bluuebunny/crossref_metadata_2025 huggingface dataset:
T Follicular Regulatory (TFR) Cells Regulate B Cell Responses in Both, T-cell Dependent and Independent Pathways
Authors: Lopez-Ocasio Maria D.
Published: January 2023

Production of high-affinity antibody-secreting plasma cells and memory B cells requires interaction of TFH cells with B cells in the follicle. TFR cells, a subset of Foxp3+ Tregs, localize into follicles, regulating GC responses. To elucidate the mechanisms and cellular targets used by TFR cells to suppress GC responses, we quantitated their function in vitro. In the absence of TFR cells, TFH from primed mice induce naive B cell differentiation into GC B cells and class switching in the presence of anti-CD3 alone or anti-IgM/anti-CD3 in a contact dependent manner. Addition of TFR cells from primed mice efficiently suppressed GC B cell differentiation and class switching in the anti-CD3 alone cultures, but moderately suppressed BCR-stimulated B cells. Under anti-CD3 conditions, IL-4−/− TFH cells did not promote B cell differentiation and class switching. In contrast, IL-21R−/− B cells differentiated into GC B cells with a reduced number of IgG1+ cells. When IL-4−/− TFH cells were co-cultured with IL-21R−/− B cells, both GC differentiation and class switching were reduced. Under anti-IgM/anti-CD3 conditions, IL-4−/− TFH cells promoted B cell differentiation, but class switching was reduced, while IL-21R−/− B cells differentiated normally into GC B cells, with reduced class switching. When B cells alone were stimulated in culture with either LPS or CpG, TFR cells did not block B cell proliferation, but their differentiation into plasma cells was reduced. These studies suggest that in vitro, TFR cells regulate GC responses primarily by acting on TFH cells by inhibiting cytokine production and CD40L expression, but can also directly suppress some aspects of B cell differentiation.

Supported by the Intramural Research Program of the NIAID.
Preoperative exercise therapy protects the liver from ischemia-reperfusion injury
Authors: Zhang Hongji , Ren Jinghua , Yazdani Hamza O. , Van Der Windt Dirk , Zhang Jinxiang , Tsung Allan , Huang Hai
Published: January 2023

Introduction

Surgical stress such as hepatic ischemia/reperfusion (I/R) injury results in an increased risk of postoperative morbidity and mortality, including liver transplantation. Preoperative exercise therapy has been shown to enhance physical capacity at the moment of hospital admission and may facilitate better recovery after surgery. However, the mechanisms behind this protection remain to be elucidated. Here we hypothesize that preoperative aerobic exercise therapy has protective effects as a result of inhibiting sterile inflammatory processes after liver I/R injury.

Methods

Eight-week-old male C57BL/6 mice were randomly divided into exercise (n=15) or sedentary (n=15) groups. The exercise group ran on a motorized treadmill for 60 min/day, 5 days/week at a speed of 12.5m/min for 4 weeks. 70% partial liver warm I/R was performed in both groups of mice at 72 hours after the final exercise session.

Results

After 4 weeks,60% exercised mice showed a 3%–6% weight loss whereas the weight of all sedentary mice were increased. The sALT levels was significantly reduced in the exercise mice than the sedentary mice after liver I/R. Significantly increased Natural Killer (NK) cells were observed, whereas less neutrophil infiltration was seen in exercise mice after I/R, suggesting a link between hepatic immune modulation and exercise. Moreover, the lactate dehydrogenase (LDH), apoptosis, reactive oxygen species (ROS) in hepatocyte were all decreased in exercise mice.

Conclusions

These data show beneficial effects of preoperative exercise therapy on the experimental model of liver I/R injury, which offer a rationale for encouraging predisposed patients of HCC or other end-stage liver diseases that need liver surgery.
The transcription factor ROR alpha preserves group 3 innate lymphoid cell lineage identity and function
Authors: Messing Melina , Lo Bernard C. , Hughes Michael R. , Hernaez Diana Canals , McNagny Kelly M.
Published: January 2023

Innate lymphoid cells (ILCs) are critical for host defense and tissue repair. The transcription factor RORα is essential for ILC2 development but it is also highly expressed by ILC3s where its function remains poorly defined. Previously, we found that Rorasg/sg bone marrow transplant (BMT) mice were protected from intestinal fibrosis in a Salmonella--induced model of Crohn’s disease and that this is due to defective cytokine production by ILC3s. In the current study, whole transcriptome sequencing analysis reveals a striking downregulation of ILC3 signature genes in RORα-deficient ILC3s isolated from Salmonella infected mice. In particular, the expression of genes involved in sensing an inflammatory milieu is attenuated. Moreover, we find that Rorasg/sg ILC3s fail to express IL-17A following ex vivo stimulation with IL-23 and IL-1β. Consistent with these observations, we also find that Rorasg/sg BMT mice are more susceptible to Citrobacter rodentium infection due to attenuated expression of IL-22 and impaired induction of antimicrobial peptides. Collectively, our data suggests that RORα plays a key role in preserving functional ILC3s by modulating their ability to sense environmental cues that stimulate the efficient production of cytokines. These observations also suggest that targeting ILC3 cells could be of therapeutic benefit in fibrosis associated with Crohn’s disease.
Modulation of oxidative burst with exposure to cytokines in neutrophil cell activation
Authors: Swain Pamela , Romero Natalia , Dranka Brian P.
Published: January 2023

Neutrophils are essential for killing microorganisms and have a significant role in regulation of inflammatory response. Generation of reactive oxygen species (ROS) is one of the critical events that modulate the immune response in phagocytic cells. Stimulated neutrophils activate membrane-associated NADPH oxidase (NOX2) resulting in a powerful oxidative burst during which a large amount of oxygen is consumed generating ROS. The Agilent Seahorse XF analyzer is used to quantify oxygen consumption rate (OCR) as a direct non-invasive measure of neutrophil activation. In this study, neutrophil activation in the presence of cytokines known to be expressed within microenvironments in normal and disease states is evaluated. Treatment of neutrophils with the inflammatory cytokine TNFα induces oxidative burst within an hour of treatment, as observed in real time using the XF analyzer. Interestingly, pretreatment with TNFα reduces the oxidative burst observed when neutrophils were activated with phorbol myristate acetate (PMA). In contrast, treatment with GM-CSF and IL1 β had no acute effect in inducing oxidative burst. Rather, pretreatment of neutrophils with GM-CSF and IL1 β primed them for an enhanced response to PMA and a reduced elapsed time to reach maximal activation. Of note, the oxidative burst observed is associated with simultaneous increased of proton efflux rate (PER), indicative of the dependence on glycolysis and pentose phosphate pathway for production of NADPH during oxidative burst. These results, show the utility of the XF neutrophil activation assay to study the effect of modulators such drug treatments, microenvironment and disease progression during innate immune response.
cIAP1/2-TRAF2- SHP1-Src-MyD88 complex regulates LPS-induced IL-27 production through NFkB activation in human macrophages
Authors: Kumar Ashok , Busca Aurelia , Gajanayaka Niranjala , Angel Jonathan
Published: January 2023

The inhibitors of apoptosis (IAP) proteins, initially described in the context of apoptosis regulation as promoting cell survival, have recently emerged as key regulators of innate immune signaling. As a result, downregulation of IAP via Smac mimetics (SMM) has both survival and immunoregulatory effects. IAPs modulate cytokine production in murine models either as a single agent or in response to LPS. However, the role of SMM and the involvement of IAPs in primary human cells and in particular macrophages with respect to cytokine production and innate immune responses remain largely unknown. IL-27, a member of the IL-12 cytokine family produced by antigen presenting cells such as macrophages, has broad immunoregulatory properties in both innate and adaptive immune responses. Herein, we show that cIAPs positively regulate LPS-induced IL-27 production in both primary human monocytes and macrophages. Investigations for the signaling mechanism of cIAPs involvement in IL-27 production in human macrophages revealed that LPS-induced IL-27 production is regulated by a novel signaling complex comprising of cIAP1/2, TNF receptor associated factor-2 (TRAF2), SHP-1, Src, and MyD88 leading to p38, C-Jun N-terminal kinases (JNK) and Akt activation and NFκB signaling. In cancer cells, SMM induce the production of cytokines by activating the non-canonical alternate NFkB pathway. However, in human macrophages, SMM do not induce the production of TNF-a and other cytokines while inhibiting LPS-induced IL-27 production by inhibiting the classical NFkB pathway. These signaling pathways may constitute novel therapeutic avenues for immune-modulation of IL-27 and provide insight into the modulatory immune effects of SMM.
Inhibition of mTORC1 activity is required for the IL-12p40 response of classical CD16neg CD14+ primary human monocytes to Toxoplasma gondii
Authors: Amancio Alice Muglia T. S. , Mittereder Lara , Carletti Alexie , Sher Alan , Jankovic Dragana
Published: January 2023

Primary human monocytes produce IL-12 and TNF in response to live Toxoplasma gondii tachyzoytes by a mechanism requiring parasite phagocytosis. Interestingly, in contrast to the patrolling CD16+ CD14+ subset, monocytes belonging to the major CD16neg CD14+ subset fail to secrete IL-12 and TNF following T. gondii exposure. Here we show that the CD16neg CD14+ subset, nevertheless, upregulates Il12b mRNA and thus is able to sense the parasite. In addition, we demonstrate that priming with IFNg, but not IFNa, enables CD16negCD14+ monocytes to produce IL-12 protein in response to T. gondii. This effect of IFNg priming was not due to augmented phagocytosis and was not associated with altered rates of glycolysis, oxygen consumption, or increased Il12b mRNA expression. To test the possible role of IFNγ on cytokine translation, we evaluated the requirement for mTOR (mTORC1/2) pathway, a key regulator of protein synthesis. The T. gondii-induced IL-12 response of IFNγ primed CD16neg monocytes was inhibited by Torin, an inhibitor of mTORC2, but surprisingly not by the classical mTORC1 inhibitor Rapamycin. Because mTORC2 has been shown to downregulate mTORC1 activity, we hypothesized that the priming effect of IFNg might result from the upregulation of mTORC2 resulting in suppressed mTORC1. Indeed, in the absence of IFNγ, suppression of mTORC1 in CD16neg monocytes with Rapamycin enhanced IL-12 secretion. Taken together, our results suggest that classical CD16neg CD14+ monocytes fail to secrete IL-12 in response to T. gondii due to increased mTORC1 activity, which can be reversed directly by Rapamycin or indirectly through stimulation of mTORC2 by IFNγ priming.
Exploring a role for Human Herpesvirus 6B-mediated mitochondrial dysfunction in dysregulating antiviral innate immunity
Authors: Birdwell Christine E. , West Phillip
Published: January 2023

Human herpesviruses are ubiquitous in the world population, in part due to the ability of this family of viruses to establish life-long, persistent infections. Human herpesvirus 6B (HHV-6B) is generally acquired early in life, and is the causative agent of the benign childhood febrile illness, Roseola Infantum, and severe encephalitis in rare instances. Mitochondria play diverse roles in cell homeostasis, including metabolism, energy production, and regulation of antiviral innate immunity. As a family, herpesviruses are known modulators of mitochondria and we have observed that HHV-6B significantly alters mitochondrial function of infected T-cells. Specifically, we have discovered that HHV-6B alters mitochondrial DNA replication and transcription, while also dramatically decreasing mitochondrial membrane potential during infection. We hypothesize that HHV-6 disrupts mitochondrial function by altering mitochondrial gene expression and oxidative phosphorylation, leading to metabolic and signaling imbalances that influence cellular homeostasis and anti-HHV-6 immunity.
Trajectory of neuro-inflammation and microglial activation assessed using Translocator protein (18kDa TSPO) and positron emission tomography (PET) imaging in bacterial meningitis
Authors: Barichello Tatiana , Giridharan Vijayasree V. , Scaini Giselli , Generoso Jaqueline S. , Simões Lutiana Roque , Dominguini Diogo , Hasbun Rodrigo , Selvaraj Sudhakar , Dal-Pizzol Felipe
Published: January 2023

Here we present novel data from acute (24 hours) and chronic (10 days) pneumococcal meningitis rat survivors evaluating microglia activation through [11C] PBR28, a tracer targeting the TSPO using PET-imaging. Wistar rats received an injection of Streptococcus pneumoniae or artificial-CSF through the cisterna magna following by ceftriaxone/100 mg/kg/7days. At 24 hours and 10 days the animals were subjected to TSPO-PET-imaging. Following the imaging IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-17, IL-18, IFN-γ, TNF-α, and microglial marker Iba-1 and astrocyte marker GFAP were evaluated in prefrontal cortex and hippocampus (n = 7). In chronic phase, the animals were subjected to open field, step-down inhibitory avoidance and in novel object recognition task, n = 10. In acute phase, the imaging results revealed that in amygdala, cortex, hippocampus, hypothalamus, midbrain, and thalamus (p < 0.05) uptake of [11C] PBR28 was greater in the meningitis group as compared to the control group. We observed increased expression of pro-inflammatory markers such as IL-1α, IL-1β, IL-6, IL-18, IFN-γ, TNF-α, Iba-1, and GFAP expression at 24 h (p < 0.05). In the chronic phase, in amygdala, hippocampus, and thalamus (p < 0.05) uptake of [11C] PBR28 was greater in the meningitis group. The control group presented retention of habituation memory, aversive memory and long-term recognition memory. However, animals in the meningitis group demonstrated memory impairment in all behavioral tasks. TNF-α, Iba-1, and GFAP expression increased in chronic phase survivors (p < 0.05). The above results support our hypothesis that increased activation of brain microglia is associated with behavioral changes in experimental model of meningitis.
AAK1 and GAK inhibitors demonstrate activity against Filoviruses
Authors: Dorosky Danielle , Prugar Laura I. , Pu Szuyan , O’Brien Cecilia , Bakken Russell , De Jonghe Steven , Herdewijn Piet , Brannan Jennifer , Dye John M. , Einav Shirit
Published: January 2023

Ebola virus (EBOV) and Marburg virus (MARV) are highly pathogenic members of the family filoviridae. The recent West Africa epidemic of Ebola virus disease was a grave reminder of the unmet need for post exposure therapeutics to combat filovirus infection. Our group has reported that multiple unrelated viruses, including EBOV, commandeer host kinases AP2-associated protein kinase-1 (AAK1) and cyclin G-associated kinase (GAK) during viral entry, assembly, and/or egress. This shared attribute makes AAK1 and GAK desirable broad-spectrum targets for the treatment of multiple viral infections without necessitating species identification. As the targets of these inhibitors are host cell factors, this may allow for the reduction in the likelihood that the virus will develop drug resistance. We have demonstrated that treatment with sunitinib and erlotinib, approved anticancer drugs with anti-AAK1 or GAK activity, is effective in inhibiting replication of Ebola, Hepatitis C and Dengue (DENV) viruses in vitro. Additionally, the combination of sunitinib and erlotinib has demonstrated efficacy in reducing morbidity and mortality following challenge with EBOV and DENV in the relevant mouse models. In an effort to improve toxicity and survival, we are developing novel, chemically distinct, more selective inhibitors of AAK1 and GAK. Here we describe the capability of these next generation AAK1 and GAK inhibitors to reduce in vitro infection of Ebola virus and Marburg virus.
Unique phenotypes and clonal expansions of human CD4 effector memory T cells re-expressing CD45RA
Authors: Tian Yuan , Babor Mariana , Lane Jerome , Schulten Véronique MJ. , Patil Veena , Seumois Grégory , Rosales Sandy , Fu Zheng , Picarda Gaelle Genevieve Alice , Burel Julie , Zapardiel-Gonzalo Jose , Tennekoon Rashika , De Silva Aruna , Premawansa Sunil , Premawansa Gayani , Wijewickrama Ananda , Greenbaum Jason , Vijayanand Pandurangan , Weiskopf Daniela , Sette Alessandro , Peters Bjorn
Published: January 2023

The expression of CD45RA is generally associated with naive T cells. However, a subset of effector memory T cells re-expresses CD45RA (termed TEMRA) after antigenic stimulation with unknown molecular characteristics and functions. CD4 TEMRA cells have been implicated in protective immunity against pathogens such as dengue virus (DENV). Here we show that not only the frequency but also the phenotype of CD4 TEMRA cells are heterogeneous between individuals. These cells can be subdivided into two major subsets based on the expression of the adhesion G protein-coupled receptor GPR56, and GPR56+ TEMRA cells display a transcriptional and proteomic program with cytotoxic features that is distinct from effector memory T cells. Moreover, GPR56+ TEMRA cells have higher levels of clonal expansion and contain the majority of virus-specific TEMRA cells. Overall, this study reveals the heterogeneity of CD4 TEMRA cells and provides insights into T-cell responses against DENV and other viral pathogens.
CD28 Induces Mitochondrial Respiration Dependent Reactive Oxygen Species (ROS) Signaling for Metabolic Fitness and Survival in Long-Lived Plasma Cells
Authors: Utley Adam , Cooper James J. M. , Chavel Colin A. , Peng Peng , Carlson Louise M. , Lightman Shivana M. , Kambayashi Taku , Green Jonathan , Lee Kelvin P.
Published: January 2023

Durable humoral immunity depends upon bone marrow resident long-lived plasma cell (LLPC) survival. We have shown that CD28, the canonical T-cell costimulatory molecule, is required for LLPC survival; however, the mechanistic basis for this is unclear. We have published that of two binding motifs in the CD28 cytoplasmic tail, only the Grb2/Vav moiety, and not the direct PI3k binding motif, supports CD28-mediated survival. CD28 is known to induce glycolysis in T-cells. In contrast, CD28 activation in both LLPCs and human PCs induces glucose metabolism through mitochondrial respiration. A major byproduct of mitochondrial respiration is the production of reactive oxygen species (ROS). Interestingly, the mitochondrial respiration-dependent production of ROS is required for CD28-mediated survival. Previously published data demonstrate the ability of ROS to signal through both NFkB and PI3k. CD28-induced ROS signal through PI3k in LLPCs for increased glucose uptake and the inhibition of PI3k prevents CD28-mediated survival. The ROS-PI3k signal drives the phosphorylation of both S6k and rpS6, downstream targets of mTORC1, and this regulation is independent of Blimp1 expression. Irf4, a critical PC survival factor, is upregulated by CD28-dependent NFkB activation. To our great surprise, ROS inhibition prevented the induction of mitochondrial respiration itself, demonstrating that signals emanating downstream of ROS govern the metabolic program for survival in LLPCs. Taken together, these data suggest a model wherein CD28 signals directly through Grb2/Vav to induce mitochondrial respiration and ROS production. ROS then reinforce signaling through PI3k and NFkB to increase Irf4 expression and metabolic fitness for LLPC survival.
The long chain fatty acid transporter, MFSD2A, is essential for memory CD8+ T cell formation and maintenance
Authors: Piccirillo Ann R. , Hawse William F. , Buechel Heather M. , Silver David L. , D’Cruz Louise M.
Published: January 2023

Access to nutrients is critical for an effective T cell immune response to infection. Although transporters for sugars and amino acids have previously been described in the context of the CD8+ T cell immune response, the active transport of exogenous esterified fatty acids has remained enigmatic. Here we discovered the long chain fatty acid transporter, Major Facilitator Super Family Domain Containing 2a (MFSD2A), is upregulated on activated CD8+ T cells and is essential for their memory cell formation. MFSD2A deficiency resulted in decreased import of long chain fatty acids (LCFAs) esterified to lysophosphatidylcholine (LPC) into activated CD8+ T cells but overall resulted in a normal primary effector T cell response. However, loss of MFSD2A led to reduced memory T cell formation and maintenance. MFSD2A deficient memory CD8+ T cells showed reduced CD127 and CD62L expression and their cell turnover was significantly impaired in contrast to their wildtype counterparts. Moreover, the secondary response to infection was severely diminished in MFSD2A deficient T cells. Mechanistically, import of LCFAs was required to maintain cell energy requirements and ‘fitness’, that when lost resulted in a decreased memory T cell pool and inability to proliferate upon secondary stimulation. Our results show that MFSD2A and LPC may be useful for future small molecule therapy design to generate a more robust T cell response for new vaccines or cancer treatments.
CD45RB Status Defines TCR Priming Affinity and CD8+ T Cell Memory Persistence
Authors: Krummey Scott , Jacobs Jesica R. , Kissick Haydn , Evavold Brian D. , Ford Mandy L.
Published: January 2023

T cell receptor (TCR) affinity plays a critical role in shaping T cell differentiation, but few studies have investigated the role of TCR affinity in the context of the endogenous CD8+ T cell memory pool. While naïve CD8+ T cells express primarily large CD45 isoforms (denoted as CD45RBhi), CD8+ T cell memory cells are traditionally described as expressing small isoforms (CD45RBlo). Interestingly, although CD8+ T cell memory to acute infection with LCMV included CD45RBlo cells, we found that over half of the CD8+ T cell memory pool remained CD45RBhi. The 2D micropipette affinity assay revealed that CD45RBhi memory cells had a 29-fold lower affinity for antigen relative to CD45RBlo cells. CD45RBloand CD45RBhi memory cells expressed similar levels of granzyme B and IFN-γ, but RNASeq analysis revealed that these populations differentially expressed over 150 genes. Phenotypically, the CD45RBhi population contained a higher frequency of the CD62LhiCD27hiCD127hiKLRG1lo persistent memory phenotype compared to CD45RBlo cells. We found that from early to late memory time-points, portions of each of the CD45RBhi and CD45RBlo populations underwent conversion to CD62Lhi, demonstrating that CD45RB status is distinct from the TCM phenotype. Adoptive transfer of purified CD45RBhi and CD45RBlo populations into naïve mice revealed that CD45RBlo memory cells underwent significant cell loss relative to CD45RBhi memory cells. Over time, the majority of CD45RBlo cells homeostatically converted to the CD45RBhipersistent memory phenotype. Together, these data demonstrate a novel role for CD45RB expression as a marker of TCR priming affinity, and describe a novel way in which CD45RB status can functionally categorize the CD8+ T cell memory pool.
Mycobacterium tuberculosis stimulates neutrophil infiltration into the lungs through activation of S100A proteins in an early secreted antigenic target of 6-kDa dependent manner
Authors: Jung Bock-Gie , Adeniyi Biodun A. , Samten Buka
Published: January 2023

Health Science Center at Tyler Neutrophils infiltrate the lungs in active tuberculosis (TB) and contribute to the exacerbation of TB pathology. However, the contributing factors and the mechanisms of neutrophil infiltration in TB are unclear. As secretion of early secreted antigenic target of 6 kDa (ESAT-6) and its molecular partner culture filtrate protein 10 kD (CFP10) by ESX-1 secretion system is essential for pathogenesis of Mycobacterium tuberculosis (Mtb), we infected C57BL/6 mice intranasally with Mtb strains H37Rv, its esat-6 deletion mutant, and esat-6 complemented strain and evaluated lung neutrophil infiltration. H37Rv and esat-6 complemented strain induced neutrophil infiltration at day 7 and peaked at day 14 post infection. Whereas esat-6 deletion mutant induced significantly less neutrophil infiltration at both time points. In line with this, intranasal delivery of recombinant ESAT-6 but not CFP10 or Hank’s balanced salt solution (vehicle) induced neutrophil infiltration of the lungs as early as 2 hours, peaked at 24 hours, and started to decline at 72 hours post treatment. RNA-sequencing analysis of total lung RNA from ESAT-6 or CFP10 treated mice identified S100A9 as one of the most significantly upregulated genes by ESAT-6. Real-time PCR and ELISA validated that ESAT-6 stimulated increased gene expression and protein secretion of S100A9 in mouse lungs. Immunohistochemistry analysis revealed that ESAT-6 induced neutrophilic lung inflammation. Consistent with this, H37Rv and esat-6 complemented strain induced significantly higher levels of S100A9 compared to their esat-6 deletion mutant in mouse lungs after intranasal infection. We conclude that ESAT-6-induced S100A9 plays a critical role in lung neutrophil infiltrations in TB infection.
CARD9 adaptor molecule is indispensable for protection against Cryptococcosis
Authors: Campuzano Althea , Castro-Lopez Natalia , Martinez Amanda , Leopold Wager Chrissy M. , Wormley Floyd L.
Published: January 2023

Caspase recruitment domain-containing protein 9 (CARD9) is a critical adaptor molecule triggered by the interaction of C-type lectin receptors (CLRs) with carbohydrate motifs found in fungi. Cryptococcus neoformans is an opportunistic fungal pathogen predominantly affecting patients with suppressed T cell-mediated immunity and is responsible for ~15% of AIDS-related deaths globally. Cryptococcus can mask itself with a unique carbohydrate capsule complex which also serves as its predominant virulence factor. Previous studies have shown that immunization with a C. neoformans strain genetically engineered to express murine IFN-γ, H99γ, or a hyphaeforming mutant, LW10, results in protection against an otherwise lethal challenge with wild type (WT) C. neoformans. We therefore analyzed the role of CARD9 during the induction of protective anti-Cryptococcus immunity in WT and CARD9 knockout (KO) mice given an experimental pulmonary infection with Cryptococcus neoformans strain H99, H99γ or LW10. CARD9 KO mice were significantly more susceptible to C. neoformans infection compared to their WT counterparts. We then evaluated the role of pulmonary macrophages, the first line of defense against cryptococcosis. Pulmonary macrophages from CARD9 KO mice were unable to control fungal burden. These results suggest that CARD9-mediated signaling is required for optimal macrophage fungicidal activity against Cryptococcus. The overall goal of these studies is to define PRR/ligand interactions and downstream signal transduction pathways that are critical for the induction of protective immunity against cryptococcosis.
Human cytomegalovirus reprograms hematopoietic progenitor cells into immunosuppressive monocyte to achieve latency
Authors: Zen Ke
Published: January 2023

The precise cell type hosting latent human cytomegalovirus (HCMV) remains elusive. Here we report that HCMV reprograms human hematopoietic progenitor cells (HPCs) into a unique monocyte subset to achieve latency. Unlike conventional monocytes, this monocyte subset possesses higher levels of B7-H4, IL-10 and iNOS, longer lifespan and strong immunosuppressive capacity. Cell sorting of peripheral blood from latently infected human donors confirms that only this monocyte subset, representing less than 0.1% of peripheral mononuclear cells, is HCMV genome-positive but immediate-early (IE)-negative. Mechanistic studies demonstrate that HCMV promotes the differentiation of HPCs into this monocyte subset by activating cellular signal transducer and activator of transcription 3 (STAT3). In turn, this monocyte subset generates high level of nitric oxide (NO) to silence HCMV IE transcription and promote viral latency. By contrast, US28-knockout HCMV mutant incapable of activating STAT3, fails to reprogram the HPCs and achieve latency. Our findings reveal that HCMV differentiates human HPCs into long-life, immunosuppressive monocyte subset for viral latency via activating STAT3/iNOS/NO axis.
Novel M-CSF-producing γδ T cells protect against recurrent malaria
Authors: Mamedov Murad R. , Scholzen Anja , Nair Ramesh V. , Cumnock Katherine , Kenkel Justin A. , Oliveira Jose H. M. , Trujillo Damian L. , Saligrama Naresha , Zhang Yue , Rubelt Florian , Schneider David S. , Chien Yueh-hsiu , Sauerwein Robert , Davis Mark M.
Published: January 2023

In 2016, there were 216 million malaria cases – 445,000 of which resulted in deaths. Despite overwhelming evidence that γδ T cells strongly respond during malaria infection and vaccination, their functional and phenotypic characteristics remain the least understood facets of the adaptive immune response. Therefore, we studied the role of these cells in human and mouse malaria. In both Plasmodium falciparum-infected subjects and in P. chabaudi-infected mice, we found γδ T cells expanding rapidly after resolution of acute parasitemia, in contrast to αβ T cells that expanded at the acute stage and then declined. Silencing the murine γδ T cells led to recurrent rounds of Plasmodium parasitemia. Single-cell T cell receptor sequencing of the expanded mouse cells revealed oligoclonal γδ T cells restricted to the TRAV15N-1 (Vδ6.3) V-region and converging complementarity-determining region 3 (CDR3) motifs. Also, RNA-seq of the expanded γδ T cells showed an unexpected transcriptional profile characterized by myeloid-modulating factors, previously unseen in γδ T cells. The expanded TRAV15N-1 γδ T cells abundantly produced M-CSF, which was necessary for preventing parasitemic recurrence. Interestingly, αβ T cells were the major source of M-CSF during acute infection, while γδ T cells filled that role during the post-acute stage. We have uncovered a novel γδ T cell subset that fills a protective role in the late stage of malaria. These cells could provide the mechanism for other observed correlations between γδ T cell and myeloid activity in cancer and infectious disease.
Bone marrow-derived RELMα regulates immunity and inflammation to gastrointestinal helminth Nippostrongylus brasilienis through effects on macrophages
Authors: Nair Meera G.
Published: January 2023

RELMα is a highly secreted protein in type 2 cytokine-induced inflammation including helminth infection and allergy. We previously showed that RELMα dampens Th2 immune responses to helminth Nippostronglyus brasiliensis (Nb). RELMα is expressed by immune cells such as macrophages, and by epithelial cells (EC), however, the functional impact of immune versus EC-derived RELMα is unknown. We generated bone marrow (BM) chimeras that were RELMα deficient (RELMα−/−) in BM or non BM cells and infected them with Nb. Non BM RELMα−/− chimeras had comparable inflammatory responses and parasite burdens to RELMα+/+ mice, suggesting that EC-derived RELMα does not significantly influence immune responses to Nb. In contrast, both RELMα−/− and BM RELMα−/−mice exhibited increased Nb-induced lung and intestinal inflammation, correlated with elevated macrophage responses, Th2 cytokines and Nb killing. CD11c+ lung macrophages were the dominant BM-derived source of RELMα and have been shown to mediate Nb killing. Therefore, we employed a macrophage-worm co-culture system to investigate whether RELMα regulates macrophage-mediated Nb killing. Compared to RELMα+/+macrophages, RELMα−/−macrophages exhibited increased binding to Nb and functionally impaired Nb development including decreased motility, growth and viability. Collectively, these studies demonstrate that BM- derived RELMα is necessary and sufficient to dampen Nb immune responses, and identify that one mechanism of action of RELMα is through inhibiting macrophage recruitment and interaction with Nb. Our findings suggest that RELMα acts as an immune brake that provides mutually beneficial effects for the host and parasite by limiting tissue damage and delaying parasite expulsion.
Splenocytes from young mice protect immunocompromised mice against death from malaria infection, but they do not proliferate well.
Authors: Opata Michael M. , Smith Margaret R. , Johnson Corey M. , Campbell Logan , Santos Hugo
Published: January 2023

The immune system plays an essential role in the elimination of malaria parasites. CD4+ T cells are imperative in the immune response to the infection, but immune response to malaria especially in young children is poorly understood, due to a lack of a better young animal model to study the pathogenesis of the disease. We have developed a young mouse (pup) model using day 15 pups to help understand the pathogenesis, and development of protective CD4 T cells in malaria infection. Here, we determined the ability of pup cells to protect immunocompromised RAGKO mice from malaria infection. We infected day 15 old pups with Plasmodium chabudi at 1×105 iRBCs, and transferred splenocytes to RAGKO mice on day 8 post-infection. RAGKO mice were then infected. Mice that received pup cells looked healthier and active throughout the experiment, compared to their counterparts who received adult spleen cells. During the peak of infection, mice that received adult cells lost more weight than the pup cell recipients. On day 60 post-transfer, we observed same numbers of CD4 T cells, central memory and effector memory T cells. There were significantly lower proportions and numbers of IFNγ and TNFα positive cells from pups compared to adult cells. We next determined if there was any difference in the proliferation of CD4 T cells and all splenocytes in pup cells. We purified CD4+ T cells using EasySep kit and cultured them on αCD3/CD28 pre-coated plates, compared to all splenocytes. There was a significant growth in purified CD4 T cells from adult than pup cells. Interestingly, all splenocytes from pups proliferated better than adults. Therefore, our data suggest that pup cells protect RAGKO mice from death, but do not develop into functional memory due to poor proliferation.
Cholera toxin requires microbiota metabolites short-chain fatty acids for its mucosal adjuvant activity
Authors: Yang Wenjing , Xiao Yi , Huang Xiangsheng , Chen Feidi , Chen Liang , Bilotta Anthony James , Xu Leiqi , Yao Suxia , Cong Yingzi
Published: January 2023

Cholera toxin (CT) is a potent mucosal adjuvant, however, the mechanisms by which CT induces both mucosal and systemic immune responses are still not completely understood. It has been shown recently that gut microbiota is critical in inducing immune response by CT, however, the mechanisms involved remain largely unknown. We demonstrated in this study that gut microbiota metabolites short-chain fatty acids (SCFA), which are solely metabolic products of gut microbiota from dietary fiber, are required for CT in inducing antigen-specific IgG and IgA responses after oral immunization. Depleting of gut bacteria by antibiotics significantly decreased mucosal and systemic IgG and IgA responses in mice orally immunized with ovalbumin (OVA) and CT. Feeding SCFA acetate and butyrate rescued antibody responses in antibiotics-treated WT mice. Furthermore, deficiency of GPR43, a SCFA binding receptor, impaired CT’s adjuvant activity. Acetate and butyrate did not directly act on B cells to promote IgG production, but promoted B cell IgG production and plasma B cell differentiation related genes in the presence of WT but not GPR43−/− DC. Furthermore, SCFA increased IgG production by both WT and GPR43−/− B cells when cultured with WT DC, indicating a crucial role for DC expression of GPR43 in SCFA induction of B cell IgG production. Mechanistically, SCFA promoted DC expressions of BAFF, a B cell activating factor, and Aldh1a2, an enzyme promoting production of retinoic acid (RA) from Vitamin A. Blockade either RA or BAFF signaling significantly inhibited SCFA induced B-cell IgG production. Therefore, our study indicated a critical role of gut microbiota and their metabolites SCFA in promoting the mucosal adjuvant activity of CT through partially GPR43.
Critical role for the microbiota in limiting inflammatory intestinal T cell responses
Authors: Kim Myunghoo , Galan Carolina , McAlester Andrea Hill , Wu Wan-Jung , Song Hyo Won , Littman Dan R. , Diehl Gretchen
Published: January 2023

Efficient functioning of the intestine and intestinal immune system depends on interactions between the host and microbiota. Breakdown in these interactions leads to reduced ability to respond to intestinal pathogens along with increased intestinal inflammation which together can lead to pathologic inflammatory conditions such as inflammatory bowel disease.

While a number of intestinal immune cells are likely regulated by the microbiota, we have found critical regulation of intestinal antigen presenting cells (APCs) expressing the chemokine receptor, CX3CR1. CX3CR1+ APCs are able to secrete both pro- and anti-inflammatory cytokines and are capable of mediating intestinal protection or pathology.

Using in vivo models, we identified a regulatory loop whereby intestinal CX3CR1+ APCs reduced induction of Th1 cells against intestinal pathogens and promoted generation of regulatory T cells against soluble food antigens and the microbiota itself. This regulatory response depends on anti-inflammatory cytokine IL-10 production by CX3CR1+ APCs. Disruption of the intestinal microbiota switched the functional capacity of CX3CR1+ APCs so they now drove inflammatory Th1 responses and mediated pathology in a model of colitis. Mechanistically, we determined that epithelial attachment by intestinal microbes was required to turn on anti-inflammatory functions by CX3CR1+ APCs. Our results identify a cellular mechanism by which the microbiota limits intestinal inflammation and promotes homeostasis. Our study demonstrates the potential therapeutic benefits of microbiota and host immune cell manipulation with the goal of amplifying or inhibiting intestinal T cell responses.
HDAC10 Targeting Regulates Foxp3 Promoter, Enhances T-regulatory (Treg) Function and Suppresses Autoimmune Colitis
Authors: Dahiya Satinder , Wang Liqing , Beier Ulf H. , Han Rongxiang , Hancock Wayne W.
Published: January 2023

Targeting histone/protein deacetylases (HDACs), especially by isoform-selective targeting, has major promise as a means to enhance the function of Tregs and thereby suppress immune-related disorders, such as colitis. We present data on the functions of HDAC10 in Treg cells. We found that HDAC10 binds to the Foxp3 promoter in the Tregs, is able to physically bind and deacetylate Foxp3 (at K31), and regulates the transcriptional activity of the Foxp3 promoter. Next, we evaluated the function of HDAC10 using HDAC10 KO mice. Overall, such mice were healthy and HDAC10 deletion: (i) enhanced Treg suppressive function both in vitro (Treg suppression assays) and in vivo (homeostatic proliferation assays), compared to wild-type (WT) Tregs; (ii) modestly enhanced the stability of monomeric Foxp3 (reducing gels) but the oligomeric forms, which are more relevant functionally, were increased by more than two-fold (native gels); (iii) increased H3K4Me3 (activating) marks on Foxp3 promoter and the key regulatory region of Foxp3 promoter-CNS2; and (iv) enhanced binding of ATF/CREB and FoxO1 at the Foxp3 promoter and CNS2 sites. We then evaluated the effects of HDAC10 deletion in Tregs in colitis models using adoptively transferred Rag1 KO mice. We found that HDAC10 KO Tregs were superior to WT Tregs in: (i) a ‘prevention of induction’ model of colitis, whereby WT T effector cells were injected with a limiting ratio of Tregs that allowed for weight loss as an indicator of colitis; and (ii) rescuing from colitis. Studies to develop and test HDAC10-specific inhibitors are currently underway. Overall, we show that HDAC10 has a key role in Foxp3+ Treg cells, and is an important new target for therapeutic intervention in autoimmune diseases, including colitis.
Epigallocatechin-3-gallate (EGCG) ameliorates dextran sulfate sodium (DSS) –induced inflammatory bowel disease
Authors: Payan Brittany A. , Vanarsa Kamala , Ding Huihua , Soomro Sanam , Baig Sahar , Du Yong , Hicks John , Mohan Chandra
Published: January 2023

Background

Given the potential side effects associated with conventional medical treatment of Inflammatory Bowel Disease (IBD), dietary supplements have been investigated by several groups. We tested if epigallocatechin gallate (EGCG) may be beneficial in IBD.

Methods

36 C57BL/6J mice were subjected to dextran sulfate sodium (DSS)-induced IBD and divided into 3 groups: model group (MD; no EGCG), low dose EGCG group (LE, 20 mg/kg/d) and high dose EGCG group (HE, 50 mg /kg/d).

Results

Both high and low dose EGCG treatment alleviated clinical manifestations of IBD including body weight loss and disease activity index (DAI): HE: 3.3 and LE: 2.8 vs. 8.3 in MD (p < 0.001, n=8–10 mice/group). Macroscopic severity score (MSS) of HE and LE groups were 2.4, and 2.2, respectively, significantly lower than in MD (5.0, P < 0.01). The mean colon length of MD group was 5.8 cm; treatment with EGCG restored colon length (HE: 6.5 cm; LE: 6.4 cm). Intestinal permeability was improved as demonstrated by FITC-Dextran assay (HE: 3.28; LE: 2.85, vs. MD group: 6.69 ng/ml, P < 0.001) .The colitis histology score of HE (1.54) and LE (0.42) groups were significantly lower than that in MD (4.3, P < 0.001). In addition, EGCG treatment reduced colonic levels of pro-inflammatory cytokines: IL-6 (HE: 0.45; LE group: 0.41 vs MD: 0.89 ng/g tissue, P =0.03), MCP-1 (HE: 1.83; LE: 1.87, vs MD: 3.5 ng/g tissue P = 0.01) and TNF-alpha (HE: 8.87; LE: 10 vs MD: 21.7 ng/g tissue, P <0.001). Lower CD3+ T cell and CD68+ macrophage infiltration was also noted in HE and LE mice (P < 0.001), compared to MD controls.

Conclusion

These results underscore the therapeutic potential of EGCG in the treatment of IBD, which appear to be mediated through multiple inflammatory pathways.
Anti-inflammatory activity of the piperazino-enaminone JODI-19 in vitro and in vivo
Authors: Szollosi Doreen , Sun Junjiang , Ghoneim Ola , Bill Ashley , Edafiogho Ivan
Published: January 2023

Data to characterize novel anti-inflammatory agent enaminone E121 have shown a marked decrease in the release of pro-inflammatory cytokines in macrophages stimulated with lipopolysaccharide (LPS) compared to the control (DMSO) group. Literature has described that when an N-alkylated piperazine motif is added to the terminal end of the lead compound, it is able to increase the CCR2 antagonistic activity. We hypothesized that incorporating the N-alkylated piperazine motif into the existing E-121 enaminone pharmacophore could lead to a ligand with dual functionality as a cytokine inhibitor and chemokine receptor antagonist. The resultant series (JODI) were studied in vitro, and data suggest they can suppress TNF-alpha and IL-6 in a dose-dependent manner, superior to that of dexamethasone. Additionally, macrophages stimulated with chemokine MCP-1 and treated with JODI compounds, (but not E121), are able to block CCR2 signaling as evidenced by decreased total ERK1/2. To determine if JODI-19 has activity in vivo, it was utilized in a mouse model of hemarthrosis to assess decreased inflammation as evidenced by local cytokine suppression. In a pilot study, FIX−/−mice were subjected to hemarthrosis induction with or without JODI administration. At different timepoints from four hours to four weeks after hemarthrosis, synovial fluid was collected by lavage for multiple cytokine analyses. Administration of JODI-19 significantly decreased TNFα, IL-1α, MCP-1 and MIP-1α levels in the synovial fluid. As such, JODI-19 may be a novel approach to better manage hemophilia arthropathy and its associated bone damage.
Syngeneic stem cell transplantation generates antigen-specific T cells that maintain myeloma-immune equilibrium
Authors: Minnie Simone , Vuckovic Slavica , Smith David , Gartlan Kate H. , Watkins Thomas S. , Kuns Rachel D. , Guillerey Camille , Chesi Marta , Markey Kate A. , Miles John J. , Smyth Mark J. , Hill Geoffrey R.
Published: January 2023

Multiple myeloma (MM) is a largely incurable hematological malignancy characterized by clonal expansion of plasma cells in bone marrow (BM). Autologous stem cell transplantation (ASCT) remains a standard treatment for MM, allows intensive cytoreduction and induces inflammation in the context of lymphodepletion. We hypothesized that SCT may overcome myeloma-induced immune defects to restore immune-equilibrium. To address this, we developed a model of ASCT for MM using Vk*MYC myeloma. Surprisingly, we demonstrate the induction of T cell-dependent MM control after SCT as recipients of syngeneic BM grafts containing T cells had prolonged survival and reduced tumor burden compared to BM alone. This anti-myeloma activity was independent of γδ T, natural killer (NK) and NK T cells and was dependent on CD4+and CD8+ T cells. MM-primed T cells, harvested from SCT recipients with controlled MM, protected against relapse in secondary recipients and were Vk*MYC clone-specific. Recipients of MM-primed T cells had increased central memory CD8+ T cells and transfer of MM-primed CD8+ T cells was sufficient to prevent relapse. Furthermore, TCR sequencing of CD8+ T cells from mice with controlled MM revealed a higher clonotype overlap relative to MM-free SCT recipients, consistent with MM-specific T cells. Donor IFNγ secretion and signaling was critical to protective immunity and CD8+ T cells from mice with relapsed MM had impaired IFNγ production compared to those with controlled disease. Importantly, IFNγ production was profoundly augmented by CD137 agonist treatment such that majority of mice controlled disease. These data provide new insights into the mechanisms of action of SCT in myeloma and suggest the use of CD137 agonists to prevent relapse.
Aquaporin 4 blockade alters T cell trafficking through a novel mechanism of S1PR1 regulation.
Authors: Nicosia Michael , Fan Ran , Miyairi Satoshi , Farr George W. , McGuirk Paul R. , Pelletier Marc F. , Beavers Ashley , Valujskikh Anna
Published: January 2023

The water channels aquaporins mediate a variety of immune functions. We previously demonstrated that aquaporin 4 (AQP4) is expressed by CD4 and CD8 T cells and blockade of AQP4 with small molecule inhibitor AER-270/271 significantly prolongs survival of heart allografts in two robust models of rejection. The goal of this study was to determine the effects of AQP4 blockade on T cells.

Administration of AER-271 into naïve non-transplanted mice (250 μg i.p. every 6 h on d. 0–4) decreased numbers of circulating CD4 and CD8 T cells, but not B cells, by >90%, compared to untreated controls. The T cell frequencies and numbers in the secondary lymphoid organs (SLOs) were not significantly affected by AER-271, suggesting the lack of circulating T cells is not due to systemic depletion but rather to altered T cell trafficking.

The effect of AER-271 treatment was transient, as circulating T cell numbers returned to pretreatment levels by d 21. AER-271 treated animals promptly rejected heart allografts transplanted 24 d after treatment cessation (MST 6 d, n=4).

We next tested the effects of AQP4 blockade on Sphingosine-1 Phosphate Receptor 1 (S1PR1), a key mediator of T cell trafficking. S1PR1 mRNA expression was reduced in isolated mouse spleen T cells within 3 h of in vitro incubation with AER-270. Decreased S1PR1 mRNA expression translated into altered chemotaxis, as AER-270 reduced T cell migration toward S1PR1 ligand, Sphingosine-1 Phosphate (S1P), in a transwell system.

Our data suggest that AER-270/271 treatment results in altered S1PR1 transcription thus changing T cell trafficking and prolonging allograft survival. Therefore, AQP4 blockade may be an attractive therapeutic strategy in transplantation and other immune-mediated diseases.