas , Bhakta Hina , Stacey Hannah D , Ang Jann C , Bramson Jonathan L , Nazy Ishac , Miller Matthew S , Denburg Judah , Costa Andrew P , Bowdish Dawn M E
Published: January 2023

Chronic infection with human CMV may contribute to poor vaccine efficacy in older adults. We assessed the effects of CMV serostatus on Ab quantity and quality, as well as cellular memory recall responses, after two and three SARS-CoV-2 mRNA vaccine doses, in older adults in assisted living facilities. CMV serostatus did not affect anti-Spike and anti–receptor-binding domain IgG Ab levels, nor neutralization capacity against wild-type or β variants of SARS-CoV-2 several months after vaccination. CMV seropositivity altered T cell expression of senescence-associated markers and increased effector memory re-expressing CD45RA T cell numbers, as has been previously reported; however, this did not impact Spike-specific CD4+ T cell memory recall responses. CMV-seropositive individuals did not have a higher incidence of COVID-19, although prior infection influenced humoral immunity. Therefore, CMV seropositivity may alter T cell composition but does not impede the durability of humoral protection or cellular memory responses after SARS-CoV-2 mRNA vaccination in older adults.
The TCR Cα Domain Regulates Responses to Self-pMHC Class II
Authors: Kim Caleb Y , Parrish Heather L , Kuhns Michael S
Published: January 2023

T cells play a central role in adaptive immunity by recognizing peptide Ags presented by MHC molecules (pMHC) via their clonotypic TCRs. αβTCRs are heterodimers, consisting of TCRα and TCRβ subunits that are composed of variable (Vα, Vβ) and constant (Cα, Cβ) domains. Whereas the Vα, Vβ, and Cβ domains adopt typical Ig folds in the extracellular space, the Cα domain lacks a top β sheet and instead has two loosely associated top strands (C- and F-strands) on its surface. Previous results suggest that this unique Ig-like fold mediates homotypic TCR interactions and influences signaling in vitro. To better understand why evolution has selected this unique structure, we asked, what is the fitness cost for development and function of mouse CD4+ T cells bearing a mutation in the Cα C-strand? In both TCR retrogenic and transgenic mice we observed increased single-positive thymocytes bearing mutant TCRs compared with those expressing wild-type TCRs. Furthermore, our analysis of mutant TCR transgenic mice revealed an increase in naive CD4+ T cells experiencing strong tonic TCR signals, increased homeostatic survival, and increased recruitment of responders to cognate pMHC class II upon immunization compared with the wild-type. The mutation did not, however, overtly impact CD4+ T cell proliferation or differentiation after immunization. We interpret these data as evidence that the unique Cα domain has evolved to fine-tune TCR signaling, particularly in response to weak interactions with self-pMHC class II.
Cutting Edge: Neutrophil Complement Receptor Signaling Is Required for BAFF-Dependent Humoral Responses in Mice
Authors: Cumpelik Arun , Cody Evan , Yu Samuel Mon-Wei , Grasset Emilie K , Dominguez-Sola David , Cerutti Andrea , Heeger Peter S
Published: January 2023

T cell–independent (TI) B cell responses to nonprotein Ags involve multiple cues from the innate immune system. Neutrophils express complement receptors and activated neutrophils can release BAFF, but mechanisms effectively linking neutrophil activation to TI B cell responses are incompletely understood. Using germline and conditional knockout mice, we found that TI humoral responses involve alternative pathway complement activation and neutrophil-expressed C3a and C5a receptors (C3aR1/C5aR1) that promote BAFF-dependent B1 cell expansion and TI Ab production. Conditional absence of C3aR1/C5aR1 on neutrophils lowered serum BAFF levels, led to fewer Peyer’s patch germinal center B cells, reduced germinal center B cells IgA class-switching, and lowered fecal IgA levels. Together, the results indicate that sequential activation of complement on neutrophils crucially supports humoral TI and mucosal IgA responses through upregulating neutrophil production of BAFF.
Cutting Edge: Neutrophils License the Maturation of Monocytes into Effective Antifungal Effectors
Authors: Espinosa Vanessa , Dutta Orchi , Heung Lena J , Wang Keyi , Chang Yun-Juan , Soteropoulos Patricia , Hohl Tobias M , Siracusa Mark C , Rivera Amariliz
Published: January 2023

Neutrophils are critical for the direct eradication of Aspergillus fumigatus conidia, but whether they mediate antifungal defense beyond their role as effectors is unclear. In this study, we demonstrate that neutrophil depletion impairs the activation of protective antifungal CCR2+ inflammatory monocytes. In the absence of neutrophils, monocytes displayed limited differentiation into monocyte-derived dendritic cells, reduced formation of reactive oxygen species, and diminished conidiacidal activity. Upstream regulator analysis of the transcriptional response in monocytes predicted a loss of STAT1-dependent signals as the potential basis for the dysfunction seen in neutrophil-depleted mice. We find that conditional removal of STAT1 on CCR2+ cells results in diminished antifungal monocyte responses, whereas exogenous administration of IFN-γ to neutrophil-depleted mice restores monocyte-derived dendritic cell maturation and reactive oxygen species production. Altogether, our findings support a critical role for neutrophils in antifungal immunity not only as effectors but also as important contributors to antifungal monocyte activation, in part by regulating STAT1-dependent functions.
IgG Fab Glycans Hinder FcRn-Mediated Placental Transport
Authors: Volkov Mikhail , Brinkhaus Maximilian , van Schie Karin A , Bondt Albert , Kissel Theresa , van der Kooi Elvera J , Bentlage Arthur E H , Koeleman Carolien A M , de Taeye Steven W , Derksen Ninotska I , Dolhain Radboud J E M , Braig-Scherer Ute , Huizinga Tom W J , Wuhrer Manfred , Toes René E M , Vidarsson Gestur , van der Woude Diane
Published: January 2023

Abs can be glycosylated in both their Fc and Fab regions with marked effects on Ab function and binding. High levels of IgG Fab glycosylation are associated with malignant and autoimmune conditions, exemplified by rheumatoid arthritis and highly Fab-glycosylated (∼90%) anti-citrullinated protein Abs (ACPAs). Important properties of IgG, such as long half-life and placental transport, are facilitated by the human neonatal Fc receptor (hFcRn). Although it is known that glycosylation of Abs can affect binding to Fc receptors, little is known on the impact of IgG Fab glycosylation on hFcRn binding and transplacental transport. Therefore, we analyzed the interaction between hFcRn and IgG with and without Fab glycans in vitro with various methods as well as in vivo by studying placental transfer of Fab-glycosylated Abs from mothers to newborns. No effect of Fab glycosylation on IgG binding to hFcRn was found by surface plasmon resonance and hFcRn affinity chromatography. In contrast, studies in a cell membrane context revealed that Fab glycans negatively impacted IgG–hFcRn interaction. In line with this, we found that Fab-glycosylated IgGs were transported ∼20% less efficiently across the placenta. This appeared to be a general phenomenon, observed for ACPAs, non-ACPAs, as well as total IgG in rheumatoid arthritis patients and healthy controls. Our results suggest that, in a cellular context, Fab glycans inhibit IgG–hFcRn interaction and thus negatively affect the transplacental transfer of IgG. As Fab-glycosylated Abs are frequently associated with autoimmune and malignant disorders and may be potentially harmful, this might encompass a regulatory mechanism, limiting the half-life and transport of such Abs.
CD40 Drives Central Nervous System Autoimmune Disease by Inducing Complementary Effector Programs via B Cells and Dendritic Cells
Authors: Lu Ying , Xu Max , Dorrier Cayce E , Zhang Ray , Mayer Christian T , Wagner David , McGavern Dorian B , Hodes Richard J
Published: January 2023

Costimulatory CD40 plays an essential role in autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), a murine model of human multiple sclerosis (MS). However, how CD40 drives autoimmune disease pathogenesis is not well defined. Here, we used a conditional knockout approach to determine how CD40 orchestrates a CNS autoimmune disease induced by recombinant human myelin oligodendrocyte glycoprotein (rhMOG). We found that deletion of CD40 in either dendritic cells (DCs) or B cells profoundly reduced EAE disease pathogenesis. Mechanistically, CD40 expression on DCs was required for priming pathogenic Th cells in peripheral draining lymph nodes and promoting their appearance in the CNS. By contrast, B cell CD40 was essential for class-switched MOG-specific Ab production, which played a crucial role in disease pathogenesis. In fact, passive transfer of MOG-immune serum or IgG into mice lacking CD40 on B cells but not DCs reconstituted autoimmune disease, which was associated with inundation of the spinal cord parenchyma by Ig and complement. These data demonstrate that CD40 supports distinct effector programs in B cells and DCs that converge to drive a CNS autoimmune disease and identify targets for intervention.
Macrophage P2Y6 Receptor Signaling Selectively Activates NFATC2 and Suppresses Allergic Lung Inflammation
Authors: Nagai Jun , Lin Junrui , Boyce Joshua A
Published: January 2023

Innate immune responses to innocuous Ags can either prevent or facilitate adaptive type 2 allergic inflammation, but the mechanisms are incompletely understood. We now demonstrate that macrophage UDP-specific type 6 purinergic (P2Y6) receptors selectively activate NFATC2, a member of the NFAT family, to drive an innate IL-12/IFN-γ axis that prevents type 2 allergic inflammation. UDP priming potentiated IL-12p40 production in bone marrow–derived macrophages (BMMs) stimulated by the house dust mite Dermatophagoides farinae (Df) in a P2Y6-dependent manner. Inhibitions of phospholipase C, calcium increase, and calcineurin eliminated UDP-potentiated Df-induced IL-12p40 production. UDP specifically induced nuclear translocation of NFATC2, but not NFATC1 and NFATC3, in BMMs in a P2Y6-dependent manner. UDP-potentiated IL-12p40 production by BMMs and Df-induced IL-12p40 gene expression by alveolar macrophages were abrogated in cells from Nfatc2 knockout mice. Pulmonary transplantation of wild-type but not Nfatc2 knockout macrophages increased Df-induced IL-12 production and IFN-γ expression in P2ry6 fl/fl/Cre/+ recipient mice. Finally, Nfatc2 knockout mice showed significantly increased indices of type 2 immunopathology in response to Df challenge, similar to P2ry6 fl/fl/Cre/+ mice. Thus, macrophage P2Y6 receptor signaling selectively utilizes NFATC2 to potentiate an innate IL-12/IFN-γ axis, a potential mechanism that protects against inappropriate type 2 immune responses.
Long Noncoding RNA HAFML Promotes Migration and Invasion of Rheumatoid Fibroblast-like Synoviocytes
Authors: Xu Siqi , Liu Di , Kuang Yu , Li Ruiru , Wang Jingnan , Shi Maohua , Zou Yaoyao , Qiu Qian , Liang Liuqin , Xiao Youjun , Xu Hanshi
Published: January 2023

The aggressive phenotype exhibited by fibroblast-like synoviocytes (FLSs) is critical for the progression of joint destruction in rheumatoid arthritis (RA). Long noncoding RNAs (lncRNAs) have crucial roles in the pathogenesis of diverse disorders; however, few have been identified that might be able to control the joint damage in RA. In this study, we identified an lncRNA, ENST00000509194, which was expressed at abnormally high levels in FLSs and synovial tissues from patients with RA. ENST00000509194 positively modulates the migration and invasion of FLSs by interacting with human Ag R (HuR, also called ELAVL1), an RNA-binding protein that mainly stabilizes mRNAs. ENST00000509194 binds directly to HuR in the cytoplasm to form a complex that promotes the expression of the endocytic adaptor protein APPL2 by stabilizing APPL2 mRNA. Knockdown of HuR or APPL2 impaired the migration and invasion of RA FLSs. Given its close association with HuR and FLS migration, we named ENST00000509194 as HAFML (HuR-associated fibroblast migratory lncRNA). Our findings suggest that an increase in synovial HAFML might contribute to FLS-mediated rheumatoid synovial aggression and joint destruction, and that the lncRNA HAFML might be a potential therapeutic target for dysregulated fibroblasts in a wide range of diseases.
Germline-Encoded Positional Cysteine Polymorphisms Enhance Diversity in Antibody Ultralong CDR H3 Regions
Authors: Jenkins Gabrielle Warner , Safonova Yana , Smider Vaughn V
Published: January 2023

Ab “ultralong” third H chain complementarity-determining regions (CDR H3) appear unique to bovine Abs and may enable binding to difficult epitopes that shorter CDR H3 regions cannot easily access. Diversity is concentrated in the “knob” domain of the CDR H3, which is encoded by the DH gene segment and sits atop a β-ribbon “stalk” that protrudes far from the Ab surface. Knob region cysteine content is quite diverse in terms of total number of cysteines, sequence position, and disulfide bond pattern formation. We investigated the role of germline cysteines in production of a diverse CDR H3 structural repertoire. The relationship between DH polymorphisms and deletions relative to germline at the nucleotide level, as well as diversity in cysteine and disulfide bond content at the structural level, was ascertained. Structural diversity is formed through (1) DH polymorphisms with altered cysteine positions, (2) DH deletions, and (3) new cysteines that arise through somatic hypermutation that form new, unique disulfide bonds to alter the knob structure. Thus, a combination of mechanisms at both the germline and somatic immunogenetic levels results in diversity in knob region cysteine content, contributing to remarkable complexity in knob region disulfide patterns, loops, and Ag binding surface.
CD96 Blockade Ameliorates Imiquimod-Induced Psoriasis-like Dermatitis via Suppression of IL-17A Production by Dermal γδ T Cells
Authors: Oh-oka Kyoko , Abe Fumie , Shibuya Akira , Shibuya Kazuko
Published: January 2023

Psoriasis is a chronic inflammatory skin disease. IL-23 plays a critical role in its pathogenesis by inducing production of IL-17A from pathological Th17 cells and IL-17A–producing γδ T cells. However, the mechanisms regulating the IL-23/IL-17 axis in psoriasis are incompletely understood. In this study, we show that, in comparison with wild-type mice, those deficient in the CD96 immunoreceptor had lower production of IL-17A in their dermal γδ T cells and milder psoriasis-like dermatitis after topical application of imiquimod (IMQ). Moreover, transfer of CD96-deficient dermal γδ T cells into the skin of Rag1-deficient mice resulted in them developing milder IMQ-induced dermatitis compared with Rag1-deficient mice transferred with wild-type dermal γδ T cells. In γδ T cells in vitro, CD96 provides a costimulatory signal for the production of IL-23–induced IL-17A. In mice given an anti-CD96 neutralizing Ab, IL-17A production from dermal γδ T cells decreased and IMQ-induced dermatitis was milder compared with mice given a control Ab. These results suggest that CD96 is a potential molecular target for the treatment of psoriasis.
Transcriptional Profiling of Phagocytic Leukocytes and Microglia Reveals a Critical Role for Reactive Oxygen Species in Biofilm Containment during Staphylococcus aureus Craniotomy Infection
Authors: Menousek Joseph , Horn Christopher M , Heim Cortney E , Van Roy Zachary , Korshoj Lee E , Kielian Tammy
Published: January 2023

Craniotomies are performed to treat a variety of intracranial pathology. Surgical site infection remains a complication of craniotomy despite the use of prophylactic antibiotics and universal sterile precautions. Infections occur in 1–3% of procedures, with approximately half caused by Staphylococcus aureus that forms a biofilm on the bone flap and is recalcitrant to systemic antibiotic therapy. We used an S. aureus-dsRed construct to compare the phagocytic capacity of leukocytes and microglia in vitro and in vivo using a mouse model of craniotomy infection. In addition, single-cell RNA sequencing (scRNA-seq) was applied to determine whether a transcriptional signature could be identified for phagocytic versus nonphagocytic cells in vivo. S. aureus was phagocytosed to equivalent extents in microglia, macrophages, neutrophils, and granulocytic myeloid-derived suppressor cells in vitro; however, microglial uptake of S. aureus was limited in vivo, whereas the other leukocyte populations exhibited phagocytic activity. scRNA-seq comparing the transcriptional signatures of phagocytic (S. aureus-dsRed+) versus nonphagocytic (S. aureus-dsRed−) leukocytes identified classical pathways enriched in phagocytic cells (i.e., reactive oxygen species [ROS]/reactive nitrogen species, lysosome, iron uptake, and transport), whereas nonphagocytic populations had increased ribosomal, IFN, and hypoxia signatures. scRNA-seq also revealed a robust ROS profile, which led to the exploration of craniotomy infection in NADPH oxidase 2 knockout mice. S. aureus burden, leukocyte recruitment, and intracellular bacterial load were significantly increased in NADPH oxidase 2 KO compared with wild-type animals. Collectively, these results highlight the importance of ROS generation in phagocytes for S. aureus biofilm containment, but not clearance, during craniotomy infection.
Memory B Cells and Memory T Cells Induced by SARS-CoV-2 Booster Vaccination or Infection Show Different Dynamics and Responsiveness to the Omicron Variant
Authors: Mise-Omata Setsuko , Ikeda Mari , Takeshita Masaru , Uwamino Yoshifumi , Wakui Masatoshi , Arai Tomoko , Yoshifuji Ayumi , Murano Kensaku , Siomi Haruhiko , Nakagawara Kensuke , Ohyagi Masaki , Ando Makoto , Hasegawa Naoki , Saya Hideyuki , Murata Mitsuru , Fukunaga Koichi , Namkoong Ho , Lu Xiuyuan , Yamasaki Sho , Yoshimura Akihiko
Published: January 2023

Although the immunological memory produced by BNT162b2 vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been well studied and established, further information using different racial cohorts is necessary to understand the overall immunological response to vaccination. We evaluated memory B and T cell responses to the severe acute respiratory syndrome coronavirus 2 spike protein before and after the third booster using a Japanese cohort. Although the Ab titer against the spike receptor-binding domain (RBD) decreased significantly 8 mo after the second vaccination, the number of memory B cells continued to increase, whereas the number of memory T cells decreased slowly. Memory B and T cells from unvaccinated infected patients showed similar kinetics. After the third vaccination, the Ab titer increased to the level of the second vaccination, and memory B cells increased at significantly higher levels before the booster, whereas memory T cells recovered close to the second vaccination levels. In memory T cells, the frequency of CXCR5+CXCR3+CCR6− circulating follicular Th1 was positively correlated with RBD-specific Ab-secreting B cells. For the response to variant RBDs, although 60–80% of memory B cells could bind to the omicron RBD, their avidity was low, whereas memory T cells show an equal response to the omicron spike. Thus, the persistent presence of memory B and T cells will quickly upregulate Ab production and T cell responses after omicron strain infection, which prevents severe illness and death due to coronavirus disease 2019.
Theilovirus 3C Protease Cleaves the C-Terminal Domain of the Innate Immune RNA Sensor, Melanoma Differentiation–Associated Gene 5, and Impairs Double-Stranded RNA–Mediated IFN Response
Authors: Miyamoto Masahiko , Himeda Toshiki , Ishihara Kazuki , Okuwa Takako , Kobayashi Daiki , Nameta Masaaki , Karasawa Yu , Chunhaphinyokul Benyapa , Yoshida Yutaka , Tanaka Nobuyuki , Higuchi Masaya , Komuro Akihiko
Published: January 2023

Melanoma differentiation–associated gene 5 (MDA5), a member of the retinoic acid–inducible gene I (RIG-I)–like receptors (RLRs), has pivotal roles in innate immune responses against many positive-stranded RNA viruses, including picornavirus and coronavirus. Upon engagement with dsRNA derived from viral infection, MDA5 initiates coordinated signal transduction leading to type I IFN induction to restrict viral replication. In this study, we describe a targeted cleavage events of MDA5 by the 3C protease from Theilovirus. Upon ectopic expression of theilovirus 3C protease from Saffold virus or Theiler’s murine encephalomyelitis virus but not encephalomyocarditis virus, fragments of cleaved MDA5 were observed in a dose-dependent manner. When enzymatically inactive Theilovirus 3C protease was expressed, MDA5 cleavage was completely abrogated. Mass spectrometric analysis identified two cleavage sites at the C terminus of MDA5, cleaving off one of the RNA-binding domains. The same cleavage pattern was observed during Theilovirus infection. The cleavage of MDA5 by Theilovirus protease impaired ATP hydrolysis, RNA binding, and filament assembly on RNA, resulting in dysfunction of MDA5 as an innate immune RNA sensor for IFN induction. Furthermore, the cleavage-resistant MDA5 mutant against the 3C protease showed an enhanced IFN response during Saffold virus infection, indicating that Theilovirus has a strategy to circumvent the antiviral immune response by cleaving MDA5 using 3C protease. In summary, these data suggest MDA5 cleavage by 3C protease as a novel immune evasive strategy of Theilovirus.
Cutting Edge: High-Dose Live Attenuated Influenza Vaccines Elicit Pulmonary Tissue-Resident Memory CD8+ T Cells in the Face of Pre-Existing Humoral Immunity
Authors: Zheng Ming Z M , Fritzlar Svenja , Wang Zhongfang , Tan Tiong Kit , Kedzierska Katherine , Townsend Alain , Reading Patrick C , Wakim Linda M
Published: January 2023

In this study, we investigated how pre-existing Ab immunity to influenza virus established from prior immunizations affects the development of CD8+ T cell responses evoked after vaccination with a live attenuated vaccine. Using a mouse model and a panel of live attenuated influenza virus vaccine candidates (cold adapted and single cycle), we show that pre-existing influenza-specific Abs directed against the vaccine backbone attenuate the size and quality of the vaccine-induced CD8+ T cell response. Importantly, we show that increasing the vaccine dose can overcome this impediment, resulting in improved vaccine-induced circulating and tissue-resident memory CD8+ T cell responses, which were protective against heterologous influenza challenge. Thus, the reduced size and quality of the T cell response elicited by a live attenuated influenza virus vaccine imparted by the influenza-specific Ab landscape of the vaccinee can be overcome by increasing vaccine dose.
Arginase 1 Expression by Macrophages Promotes Cryptococcus neoformans Proliferation and Invasion into Brain Microvascular Endothelial Cells
Authors: Hansakon Adithap , Ngamphiw Chumpol , Tongsima Sissades , Angkasekwinai Pornpimon
Published: January 2023

Cryptococcal meningoencephalitis caused by Cryptococcus neoformans infection is the most common cause of death in HIV/AIDS patients. Macrophages are pivotal for the regulation of immune responses to cryptococcal infection by either playing protective function or facilitating fungal dissemination. However, the mechanisms underlying macrophage responses to C. neoformans remain unclear. To analyze the transcriptomic changes and identify the pathogenic factors of macrophages, we performed a comparative transcriptomic analysis of alveolar macrophage responses during C. neoformans infection. Alveolar macrophages isolated from C. neoformans–infected mice showed dynamic gene expression patterns, with expression change from a protective M1 (classically activated)–like to a pathogenic M2 (alternatively activated)–like phenotype. Arg1, the gene encoding the enzyme arginase 1, was found as the most upregulated gene in alveolar macrophages during the chronic infection phase. The in vitro inhibition of arginase activity resulted in a reduction of cryptococcal phagocytosis, intracellular growth, and proliferation, coupled with an altered macrophage response from pathogenic M2 to a protective M1 phenotype. In an in vitro model of the blood–brain barrier, macrophage-derived arginase was found to be required for C. neoformans invasion of brain microvascular endothelium. Further analysis of the degree of virulence indicated a positive correlation between arginase 1 expression in macrophages and cryptococcal brain dissemination in vivo. Thus, our data suggest that a dynamic macrophage activation that involves arginase expression may contribute to the cryptococcal disease by promoting cryptococcal growth, proliferation, and the invasion to the brain endothelium.