bluuebunny/crossref_metadata_2025 huggingface dataset:
Diuretics combined with captopril modulate macrophage-induced humoral and contact hypersensitivity responses in mice
Authors: Bryniarski Pawel , Nazimek Katarzyna , Marcinkiewicz Janusz
Published: January 2023

Introduction

The over-activated inflammatory responses of macrophages (Mph) likely increases the risk of hypertension development, while clinically relevant diuretics administered alone or in combination with antihypertensive drugs may possibly exert the immunomodulatory effects. Thus, we investigated the impact of these drugs on macrophage-mediated immunity in mice.

Methodology

CBA mice were treated i.p. with captopril (5 mg/kg) with or without hydrochlorothiazide (10 mg/kg) or furosemide (5 mg/kg) by 8 days. On the third day, mice were i.p. injected with mineral oil and 5 days later Mph were harvested to assess the generation of reactive oxygen species (ROS) and nitric oxide (NO). Mph were also pulsed with sheep red blood cells (SRBC) or hapten and transferred to naive mice for evaluation of their ability to induce humoral or contact hypersensitivity (CHS) reactions, respectively.

Results

Diuretics, when administered alone or with captopril, enhanced ROS, but decreased NO production by Mph. SRBC-pulsed Mph from mice treated with captopril combined with diuretics increased the secretion of antigen-specific antibodies by recipient B cells, while Mph of mice treated with hydrochlorotiazide or furosemide with captopril increased the number of antigen-specific B cells. Captopril reduced CHS ear swelling response and this effect was augmented by diuretics.

Conclusions

Our results showed that diuretics with or without captopril modulate the humoral and allergic cell-mediated immune responses by affecting the function of Mph. Further studies should investigate the clinical effect of these observations.

The study was supported by budget funds for science in 2017-2021 under the “Diamond Grant” program (0168/DIA/2017/46).
Nrf2 suppresses allergic lung inflammation by attenuating the type2-innate lymphoid cell response
Authors: Nagashima Ryuichi , Tanaka Nobuyuki
Published: January 2023

The Keap1-Nrf2 system has an essential role in oxidative-stress responses by inducing a number of cytoprotective genes. Under stress, damaged epithelial cells release cytokines that activate Type 2 Innate lymphoid cells (ILC2s), which mediate the allergic immune response. Here we investigated the role of the Keap1-Nrf2 pathway in ILC2 homeostasis and allergic inflammation, using Nrf-2-knockout mice. ILC2s from Nrf2-deficient mice showed a transient, upregulated IL-33 response, and underwent hyper-proliferation in response to combined stimulation by IL-33 with IL-2, IL-7, or TSLP. This enhanced proliferation was correlated with an increased activation of downstream signals, including JAK1, Akt, and Erk1/2. In contrast, activating Nrf2 with a chemical inducer (CDDO-Im) decreased the viability of wild-type, but not of the Nrf2-deficient ILC2s. The reduced ILC2 viability resembled that exerted by the corticosteroid dexamethasone; however, unlike the latter, the Nrf2-dependent cell death was mediated by neither caspase 3-dependent apoptosis nor necroptosis. Using a mouse intratracheal IL-33-administration allergy model, we found that the activation of Nrf2 by CDDO-Im in vivo decreased the numbers of pulmonary ILC2s and eosinophils. These findings indicated that Nrf2 is an important regulator of the allergic response, by determining the survival and death of ILC2s, and suggest that Nrf2 activation is a potential therapeutic strategy for steroid-resistant allergy alleviation.
Defense mechanisms during respiratory infections in hosts with underlying asthma
Authors: Samarasinghe Amali , LeMessurier Kim , Tiwary Meenakshi
Published: January 2023

Influenza can often be complicated by superinfections by bacteria such as Streptococcus pneumoniae. While these opportunistic bacterial pathogens do co-infect during seasonal influenza epidemics, the relationship is exaggerated during pandemic influenza virus infections as documented during all four previous influenza pandemics. Approximately 29–55% of hospitalized patients with Swine Flu with severe disease had a bacterial co-infection; however, asthmatics that were hospitalized during this influenza pandemic were less likely to have co-infections. We built a mouse model that recapitulated these intriguing epidemiologic findings that underlying asthma protected the host from severe morbidity and mortality associated with IAV and S. pneumoniae co-infection. These mice had significantly reduced bacterial load in the lungs, increased eosinophils and macrophages in the airways, but reduced tissue inflammation and scarring compared to non-allergic co-infected mice. We hypothesized that TH2-biased programming of eosinophils within the lungs of allergic mice heightened their immune responses to invading respiratory pathogens. We found that allergen sensitization and IAV exposure activated eosinophils marked by upregulation of CD69, ICAM-1, and IL-5Rα, maintained their viability, and enhanced bacterial uptake through receptor-mediated phagocytosis. Activated eosinophils prevented IAV-induced cytopathology on respiratory epithelia and were able to inhibit the growth and kill S. pneumoniae without undergoing cytolysis. Our data suggest a novel pathway by which eosinophils in hosts with asthma may protect the host from severe diseases induced by respiratory pathogens by inhibiting viral-bacterial synergy.
Neutrophils are actively involved in the regulation of inflammation and the maintenance of surfactant homeostasis during cigarette smoke exposure
Authors: Milad Nadia , Pineault Marie , Lechasseur Ariane , Routhier Joanie , Beaulieu Marie-Josée , Aubin Sophie , Morissette Mathieu
Published: January 2023

Rationale

Exposure to cigarette smoke induces inflammation marked by rapid and sustained neutrophil infiltration as well as pulmonary surfactant damage. However, the extent to which neutrophils contribute to the clearance of surfactant and the inflammatory response to cigarette smoke exposure is not fully understood.

Objective

We aim 1) to assess the capacity of neutrophils to internalise surfactant and 2) to determine whether neutrophil depletion has an effect on surfactant homeostasis and the inflammatory response induced by cigarette smoke exposure.

Methods/Results

1) By incubating cells isolated from the bronchoalveolar lavage of cigarette smoke exposed mice with fluorescently labelled surfactant (NBD-phosphatidylcholine), we observed via microscopy significant in vitro surfactant internalisation by neutrophils. Similarly, neutrophils absorbed labelled surfactant delivered intranasally in vivo as measured by flow cytometry. 2) Neutrophil depletion during cigarette smoke exposure via injection of depleting antibodies (anti-Ly6G or anti-Gr-1) led to exaggerated pro-inflammatory cytokine release (IL-1α, CCL2, GM-CSF and G-CSF) and increased macrophage protease expression (Mmp12). Depletion of neutrophils also led to increased surfactant protein levels (SP-A and SP-D).

Conclusions

Our results show that neutrophils recruited to the lungs during cigarette smoke exposure internalise pulmonary surfactant and that their absence leads to exacerbation of the pro-inflammatory response and alterations to surfactant composition. Therefore, neutrophils seem to play a crucial role in the maintenance of pulmonary homeostasis following cigarette smoke exposure.
Plasmacytoid dendritic cells (pDCs) and IFN-α production in aged squirrel monkeys (Saimiri boliviensis boliviensis) stimulated with Class C CpG Oligodeoxynucleotides
Authors: Nehete Pramod N , Chitta Sriram , Nehete Bharti P , Williams Lawrence E , Wisniewski Thomas , Scholtzova Henrieta
Published: January 2023

Synthetic CpG oligodeoxynucleotides (ODNs) with defined CpG motifs and immunomodulatory properties signal through an endosomal membrane-based type of pattern recognition receptor (PRR), the Toll-like receptor 9 (TLR9), that has been identified in dendritic cells (DCs), B cells, and other human immune cell types. CpG-ODNs influence several signaling pathways, leading to cytokine production in many mammalian species that make CpG ODNs suitable as therapeutic interventions in a variety of human disease conditions. The squirrel monkey (SQM), a well-established New World non-human primate (NHP) model is known to be used for studying various diseases such as Alzheimer’s disease (AD), malaria, and transmissible spongiform encephalopathies (Creutzfeldt-Jacob disease). In the present study, we generated the first report of immunoregulatory activity of class C CpG-ODN in aged squirrel monkey. CpG-ODN exhibits very potent effects on squirrel monkey immune cells by inducing IFN-γ/IFN-α from peripheral blood mononuclear cells and demonstrates accelerated kinetics of activity. In addition, class C CpG-ODN specifically activates pDCs to undergo maturation and secrete cytokines, including high levels of IFN-α. To best to our knowledge, this is the first documentation describing immune status in squirrel monkeys immunized with C class CpG-ODN by concentrating on lymphocyte surface antigen expression and function. Overall, our findings provide critical data regarding the immunomodulatory potential of CpG-ODN in this NHP model allowing for future therapeutic trials of innate immunity stimulation via TLR9 agonists for diverse indications including AD therapeutics.
CD71+ erythroid cells inhibit inflammatory cytokines secretion of monocytes during extracorporeal membrane oxygenation
Authors: Han Junyan , Cao Yu , Hao Xing , Fan Lina , Du Zhongtao , Hou Xiaotong , Zeng Hui
Published: January 2023

Extracorporeal membrane oxygenation (ECMO) has been widely used clinically to support heart and/or lung function. ECMO exposes the blood to non-endothelial cell surface and trigger systemic and local inflammatory responses. Monocytes are proved to contribute to ECMO-induced inflammatory responses and infection. In this study, we investigated the inflammatory cytokines secretion of monocytes and explored the possible mechanisms.

Materials and Methods

Fifty seven patients undergoing ECMO support were enrolled at Beijing Anzhen Hospital, Capital Medical University. Peripheral blood samples from patients were collected into EDTA tubes after the ECMO support days 1, 2, 3, 5 and 7 and post weaning. The ability of monocytes to secret cytokines and the percentage of CD71+ cells were examined by flow cytometry. The effects of CD71+ erythroid cells on the ability of secrete cytokines of monocytes were explored by depleting CD71+ erythroid cells with magnetic beads sorting. The mechanisms of inhibitory effect of CD71+ erythroid on cytokines secretion of monocytes were investigated with Transwell coculture system.

Results

In patients supported with ECMO, the ability of monocytes to secrete cytokines, TNF-a, IL-6 and IL-1b, was significantly decreased compared with healthy controls. It was accompanied by the increased percentage of CD71+ erythroid cells. Depletion of CD71+ erythroid cells rescued the ability of monocytes to secrete cytokines. CD71+ erythroid cells inhibited the cytokines secretion of monocytes in cell-to-cell contact manner.

Conclusion

The enriched CD71+ erythroid cells in peripheral blood inhibited inflammatory cytokines secretion of monocytes in cell-to-cell contact manner in patients supported with ECMO.
Mast cells play a critical role in driving prostatic inflammation in Lower Urinary Tract Symptoms associated with Benign Prostatic Hyperplasia
Authors: Pattabiraman Goutham , Bell-Cohn Ashlee Justine , Murphy Stephen Fintan , Schaeffer Anthony J , Thumbikat Praveen
Published: January 2023

Background

Prostatic inflammation is a key contributor to lower urinary tract symptoms (LUTS). An intraurethral infection with a uropathogenic isolate of E. coli (CP1) results in clinical signs of benign prostatic hyperplasia (BPH) and LUTS as demonstrated by increased urinary dysfunction, prostate inflammation and fibrosis. In this study we study the importance of mast cells in driving prostate inflammation in BPH/LUTS and the therapeutic benefit of mast cell neutralization on the disease.

Results

We observed increased mast cell infiltration and activation associated with increased fibrosis in prostate tissues from BPH patients and CP1 infected mice as identified by toluidine blue and picrosirius staining. To assess the role of mast cell activation, inhibition was performed using a combination of a mast cell stabilizer and histamine 1 receptor (H1R) antagonist at an early and late time point post CP1 infection. Treatment with this combination resulted in a decrease of activated mast cells in prostate tissues of infected mice. Furthermore, the prostate of these mice showed decreased prostate fibrosis and inflammation as assessed by picrosirius red staining and flow cytometry for immune cell infiltrates. Finally, we observed that this combination treatment can ameliorate the urinary dysfunction previously observed in response to CP1 infection, as assessed using unconscious terminal cystometry and bladder voiding function measurements.

Conclusions

These results provide evidence of the critical role played by mast cells in the development of prostate inflammation in BPH/LUTS. A combination therapy of mast cell stabilizer and H1R antagonist maybe a useful target for therapeutic intervention in men with BPH/LUTS.
NK cells support host survival and release IL-10 following polymicrobial sepsis
Authors: Jensen Isaac J , McGonagill Patrick W , Butler Noah S , Griffith Thomas S , Badovinac Vladimir P
Published: January 2023

The sepsis-induced cytokine storm, composed of both pro- and anti-inflammatory cytokines, leads to significant morbidity/mortality. However, the contrasting relationship between the biphasic cytokine storm components remains elusive. NK cells, whose pro-inflammatory IFNγ producing capability is well established, can also prevent immunopathology during systemic infection through anti-inflammatory IL-10 production. This bipolarity of NK cells positions them as a potential throughline for the biphasic components of sepsis. Thus, to precisely delineate the role of NK cells in sepsis utilizing a cecal ligation and puncture (CLP) model we observed diminished survival in NK-depleted mice, attributable to a prolonged cytokine storm. Notably, an increase in IL-15 was observed in NK-depleted mice suggesting that NK cells consume IL-15 during the cytokine storm. To interrogate the potential effect of IL-15, NK cells were treated in culture and with time switched from IFNγ production to IL-10 production. Further, NK cells were shown to have IL-15-dependent IL-10 production during sepsis and that IL-10 deficiency increased sepsis mortality. Importantly, we also observed elevated IL-10 production by NK cells from septic patients relative to healthy controls. Intriguingly, patients with the highest degree of sepsis-induced lymphopenia also had the greatest proportion of NK cell IL-10 production potentially indicating a relationship between the severity of sepsis and the need to counterbalance septic inflammation. These data demonstrate that NK cells limit sepsis-induced immunopathology by producing IL-10 in response to IL-15 during the cytokine storm and suggests a crucial role in balancing the biphasic components of sepsis.
The role of Toll-like receptor 2 and 9 in osteoclast responses to Staphylococcus aureus osteomyelitis
Authors: Petronglo Jenna Rose , Putnam Nicole , Ford Caleb , Curry Jacob , Cassat Jim
Published: January 2023

Staphylococcus aureus is the most common etiology of bone inflammation, or, osteomyelitis (OM). During S. aureus OM, difficulty of treatment is compounded by dysregulation of skeletal homeostasis, which alters the differentiation and function of skeletal cells, namely bone-building osteoblasts and bone-resorbing osteoclasts (OCs), to favor bone loss. Altered cytokine milieu and skeletal cell sensing of bacterial components contribute to this process. In systemic infection, Toll-like receptor (TLR) 2 and TLR9 are critical to antistaphylococcal immunity. Thus, in this study, we sought to determine the importance of TLR2 and TLR9 to the host response to S. aureus OM and to concomitant bone loss. Using in vitro assays, we discovered that S. aureus-induced OC differentiation proceeds in a TLR2-and TLR9-dependent manner. To confirm these findings in vivo, we surgically induced S. aureus OM in Tlr2−/− and Tlr9−/− mice. Despite increased mortality in Myd88−/− mice, we found TLR2 and TLR9 to be individually dispensable during S. aureus OM, with no difference in bacterial burdens nor in trabecular bone loss in femurs post-infection. Overall, our data suggest that redundancy in innate immune responses to S. aureus in bone renders TLR2 and 9 individually expendable in vivo. Because potentiation of these receptors in OCs may share compensatory downstream effects, we have begun in vivo experiments to examine bone loss and bacterial clearance in Tlr2/Tlr9−/− mice and in vitro assays to analyze S. aureus-induced cytokine production in Tlr2−/−, Tlr9−/−, and Tlr2/Tlr9−/− OCs. These findings support further studies into how bacterial stimulation and altered cytokine milieu contribute to dysregulated bone homeostasis during infection.
Immune fibroblasts are unconventional antigen presenting cells and nonprofessional phagocytes in Lyme arthritis
Authors: Lochhead Robert B , Danner Rebecca , Wahhab Amanda , Rouse Joseph
Published: January 2023

Synovial fibroblasts are the primary cell type in synovial tissue and are one of the first cell types involved in immune responses to Borrelia burgdorferi invasion of joint tissue. Recent studies of human Lyme arthritis have shown that synovial fibroblasts up-regulate numerous genes involved in T cell activation upon exposure to the Th1 cytokine IFN-gamma. However, animal model studies are required to better understand their role in host defense and pathogenesis in Lyme arthritis. Using primary synovial fibroblasts isolated from the tibiotarsal joint of C57BL/6 mice, we have found that fibroblasts stimulated with IFN-gamma and B. burgdorferi express MHC class II molecules, a number of costimulatory molecules, and effector cytokines involved in lymphocyte activation, including activation of CD4+ T cells and NK cells. MHC class II molecules isolated from stimulated fibroblasts contained MHC-II peptides, indicating that these cells are capable of presenting antigen to CD4+ T cells. Stimulation with IFN-gamma also reduced fibroblast migration, indicating these cells have impaired ability to repair damaged tissue. Synovial fibroblasts were also able to internalize B. burgdorferi in cell culture, indicating that these cells are capable of acting as nonprofessional phagocytes. Ongoing experiments will determine which lymphocyte subsets interact with inflammatory synovial fibroblasts, and whether these subsets are protective against B. burgdorferi invasion of joint tissue and/or arthritogenic. Together, these data indicate that immune fibroblasts are important in shaping tissue-specific immune responses to B. burgdorferi infection and subsequent inflammation.
Low density neutrophils as a potential biomarker of leprosy severity
Authors: Tavares Isabella Forasteiro , dos Santos Jessica Brandão , dos Santos Pacheco Fabiana , Gandini Mariana , Meyer Rafael Mariante , Moraes Milton Ozório , Schmitz Veronica
Published: January 2023

Erythema Nodosum Leprosum (ENL) is a recurrent acute inflammatory complication of leprosy affecting up to 50% of patients with multibacillary (MB) leprosy, and is treated with thalidomide. Although ENL is described as an immune reaction mediated by neutrophils, studies showing the direct role of neutrophils in ENL are still neglected. One subpopulation of low-density neutrophils (LDNs), present within the fraction of peripheral blood mononuclear cells (PBMC), is associated in the pathogenesis and severity of diseases like sepsis, lupus and tuberculosis. In this study, we characterized circulating LDNs in leprosy, highlighting their involvement in the development of ENL. First, we observed segmented/hypersegmented cells morphologically compatible to neutrophils in PBMC of nonreactional and ENL patients. By flow cytometry, we confirmed the elevated frequency of circulating LDNs (CD14−CD15+) in ENL patients compared to nonreactional. Also, the pattern of expression of neutrophil activation markers suggests a greater activation of LDNs during ENL. Treatment with thalidomide did not interfered the frequency of LDNs, but reduced their activation. Serum levels of MMP-9 (neutrophilic degranulation marker) were correlated with the frequency of LDNs. Finally, we revealed that Mycobacterium leprae (ML) induce generation of LDNs in whole blood and purified normal density neutrophils of healthy individuals in vitro. In brief, we showed that activated LDNs are elevated in the circulation of ENL patients, and may be originated by degranulation caused by ML stimulus. These results point the neutrophilic involvement in leprosy especially on ENL favoring the systemic inflammation and suggest LDNs as a biomarker in ENL development.
Regulation of human macrophage phenotype and GPBAR1 expression by cell seeding density and glucocorticosteroids
Authors: Hammerbeck Christopher , Goetz Christine , Peng Li , Constans Megan , Peterson Ryan , Astry Brian , Hudacek Andrew , Bonnevier Jody L
Published: January 2023

Macrophages are key mediators of tissue homeostasis and inflammation and do so by virtue of differential expression of secreted and cell surface proteins. Cytokines such as IFNγ or IL-4 and IL-13 are known to generate classical proinflammatory M1 and alternatively activated M2 macrophages, respectively, but it is less clear how cell density, cell-cell contact and corticosteroids work together to affect macrophage function. Corticosteroids such as cortisol are known to downregulate the inflammatory signaling but they can also alter the expression of cell surface proteins, such as CD163, on steady-state macrophages. Here we examined the effect of monocyte seeding density and cortisol on the expression of cell surface proteins on human macrophages.

The expression of the macrophage markers CD16, CD163, CD204 and VSIG4 were found to correlate with monocyte seeding density such that at low seeding densities, monocyte-derived macrophages were found to express minimal amounts of CD16, CD163, CD204 and VSIG4 while at high seeding densities, expression of these markers was restored. When cultured in the presence of cortisol, macrophages seeded at low densities were found to upregulate expression of these markers, but not CD204, to levels comparable to macrophages seeded at high densities grown in the presence of MCSF. Additionally, in the presence of cortisol, macrophages expressed higher levels of GPBAR1 which has also been shown to reduce classical proinflammatory macrophage activation in colitis models.

This data suggests that corticosteroids may regulate macrophage function by modulating the expression of cell surface proteins such as GPBAR1 and not just interfering with morer conventional inflammatory cell signaling pathways..
Leishmania donovani metacyclic promastigotes colonize inflammatory monocytes by impairing phagosome function
Authors: Matte Christine , Descoteaux Albert
Published: January 2023

Protozoan parasites of the Leishmania genus are the etiological agents of leishmaniasis, a debilitating disease with a wide spectrum of clinical symptoms ranging from self-healing ulcers to life-threatening visceral pathologies. These vacuolar pathogens are transmitted to mammalian hosts as metacyclic promastigotes by the bite of an infected sandfly. Parasites are then internalized by various phagocyte populations such as inflammatory monocytes. The nature of Leishmania-monocyte interactions over the course of infection and the specific role of these cells in pathogenesis are still unclear. Here, we aimed to investigate the impact of Leishmania donovani metacyclic promastigotes on inflammatory monocyte antimicrobial responses. Our results first show that these cells are highly permissive to L. donovani infection, while bacteria are rapidly eliminated. Upon internalization, L. donovani promastigotes inhibit phagosome acidification, as detected by confocal microscopy using the acidotropic probe LysoTracker. Furthermore, these parasites inhibit NOX2 assembly at the parasitophorous vacuole. Indeed, we found that the membrane-bound subunit gp91phox and the cytosolic subunit p47phox are excluded from the PV membrane. As a result, L. donovani metacyclic promastigotes prevent the production of reactive oxygen species within the PV, as detected using NitroBlue Tetrazolium. Permissiveness of inflammatory monocytes to L. donovani may thus be related to the ability of this parasite to impair the microbicidal properties of phagosomes. The virulence factors involved in these processes are currently being investigated.
Innate immune response changes during the progression of Alzheimer’s disease
Authors: Fulop Tamas , Munawara Usma , Catanzaro Michele , Frost Eric H
Published: January 2023

Alzheimer’s disease (AD) is the most frequent type of dementia. It is primarily a neurodegenerative disease but neuroinflammation plays an important role in the pathogenesis. The dementia develops over decades and progresses from health to AD via a Mild cognitive impairment (MCI) stage. Neuroinflammation precedes the development of the other pathological hallmarks like amyloid plaque formation. Monocytes and macrophages play an important role in neuroinflammation. The aim of the present work was to determine the phenotypes and functions of peripheral monocytes and macrophages, differentiated from these monocytes, through various stages of the disease from health via subjective memory complaints and MCI to mild AD. After informed consent, we enrolled 9 individuals into each of the four groups. We studied phagoburst (flow cytometry), chemotaxis (Boyden chamber), cytokine production (Luminex) and phenotypes (flow cytometry). The results showed a progressive decrease from the healthy group to the mild AD group in both free radical production as well as chemotaxis concomitantly with an increase in inflammatory cytokine production. There was also a shift towards the more inflammatory phenotype (intermediate) of monocytes from healthy to AD. In contrast, monocytes showed increased M2 phenotype in AD compared to healthy. The results demonstrated an increased inflammatory status during progression towards AD, however the various parameters of the innate immune system showed differential changes during this progression depending on their differentiation status.
Hippocampal damage and seizure severity during acute viral encephalitis are determined by the intensity of the infiltrating inflammatory monocyte response
Authors: LaFrance-Corey Reghann , Howe Charles L
Published: January 2023

The immunological response to CNS viral infection must balance the need for effective viral clearance with the need to preserve irreplaceable neurons. Failure to achieve this balance may result in death or severe cognitive impairment. We have observed hippocampal injury, compromised cognitive function, and seizures in C57BL/6 mice following acute infection with TMEV. We established that an initial innate immune response controlled by inflammatory monocyte infiltration into the brain is responsible for this neural injury. To better understand the pathogenic mechanisms involved in monocyte recruitment to the CNS we asked whether reducing the initial viral inoculum impacted the evolution of neural injury. We found that mice infected with standard (2×105 PFU) or low inoculum (1.25×104 PFU) TMEV exhibited a graded inflammatory response. Notably, a 16-fold reduction in viral inoculum resulted in 80% attenuation of the inflammatory monocyte response, concomitant with a 90% attenuation in acute production of CCL2 and complete abrogation of acute increases in TNFα and IL-6 in the hippocampus. Cognitive performance was preserved in low inoculum mice and seizure burden was highly attenuated. Despite the introduction of 16-times less virus, viral load in the brain was equivalent at 3 dpi between groups, indicating that the amount of replicating virus was not associated with reduced neuropathology. Our findings support a model in which the earliest host response to pathogen in the CNS shapes the size of the inflammatory response and constrains the downstream pathological sequelae.
CD169+ macrophages subsets are critical for regulating inflammation during sepsis
Authors: Yeung Stephen T , Khanna Kamal M
Published: January 2023

Sepsis affects more than 30 million people and accounts for approximately 6 million deaths worldwide each year. Current therapy consists of supportive care to keep organs functional and antibiotics to combat infections, with survival contingent on early detection. Our current knowledge of the cellular/molecular mechanisms that regulate the inflammatory pathways resulting in septic shock is limited. Notably, we know little about the precise populations of immune cells that play a central role during sepsis. To bridge these gaps in our knowledge, we examined the role of CD169+ macrophages in septic shock. We utilized both CD169-cre and CD169-diphtheria toxin receptor (DTR) mice to conditionally knockout (CKO) genes and selectively and temporally deplete CD169+ macrophage, respectively. We report that CD169+ macrophages, signaling pathways, and even the CD169 receptor is critical for protection against LPS shock. We show that in the absence of these macrophages, sensors, or the receptor; mice treated with sublethal LPS fail to survive and exhibit increased inflammatory cytokines and impaired IL-10 production. We also show that in the absence of these macrophages, mice exhibit increased dissemination of endotoxin to organs. Protection against LPS induced lethality is rescued by supplemental treatment with rIL-10. Lastly, we show that following LPS treatment, CD169+ macrophages are the initial targeted cell. These findings not only reveal a pivotal role for CD169+ macrophages and its sensing of LPS but also the role for the CD169 receptor as a critical mediator of protection from sepsis induced lethality, and provide new therapeutic targets against sepsis.
A genome-wide CRISPR/Cas9 screen identifies novel regulators of GSDMD pore formation in engineered macrophages
Authors: Evavold Charles Lee , Hafner-Bratkovic Iva , Jerala Roman , Kagan Jonathan C.
Published: January 2023

Cleavage of gasdermin D (GSDMD) by inflammatory caspases results in pore formation at the plasma membrane. GSDMD pores are recognized to mediate pyroptotic lysis of the cell or direct translocation of IL-1 family cytokines from the cytosol into the extracellular space depending on the quantity of pores or duration of plasma membrane occupancy. Hyperactive cells display evidence of fewer GSDMD pores compared to their pyroptotic counterparts, and recent work suggests that ESCRT-dependent membrane repair pathways oppose the cell fate towards pyroptosis through removal of GSDMD pores from the plasma membrane. To identify regulators of GSDMD pore formation at the plasma membrane, we utilized a genome-wide CRISPR/Cas9 screening platform in immortalized bone marrow derived macrophages (iBMDMs). This screen uncovered novel regulators of GSDMD pore formation in the plasma membrane.
Exosomes secreted from microglia during chronic virus infection can induce demyelinating disease
Authors: Luong Nhungoc H , Olson Julie K
Published: January 2023

Theiler’s murine encephalomyelitis virus (TMEV) induced demyelinating disease is a murine model for multiple sclerosis (MS). TMEV persists in microglia and leads to development of chronic progressive demyelinating disease associated with an inflammatory immune response in the central nervous system (CNS) of susceptible mice. TMEV-infected microglia secrete innate immune cytokines, chemokines, and effector molecules associated with development and progression of demyelinating disease. Recently, we have found that TMEV-infected microglia secrete nanovesicles called exosomes that contain and transfer viral RNA to bystander cells in the CNS. Our present studies show that exosomes secreted by microglia during TMEV-induced demyelinating disease (63–98dpi) contain viral RNA/genome that can be taken up by other CNS resident cells, transferring the viral RNA. More importantly, these exosomes could induce the bystander CNS cells to express inflammatory cytokines and chemokines. When administered into naïve mice, exosomes secreted by microglia during TMEV-induced demyelinating disease promoted MS-like pathology including microglia activation and demyelination in the spinal cord. These results suggest that exosomes secreted by infected microglia may directly contribute to the development and progression of demyelinating disease.
A road map of memory CD8+ T cell functional imprinting and fitness
Authors: Chin Shu Shien , Chorro Laurent , Achar Sooraj , Zehn Dietmar , Altan-Bonnet Grergoire , Delahaye Fabien , Lauvau Gregoire
Published: January 2023

Previous studies established that TCR signaling strength determines the size of CD8+ T cell expansion and memory pool. Here we hypothesized that TCR signaling strength further modulates the functional features of memory CD8+ T cells. We immunized mice transferred with naïve OT-I cells, which recognize the Ovalbumin (Ova)-derived epitope SIINFEKL, with Listeria monocytogenes expressing different variants, exposing OT-I cells to various TCR signaling strength. Transcriptomic analysis at early times after priming revealed that compared to OT-I cells primed with strong TCR signals, those primed with weak TCR signals upregulated genes involved in cell cycle (IL-2) and type I IFN response, suggesting that TCR signaling strength could determine how CD8+ T cells respond to cytokines at the time of priming. We postulated that disrupting cytokine signaling in T cells primed with weak TCR signals could alter memory T cell fate. As proposed, OT-I memory cells primed with weak TCR signals and lacking IL-2 or type I IFN cytokine signaling (Ifnar−/−, Il2ramut/mut) failed to re-expand upon secondary challenge compared to WT counterparts. Moreover, transcriptomic and epigenetic analyses of WT or Il2ramut/mut OT-I memory cells primed with strong versus weak TCR signals revealed that while gene expression remained similar, chromatin accessibility was significantly different. TCR and IL-2 signaling together regulate an epigenetic landscape reflecting a broad functional fitness of memory CD8+ T cells, whereas TCR signaling selectively imprinted an epigenetic landscape related to metabolism. These results suggest that epitope selection for effective CD8+ T cell vaccine design is pivotal to modulate the functional programming of memory T cells.
Enhanced ER stress responses accompany greater effector T cell functions with deficiency of PSGL-1
Authors: Bae Eun-ah , Hope Jennifer L , Otero Dennis C , Stairiker Christopher J , Faso Hannah A , Palete Ashley B , Bradley Linda M
Published: January 2023

Endoplasmic reticulum (ER) stress is induced by the accumulation of misfolded proteins and other cellular stresses, which lead to the unfolded protein response to restore ER homeostasis. Previous studies have shown that the ER stress response is required for T cell activation and differentiation. However, recent studies demonstrated that inhibition or deletion of ER stress mediators in T cells actually promotes anti-tumor immunity. Therefore, this incongruity as to the role of ER stress response in effector T cell function warrants further investigation. Our lab previously identified that the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1), acts as a T cell immune checkpoint regulator. PSGL-1-deficiency enhances T cell function, resulting in the clearance of chronic viral infection and dramatic tumor control. To better understand the contribution of ER stress to T cell function, we assessed T cell phenotype and function together with the ER stress mediators, XBP1s and ATF4, in highly-functional PSGL-1−/− T cells as well as exhausted wild-type T cells in both tumor and virus models. In melanoma models, ER stress was highly induced in activated tumor-infiltrating T cells (TILs). Effector CD8+ TILs producing IFNg displayed elevated XBP1s and ATF4 compared to IFNg− TILs. Upon chronic LCMV Cl13 viral infection in PSGL-1−/− mice, we found increased virus-specific T cells with enhanced multi-functional effector responses and increased memory/stem cell-like T cell populations characterized by the expression of the transcription factor TCF-1, as well as increased ER stress responses. Together, these data suggest that ER stress plays an essential role in effector T cell functions in settings of chronic antigen stimulation.
Elucidating the role of ATM in BER and MMR during B cell CSR
Authors: Sible Emily , Fiorica Giuseppe , Rahman Sadia , Attaway Mary , Vuong Bao Q.
Published: January 2023

Secondary immunoglobulin isotypes are produced by class switch recombination (CSR), which requires AID-dependent DNA deamination of intronic switch (S) regions within the immunoglobulin heavy chain gene. Non-canonical repair of deaminated DNA by mismatch repair (MMR) or base excision repair (BER) creates staggered DNA breaks that promote recombination between S regions. ATM-dependent phosphorylation of AID at serine-38 (pS38-AID) promotes its interaction with a BER protein, suggesting that ATM regulates CSR through BER. However, the mechanism by which ATM participates in BER remains unknown. ATM−/− mice were bred to knockout mice of the MMR gene MSH2. Surprisingly, the predicted Mendelian frequencies of ATM−/−MSH2−/− adult mice were not obtained and preliminary data indicate a late embryonic lethal phenotype. To bypass the observed lethality, ATM was conditionally deleted on an MSH2−/− background using a floxed ATM allele [ATMF] and B cell-specific Cre recombinase expression. These ATMd/dMSH2−/− mice and B cells display a CSR phenotype comparable to ATM−/− B cells, which suggest that ATM may function genetically upstream of MSH2 and implicate a role for ATM in both MMR and BER.
H3K9 dimethyltransferase G9a is an important epigenetic modulator of B cell differentiation
Authors: George-Alexander Lou-Ella , Kania Anna , Hicks Sakeenah , Mi Tian , Scharer Christopher D. , Boss Jeremy
Published: January 2023

B cell differentiation is a tightly regulated process coordinated by the timed expression of various transcription factors and chromatin accessibility changes mediated by histone modifying enzymes. The histone methyltransferase (HMT) G9a, dimethylates histone H3 lysine 9 (H3K9) at promoters and inhibits gene expression via recruitment of proteins that impair chromatin accessibility. HMTs are expressed ubiquitously but display distinct enzymatic activities and patterns of chromosomal localization. During B cell differentiation, G9a was found to co-localize with Blimp-1 which is required to silence genes associated with a B cell phenotype and cellular proliferation. However, the B cell processes that are modulated by G9a mediated dimethylation remain to be elucidated. To assess the role of G9a in B-cell differentiation, we crossed G9afl/fl mice onto the CD19Cre/+ background (G9aKO mice). Stimulation of CD19Cre/+ (CreCtrl) and G9aKO mice with the T cell independent antigen LPS resulted in a significant increase of activated B cells and plasmablast in G9aKO mice. Further characterization of this phenotype, identified a skewing within the mature B cell population towards marginal zone (MZ) B cells in G9aKO mice. Regions with chromatin accessibility and expression changes identified by ATAC-Seq and RNA-Seq elucidated the B cell processes that are modulated in G9a deficient mice. B cell processes that are subject to direct modulation by G9a will be determined using CUT&RUN. Together, our data shows the importance of G9a in epigenetic modulation required for B cell function.
Molecular mechanisms by which interferon regulatory factor 5 (IRF5) transduces signaling downstream of BCR and BCR/TLR9 pathways
Authors: Rice Matthew R , Brune Zarina , Guo Qin , Barnes Betsy J
Published: January 2023

The transcription factor IRF5 modulates human B cell activation, proliferation, and plasmablast differentiation via an intrinsic mechanism. Targeted knockdown of IRF5 in human primary naïve B cells revealed a significant reduction in CD19+CD86+ B cells, which demarcate early activated B cells, after BCR/TLR9 stimulation, resulting in reduced clonal expansion and plasmablast differentiation. The mechanism(s) by which IRF5 modulates early B cell activation is not known. We previously found that BCR stimulation alone was insufficient to induce IRF5 nuclear translocation (activation), while BCR/TLR9 stimulation induced robust IRF5 activation. Thus, the focus of this study is to determine the mechanism(s) by which IRF5 functions downstream of BCR and dual BCR/TLR9 signaling at the early time points of activation (5 and 15 min). Experiments were performed in human B cells and B cells from wild-type and Irf5−/− mice. Signaling and protein interactions were examined by Western blot, immunoprecipitation, flow cytometry, and biochemical assay. We identified Bruton’s tyrosine kinase (Btk) as a novel interacting partner of IRF5 in both unstimulated and stimulated human and murine B cells. Of note, active phosphorylated Btk was bound to IRF5 only in stimulated cells. Preliminary data also suggest a defect downstream of Btk activation in Irf5−/− B cells. Together, these data indicate a conserved role for IRF5 in the early stages of B cell activation via BCR/TLR9 signaling.
TLR adaptors MyD88 and TRIF are critical for extrafollicular B cell responses to influenza
Authors: Lam Jonathan , Baumgarth Nicole
Published: January 2023

Antibodies produced during primary influenza infection can be protective and form rapidly. This occurs independently of germinal centers (GCs) in reactions known as extrafollicular B cell (EF) responses, where antibody-secreting cells (ASCs) blast and produce antibodies in the medulla of the mediastinal lymph node. While the EF response to influenza is known to be mostly T-dependent, it is unknown why B cells are shunted away from a GC fate and what factors lead to generation of EF ASCs. We show that absence of both Toll-like receptor (TLR) adaptors MyD88 and TRIF (double knockout, DKO) cause severe, B cell-intrinsic reductions in EF responses to influenza. Observing a reduction of the B cell differentiation factor IRF4 in DKO plasmablasts in vivo, we used in vitro culture systems to assess BCR signaling and T cell help by stimulating B cells with anti-IgM(Fab)2 and CD40L and/or BAFF, respectively. DKO B cells showed reduced survival in culture, which was rescued by providing either CD40L or BAFF. Strikingly, DKO B cells barely proliferated in response to any dose of anti-IgM, with or without CD40L/BAFF. High-dose BCR stimulation further reduced the viability of DKO B cells, which correlated with reduced IRF4 induction and altered NFkB signaling. Specifically, we show by image flow cytometry reduced nuclear localization of NFkB c-Rel, a positive regulator of IRF4, following BCR stimulation of DKO compared to wild type B cells. We propose that EF responses during influenza infection require not only antigen and T cell help, but also MyD88 and/or TRIF signaling to drive activation of c-Rel. This enhances antigen-specific B cell survival, proliferation, and the strong upregulation of IRF4 required for plasmablast differentiation.
Toll-like receptor signaling directly modulates B cell antibody responses
Authors: Nelson Alexander J , WU Yee Ling
Published: January 2023

Toll-like receptor (TLR) signaling alone or in concert with other cellular or molecular signals helps shape responses by different cell types. Murine B lymphocytes express cell surface-bound TLRs 1, 2, 4, 6 and endosomal TLRs 3, 7, 9. This study aims to delineate the effects of these TLRs on B cell responses including proliferation, activation and antibody class-switch recombination (CSR).

We stimulated mature, naïve B cells in vitro with individual TLR agonists alone or together with T cell help (via ligation of CD40) and the TH2 cytokine interleukin-4 (IL-4). We measured proliferation by CellTrace Violet assay, cell activation by surface expression of B cell activation markers, and analyzed antibody class switching by surface expression and secreted levels of antibody isotypes by flow cytometry.

We found that in the presence of a minimal level of anti-CD40, stimulation with TLRs 1/2, 2/6, 4, and 9 agonists induced cell proliferation and up-regulation of activation markers including CD69 and CD80. While both types of TLR agonists down-regulated class switching to IgG1, stimulation of the surface-bound TLRs (1/2, 2/6 and 4) increased class switching to IgG3 and IgG2b. In contrast, stimulation of endosomal TLR9 inhibited switching to all isotypes. The addition of IL-4 induced robust cell proliferation and class switching to IgG1, and TLR 1/2, 2/6, 4, and 9 agonists further enhanced these IL-4-mediated B cell responses.

Altogether, our data demonstrate differential effects of surface bound and endosomal TLRs on B cell responses. Activation of surface-bound TLRs, but not endosomal TLR9, triggers CSR to TH1-associated antibody isotypes, while both groups of TLRs synergize the effect of IL-4 on driving CSR to IgG1.
TLR7/8 and TLR9 ligands induce phenotypic and transcriptional changes in human transitional B cells
Authors: Posner Jourdan K , Chang Sandra P
Published: January 2023

The maturation of human transitional B cells into mature, naïve follicular or marginal zone B cells is a key step in B cell development. Transitional B cells are a prominent B cell population in newborns and very young children who receive many vaccines during early childhood. Pattern recognition receptor (PRR) ligands are attractive candidate vaccine adjuvants due to their targeted mechanisms of action. However, the effects of PRR ligands on transitional B cell fate are not fully understood. We have demonstrated that human cord blood transitional B cells predominantly mature into follicular-like B cells when stimulated in vitro with R848 (TLR7/8 ligand) or CpG (TLR9 ligand) in the presence of IL-4. Additionally, we have profiled the transcriptome of human transitional, follicular and marginal zone-like B cells and identified genes uniquely associated with each B cell subset. Several of the genes identified during our transcriptome analysis were expressed in our in vitro-stimulated transitional B cells. These gene expression changes following PRR ligand stimulation indicate that differing stimuli can skew transitional B cell fate. Our characterization of PRR ligand-induced phenotypic and transcriptional changes associated with mature follicular and marginal zone B cell development may influence the rational approach for vaccine adjuvant development, especially for newborns and very young children.