bluuebunny/crossref_metadata_2025 huggingface dataset:
Clearance of Plasmodium falciparum-infected Red Blood Cells by Natural Killer cells and Monocytes
Authors: Sekar Padmapriya , Arora Gunjan , Schureck Marc , Desai Sanjay , Long Eric O
Published: February 2023

Antibodies naturally acquired during repeated exposure to the parasite Plasmodium falciparum are essential for control of blood-stage malaria. Natural killer (NK) cells are innate immune cells with an important role in containing viral infections and tumor growth, mainly by direct cytotoxicity and inflammatory cytokine production. Recently, the lab reported that primary human NK cells lyse Plasmodium falciparum-infected red blood cells (iRBCs) via antibody dependent cellular cytotoxicity (ADCC) in presence of IgG isolated from adults living in a malaria-endemic region of Mali. Using live imaging, I observed release of parasitophorous vacuoles (PV) from iRBCs after incubation with NK cells in the presence of rabbit anti-RBC Abs. I confirmed the imaging results by flow cytometric analysis. Direct interactions of NK cells with iRBCs was imaged by scanning electron microscopy, which revealed tight immunological synapses and extensive damage to iRBC membranes. This membrane damage results in formation of ghost RBCs and eventually release of PVs. We hypothesized that monocytes clear PVs, which could reduce the inflammatory response. I isolated PVs by mild saponin treatment and mechanical disruption of iRBC membranes. Binding of IgG isolated from plasma of Malian adults to PVs was orders of magnitude higher than binding to iRBCs. Phagocytosis of PVs by primary human monocytes was detected and was enhanced in the presence of Mali IgG. Similar results were obtained using PVs released by NK-dependent lysis of iRBCs in the presence of Mali IgG. These results suggest that NK-mediated ADCC inflicts severe damage to the plasma membrane of iRBCs, leading to release of PVs which bind immune serum IgG and are cleared through phagocytosis by monocytes.
SARs-CoV-2 Infection Impacts the Function and Phenotype of Plasmacytoid Dendritic Cells
Authors: Codrington Alicia L , Dewald Hannah , Elson David , Chopoorian Abby , Zembrziglel Krzysztof , Park Claire , Singh Sukhwinder , Xie Yingda , Fitzgerald-Bocarsly Patricia
Published: February 2023

Plasmacytoid dendritic cells (pDC) are innate immune cells and potent producers of interferon alpha (IFN-α). Sars-CoV-2, an RNA virus that causes Coronavirus Disease 2019 (COVID-19), has taken the lives of more than 400,000 people in the United States. Reports indicate that COVID-19 patients have reduced plasma IFN-α, suggesting a potential use of IFN-α as a disease therapeutic. However, investigations on pDC function and phenotype in COVID-19 patients are needed. We isolated peripheral blood mononuclear cells from fifty hospitalized COVID-19 patients. PBMC were stimulated with HSV-1 or Influenza A virus and IFN-α production and phenotype were assessed by flow cytometry. After stimulation, there were fewer IFN-α+ pDC from COVID-19 patients compared to controls. To reduce inflammation, COVID-19 patients may be treated with dexamethasone, a corticosteroid that has negative effects on pDC function. Although there was more impairment of pDC numbers and function in dexamethasone-treated subjects, the pDC dysregulation was also seen prior to dexamethasone treatment. Phenotypically, we identified reduced expression of pDC markers BDCA2 and CD123, and an upregulation of co-stimulatory markers on pDC from COVID-19 patients. We also observed an increased proportion of Ki67+ pDCs, which indicates increased turnover of pDCs during moderate to severe COVID-19 disease. In summary, pDC from COVID-19 patients produce significantly less IFN-α, express costimulatory ligands used to stimulate adaptive immunity, and may be undergoing rapid turnover. Overall, these changes may compromise the antiviral and adaptive immune response or represent pDC exhaustion during SARS-CoV-2 infection.
NLRP3 inflammasome inhibitor as an adjunct host-directed therapy in a hyper susceptible mouse model of Group A Streptococcal Necrotizing Fasciitis.
Authors: Mukundan Santhosh , Satpati Abhijit , Ambigapathy Ganesh , Combs Colin , Nookala Suba
Published: February 2023

Despite prompt intervention with antibiotics and surgical debridement, morbidity and mortality due to Group A Streptococcal Necrotizing Fasciitis (GAS-NF), frequently associated with streptococcal toxic shock, remain as high as 50%. The pathways driving the sequelae of effector responses that include an uncoordinated activation of the host immune system and inflammatory cytokine storm are not completely elucidated. Our previous studies demonstrated a central role for the pro-inflammatory mediator, IL-1b, in modulating the severity and survival in a mouse model of GAS-NF. To fine-tune IL-1b levels while simultaneously attenuating GAS multiplication and toxin synthesis, here, we investigated the effect of MCC950 (MCC), a small molecule inhibitor for NLRP3 inflammasome activation as a host-directed adjunct to clindamycin (CLN) (10mg/kg each, everyday X 5 days) in a mouse model of GAS NF. Survival outcomes did not differ between CLN and CLNMCC groups. Yet, CLNMCC significantly reduced skin mRNA levels of IL-1a, IL-1b, IL-33, and TNF-a, protein levels of the active fragment of caspase-1 and increased the ratio of pro: active caspase-1 (but not ASC or NLRP3) that coincided with reduced levels of circulating neutrophils. CLNMCC treatment significantly reduced tissue damage, levels of plasma IL-10, IL-1β, and circulating CD11b+LY6G+ granulocytic myeloid subsets. These results indicate that NLRP3 inflammasome activation may be primarily involved in excess IL-1b release, neutrophil influx, and tissue damage during GAS-NF. The evidence of improved outcomes suggests, in part, a beneficial efficacy of MCC950 to target NLRP3 as a feasible adjunct to CLN and mitigate inflammation and pathology with no adverse effects or delayed toxicity
BCG vaccination provides cross-protection against influenza infection through trained adaptive immunity.
Authors: Tran Kim , Divangahi Maziar
Published: February 2023

The ongoing COVID-19 pandemic highlights the threat emerging viruses pose to the world. While the development of a new vaccine can take at least 1–2 years, alternative approaches are urgently needed to alleviate the burden of rapid transmission. The Bacillus Calmette-Guerin (BCG) vaccine is currently used worldwide to prevent tuberculosis but has also been shown to protect against a wide range of infections. Several studies have attributed this broad protection to trained immunity. While innate immune cells usually act in a non-specific manner, in the context of trained immunity, these cells acquire an enhanced capacity to protect against different pathogens. However, preliminary work suggests that BCG may be mediating trained immunity by enhancing the adaptive immune system as well. Before the current pandemic, Influenza A virus (IAV) was responsible for the most devastating pandemic in history and remains the cause of yearly epidemics. Thus, investigating the cross-protection of BCG against IAV could determine whether BCG vaccination could offer an alternative preventive strategy against viruses with pandemic potential. To determine the protective effects of BCG against IAV infection, C57BL/6 mice were vaccinated intravenously (-iv) or subcutaneously (-sc) with BCG then infected with H1N1. We showed that BCG-iv vaccinated mice exhibited increased survival against IAV and a decreased viral load. Interestingly, BCG-iv uniquely induces the enrichment of CX3CR1+ αβ memory T cell populations which may be contributing to protective immunity against IAV infection. Overall, the present study evaluates whether beneficial effects from a widely distributed vaccine could provide some protection against emerging pulmonary pathogens.
Clearance of skin-homing CCR7+ neutrophils by conventional type1 dendritic cells regulates cutaneous local autoimmunity and infection
Authors: Kolios Antonios , Diaz Victor , Egholm Cecilie , Halin Cornelia , Boyman Onur , Özcan Alaz
Published: February 2023

A rapid neutrophil response is essential for immunity against infections, however impaired elimination or migration can inflict significant tissue damage by exacerbated inflammation. Here we show that neutrophils recruited to inflamed or infected skin are rapidly redirected to skin-draining lymph nodes (dLNs) and non-draining secondary lymphoid tissues. Using transgenic mouse models and bone marrow chimeras, cutaneous inflammation is induced with imiquimod, a TLR7-mediated psoriasis-like model, or skin infection is induced by Staphylococcus aureus. We show that neutrophils migrate to the dLNs through lymphatic vessels in a CC-motif chemokine receptor 7 (CCR7)-dependent manner and are phagocytosed by resident conventional type1 dendritic cells in the dLNs when entering the lymph node, a mechanism contributing to neutrophil clearance. Deficiency of CCR7 impairs neutrophil elimination in the dLNs and leads to neutrophilic accumulation in the skin, which in turn leads to an exacerbated phenotype in a model of psoriasis-like local inflammation as well as improved clearance and immunity against cutaneous Staphylococcus aureus infection. Overall, our findings explain how impaired neutrophil migration and clearance in the dLNs could regulate cutaneous autoimmunity and infection.
TLR9 modulates peritoneal immunity via regulating the biology of CD9hi fibroblastic reticular cells
Authors: Jiang Ting , Li Yiming , Chen Tianmeng , Wu Junru , Chen Kong , Billiar Timothy R , Deng Meihong
Published: February 2023

Fibroblastic reticular cells (FRC) are highly heterogeneous. Distinct FRC subsets play unique roles in the formation of secondary lymphoid organs and immune responses. We have previously shown that TLR9 signaling plays a critical role in regulating peritoneal immunity via suppressing chemokine production in FRC in the fat associated lymphoid clusters (FALC). However, the subset-specific roles of TLR9 in FRC of FALCs remain unknown. Using single-cell RNA-sequencing, we identified three distinct subsets of FRC (CD55hi, CD9hi and CD55loCD9lo) in mouse mesenteric FALCs at baseline. Based on the gene set enrichment analysis, the Cd55hi subset was enriched in gene expression related to cell differentiation. The CD9hi subset was enriched in gene expression related to immune response. The CD55loCD9lo subset was enriched in the gene expression related to extracellular matrix formation. Furthermore, we found that CD9hi FRC from Tlr9−/− mice increased gene expression associated with inflammation. Using flow cytometry and bulk-RNA seq, we successfully isolated and validated these three subsets in mouse FALCs. Interestingly, activation of TLR9 signaling using ODN1585 significantly decreased CD9 expression in FRC in vivo and in vitro. Furthermore, treatment of ODN1585 suppressed the proliferation of CD9hi FRC, evidenced by decreased expression of Ki67. These results indicate that CD9hi FRC in FALC are immunoregulatory. TLR9 signaling may modulate peritoneal immunity via regulation of the proliferation of CD9hi FRC. Understanding the mechanism of how TLR9 regulates CD9hi FRC may lead to discovering new therapies for inflammatory diseases.
T-box transcription factors Eomes and T-bet regulate primary human NK cell IFNγ response and in vivo persistence
Authors: Wong Pamela , Chang Lily , Neal Carly C , Wagner Julia A , Berrien-Elliott Melissa M , Cubitt Celia C , Tran Jennifer , Marsala Lynne , Schappe Timothy , Fehniger Todd A
Published: February 2023

T-box transcription factors (TF) Eomes and T-bet are vital for NK cell development. However, their role in mature NK cell homeostasis and function remains unclear. Recently we reported that Eomes regulates mouse NK cell cytotoxicity and stage-specific survival using an NK-specific, tamoxifen-inducible Eomes KO model. However, the role of Eomes and T-bet have not been investigated in primary human NK (hNK) cell biology using genetic loss-of-function approaches. We hypothesized that these TFs play a critical role in maintaining NK cell functional molecular programs.

Here we used CRISPR-Cas9 to genetically delete Eomes and T-bet expression in primary hNK cells. In vitro stimulation assays revealed that IFNγ response is impaired in CD56bright NK cells only when Eomes is deleted (p<0.01) but not T-bet. This impairment is even more striking when both TFs are deleted, resulting in both CD56bright and CD56dim NK cells having reduced IFNγ (p<0.01 and p<0.05, respectively).

Using NSG xenografts, we found that Eomes/T-bet double CRISPR-edited (DKO) hNK cells have impaired persistence/expansion in vivo. After 3 weeks, we recovered in the spleen on average 3E5 control compared to only 6E4 DKO hNK cells. A similar ~80% decrease was also observed in the blood. To determine the mechanism of this, we assessed proliferation and apoptosis of control compared to DKO cells. Ki-67 and dye dilution revealed impaired proliferation of DKO cells in vivo, and we also observed increased apoptosis measured by Annexin V/7-AAD staining (p<0.05).

Our data suggest that Eomes and T-bet are critical for the function and homeostasis of mature hNK cells. Understanding their function in mature NK cells will provide insights to improve NK cell therapy for diseases such as cancer.
Multiplexed proteomics of autophagy deficient macrophages reveals enhanced anti-microbial immunity via the oxidative stress response
Authors: Maculins Timurs , Verschueren Erik , Hinkle Trent , Chang Patrick , Chalouni Cecile , Lim Junghyun , Katakam Anand Kumar , Kunz Ryan C. , Erickson Brian K , Huang Ting , Choi Meena , Tsai Tsung-Heng , Vitek Olga , Reichelt Mike , Rohde John , Dikic Ivan , Kirkpatrick Donald S. , Murthy Aditya
Published: February 2023

Macrophages play a critical role in clearance of cytosolic pathogens. Autophagy functions at the intersection of antimicrobial innate immunity, metabolism and protein quality control; however, the impacts of infection and autophagy on shaping the macrophage proteome are poorly understood. Here, we describe a deep multi-dimensional proteomic analysis of primary murine macrophages infected with Shigella flexneri (S. flexneri). Tandem mass tagging (TMT) revealed dynamic genotype- and infection-dependent differences in host and pathogen proteins, phosphorylation and ubiquitination. These data catalogue the complex circuitry connecting autophagy, inflammatory signaling and the oxidative stress response. Loss of the autophagy gene Atg16l1 induced basal oxidative stress, activated the compensatory glutathione biosynthetic machinery, and surprisingly, enhanced clearance of S. flexneri. Pathogen clearance was similarly enhanced in wild type macrophages upon pharmacological inhibition of cysteine import. Our study provides a resource for innate immunity research and unexpectedly reveals that ATG16L1 dampens antimicrobial immunity by regulating oxidative stress.
The effect of lipopolysaccharide (LPS) on post-viral airway disease is independent of the Type I interferon receptor
Authors: Resiliac Jenny , Rohlfing Michelle , Santoro Jennifer , Hussain Syed-Rehan A. , Grayson Mitchell
Published: February 2023

Rationale

Sendai virus (SeV, murine parainfluenza virus type 1) infection of C57BL6 (WT) mice leads to post-viral airway disease, characterized by airway hyperreactivity (AHR). Previous data suggested pretreatment of WT mice with lipopolysaccharide (LPS) decreased post-viral airway disease in a dose dependent manner. LPS binding to its receptor can trigger release of type I interferon (IFN), which is a critical anti-viral cytokine. Type I IFN is known to restrict viral replication and spread. However, the role type I IFN mediates in LPS protection from post-viral airway disease is not known. This study was undertaken to determine if the LPS mediated reduction of AHR depended upon type I IFN signaling.

Methods

WT and Ifnar−/− mice (mice with genetic deletion of the type I IFN receptor) were inoculated intranasally with 30 ul 0.1 μg LPS (dose that inhibited post-viral disease in prior studies) or PBS 24 hours before SeV (2e5 pfu) or UV inactivated SeV. Twenty-one days post-viral inoculation, AHR was determined by invasive measurement of airways’ resistance to methacholine.

Results

SeV induced a similar level of AHR in both Ifnar−/−and WT mice when they were pretreated with PBS. Similarly, pretreatment of Ifnar−/− mice with LPS significantly reduced AHR compared to PBS treated Ifnar−/− mice (p<0.0001, ANOVA, n≥3). LPS treated Ifnar−/− mice demonstrated significantly reduced AHR compared to PBS treated WT mice, as well (p=0.001, n≥3).

Conclusion

LPS mediated reduction of post-viral airway disease is independent of type I IFN receptor signaling. Our results suggest LPS prevents post-viral AHR independent of IFNAR signaling. Future studies will determine other mechanisms through which LPS pre-treatment prevents post-viral AHR.
IFNβ is a critical regulator of Lyme arthritis severity in mice and is regulated by the tumor suppressor gene Cdkn2a on murine Chr4
Authors: Li Jinze , Ma Ying , Paquette Jackie K. , Zachary James F. , Teuscher Cory , Weis Janis J.
Published: February 2023

Lyme disease is caused by infection with the tick-borne spirochete Borrelia burgdorferi, and results in a spectrum of clinical disorders. Infected C3H mice develop severe Lyme arthritis while B6 mice display mild disease, allowing analysis of host genetic contribution to disease. We used forward genetics to identify a region on Chr4, Bbaa1, that includes the Type I IFN cluster and regulates arthritis severity. The development of congenic mice, B6.C3-Bbaa1, isolating the C3H allele of Bbaa1 onto the background of B6 mice demonstrated that Bbaa1 regulates arthritis severity by modulating Type I IFN expression. IFNβ was identified as the key effector for Lyme arthritis severity, however, paradoxically there are no polymorphisms between C3H and B6 in this gene. To identify regulators of IFNβ in the Bbaa1 locus, candidate genes were subjected to siRNA silencing. A single gene, Cdkn2a, was identified as the modulator of IFNβ expression, and its encoded protein ARF was found to regulate the magnitude of IFNβ expression. Reconstitution of Arf−/− mice with hematopoietic cells expressing the C3H allele of Arf developed severe arthritis, whereas mice reconstituted with cells expressing the B6 Arf allele developed mild arthritis. ARF was found to regulate B. burgdorferi induced IFNβ through p53 and BCL6 in myeloid cells. Indeed, targeted blocking of BCL6 enhanced IFNβ activation in the joint tissue of B6 mice and resulted in increased severity of Lyme arthritis. Results were similar in Rag-1-deficient mice, indicating dependence on myeloid cells expression of BCL6. Thus, we have identified novel pathways modulating IFNβ expression in Lyme arthritis development which could lead to therapeutic discovery.
Tpl2 Signaling Regulates Dendritic Cell Activation and Responding T Cell Differentiation Following Mycobacterium tuberculosis Infection
Authors: Groft Sarah Grace , Suzart Vinicius , Nagy Nancy , Boom W Henry , Harding Clifford V
Published: February 2023

Mycobacterium tuberculosis (Mtb) utilizes a number of immune evasion mechanisms in order to persist inside of host antigen-presenting cells. Dendritic cells (DCs) are important in restricting Mtb growth by migrating to draining lymph nodes and activating antigen-specific T cell responses, but the roles of DCs in Mtb infection require further study. This study investigated DC activation and functional outcomes following Mtb H37Ra-driven tumor progression locus 2 (Tpl2) signaling. The role of Tpl2 was interrogated genetically, utilizing bone marrow-derived DCs from Tpl2−/− mice. We assessed cytokine production via ELISA, mRNA levels via qRT-PCR, and expression of cell surface molecules via flow cytometry. In Mtb-treated DCs, genetic depletion of Tpl2 increased production of pro-inflammatory cytokines such as IL-12p40 and IL-6. Loss of Tpl2 in Mtb-treated DCs also led to decreased E-cadherin expression, and increased expression of Icam-1 (Cd54) and Mmp2, which are molecules involved in DC transmigration. Expression of Ccr7 and Ccr4 was also enhanced in Mtb-treated Tpl2−/− DCs, which correlated with improved migration of Tpl2−/− DCs towards CCL19 and CCL21 in vitro (assessed using a trans-well assay). When antigen-specific CD4+ T cells were co-cultured with Mtb-infected DCs, deletion of Tpl2−/− in the DCs resulted in increased T cell production of IFNγ and IL-2, as well as increased Tbet expression, indicative of enhanced Th1 polarization. Together, these data indicate that Mtb-induced Tpl2 signaling suppresses certain aspects of DC activation, leading to blunted Th1 responses against the pathogen.
Enhanced responsiveness to subsequent bacterial infection in macrophages stimulated with conserved protein patterns
Authors: Luu Abby , Shepardson Kelly , Hatton Alexis Alexandria , Robinson Ema , Goodall Cheri , Elkins Morgan , Douglas Trevor , Rynda-Apple Agnieszka
Published: February 2023

Immune modulation via exposure to a primary stimulus can modify innate immune cell responses to a subsequent unrelated infection. Previously, we found that conserved mammalian proteins which display a pattern of repeating protein subunits (RPSP) enhance clearance of a secondary respiratory bacterial infection in a TLR2/1-dependent manner. RPSPs like filamentous actin or ferritin are expressed intracellularly in a steady-state, thus their detection by extracellular pattern recognition receptors should elicit danger-like signals. Unlike classical DAMPs, which prompt an inflammatory response, TLR2/1-based recognition of RPSPs did not result in proinflammatory cytokine production or neutrophil lung infiltration. Yet, upon challenge with Staphylococcus aureus, RPSP inoculated mice exhibited a more robust inflammatory response and better bacterial clearance than control mice. This TLR2/1-based RPSP recognition was not limited to mammalian proteins. Enhanced S. aureus clearance was achieved with a synthetic peptide (Q11; QQKFQFQFEQQ) that organizes into RPSP fibers and is non-immunogenic. Further, we found that RPSP enhanced S. aureus clearance was dependent on non-classical Type I IFN signaling whereby IFNβ production was required, but absence of the IFNAR1 subunit of the Type I IFN receptor did not abrogate S. aureus clearance. In vivo depletion and bacterial clearance assays showed that macrophages were essential for this subsequent improved response, which persisted after resting cells for days post RPSP stimulation. Our results indicate that RPSP stimulation of macrophages enhances these cell’s abilities to respond to a secondary infection in an IFNβ dependent manner and has features suggestive of trained immunity.
Loss of myeloid S1PR1 makes dysfunctional alveolar macrophages and vascular injury by inducing myeloid bias
Authors: Banerjee Somenath , Joshi Jagdish C , Yalagala Vijayalakshmi , Mehta Dolly
Published: February 2023

Alveolar macrophages (AM) are the most abundant resident innate immune cells in the lungs and directly communicate with both the non-sterile external environment and the pulmonary epithelium. AM initially trigger protective inflammation upon sensing pathogen but later induce lung repair by clearing dead cells, known as efferocytosis, and dampening of inflammation. Studies show that monocytes recruited in the airspace during injury gain AM signature and contribute in lung repair. Sphingosine 1 phosphate receptor 1 (S1PR1), is widely studied in lung endothelium as a barrier protective mechanism and in immune cells in context of trafficking and inflammation but has not yet extensively studied in respect to origin and function of AM. Here, we deleted S1PR1 in mice by crossing S1PR1f/f with LysMCre (S1PR1ΔLyz) mice to investigate the role of S1PR1 in myeloid cells in the mechanism of lung injury. We show that loss of S1PR1 in myeloid cells of mice (S1PR1ΔLyz mice) has 3-fold higher AM pool but surprisingly these mice showed defective lung fluid balance basally or post LPS challenge (intratracheal 0.5 mg/kg). We found that S1PR1−/−AM produced markedly less pro-inflammatory cytokines (IL1b and TNFa) and could not efferocytose. Intriguingly, S1PR1 deficiency induced myeloid biased haematopoiesis as evident from less erythroid progenitor (~0.75 fold) and B cells (~0.82 fold) in bone marrow of S1PR1ΔLyz mice leading to increased circulating monocytes. panRNAseq of S1PR1−/− AM showed markedly reduced Sphk1 and NFκB1 expression and increased expression of KLF4, Myb, Myc, TLR7 & 8. These findings indicate that loss of S1PR1 misfires the generation of incompetent monocytes leading to dysfunctional AM and defective lung repair.
Innate Immune Memory in Lung Alveolar Macrophages Promotes Resilience and a Pro-Resolving Phenotype
Authors: Chakraborty Sreeparna , Singh Abhalaxmi , Maienschein-Cline Mark , Mukhopadhyay Amitabha , Rehman Jalees , Malik Asrar B.
Published: February 2023

Immune memory has been well-established in adaptive immunity but recent studies suggest a key for memory in innate immunity. However, the precise mechanisms and phenotypes of innate immune memory in macrophages remain poorly understood. Here, we studied the memory responses of lung alveolar macrophages (AMs) exposed consecutively (2 exposures, one week apart) intratracheally to the bacterial endotoxin lipopolysaccharide (LPS). We observed a 60%–80% loss of AMs following the initial LPS exposure, consistent with the known cell death of resident AMs during severe inflammatory injury followed by gradual replenishment. Importantly, AM loss following the second LPS injury was markedly attenuated. Lineage tracing using a CX3CR1-monocyte reporter showed no increase in monocyte-derived AMs, thus suggesting that the higher AM numbers after the second LPS exposure were due to greater resilience of AMs and not due to rapid replenishment by circulating monocytes. RNA-Seq analysis of showed higher expression of pro-survival genes and anti-inflammatory cytokines such as IL-10 in memory AMs (post 1 week LPS) than in naïve AMs. Ex vivo and in vivo functional assay showed that these memory AMs demonstrated enhanced efferocytosis of cellular debris, consistent with a pro-resolving phenotype. We also found upregulation of the transcription factor KLF4 which promotes cell survival as well as pro-resolving macrophage polarization. Our findings suggest that alveolar macrophages are reprogrammed by an initial inflammatory exposure to increase their resilience to pro-apoptotic stimuli and to promote resolution of inflammation. Such a memory mechanism could be leveraged therapeutically in severe inflammatory diseases.
Ethanol suppresses the pulmonary response to Streptococcus pneumoniae: Role of NOD2 and NF-kB signaling in alveolar macrophages exposed to muramyl dipeptide.
Authors: Hulsebus Holly J , Najarro Kevin M , McMahan Rachel H , Kovacs Elizabeth J
Published: February 2023

Alcohol use decreases host immune defense and increases susceptibility to pathogens such as Streptococcus pneumoniae. NOD2 is an intracellular pattern recognition receptor that binds muramyl dipeptide (MDP), a cell wall component of S. pneumoniae, and activates NF-kB signaling. To limit inflammation, NF-kB signaling is negatively regulated in part by TNFAIP3 (TNF Alpha Induced Protein 3), or A20. We hypothesize that ethanol impairs NOD2 signaling and A20 expression in alveolar macrophages, leading to excessive inflammation and reduced bacterial clearance after infection. For in vivo experiments, we gavaged mice once daily for 3 consecutive days with water or ethanol (blood alcohol concentration ~ 80 mg/dL at 30 min), and infected with 104 cfu S. pneumoniae. In parallel experiments, MH-S cells were treated with 25 mM ethanol and 10 μg/ml MDP. We found that ethanol treated animals, compared to vehicle treated, had impaired respiratory function (17% lowered respiration rate [p < 0.01] and 33% increased penh [p = 0.09]) and increased lung bacteria (log10 increase of 0.8 [p = 0.11]) at 24 hours. RNA sequencing of alveolar macrophages from vehicle- or ethanol-gavaged mice revealed that ethanol intake led to 25% less Nod2 and 60% less A20 (p < 0.05) in the absence of infection. These results were confirmed by qPCR in ethanol-treated MH-S cells (40% decrease in Nod2 and 50% reduction in A20 [p < 0.01]), along with 15% lower A20 protein levels, which was sustained after MDP stimulation (29% less A20 compared to vehicle-treated, stimulated cells). Future experiments aim to further investigate ethanol’s effect on A20 regulation of NF-kB signaling, and will provide insight into immune dysregulation seen in alcohol-consuming patients with pneumonia.
Thymic Stromal Lymphopoietin induces adipose loss through sebum hypersecretion
Authors: Choa Ruth , Tohyama Junichiro , Wada Shogo , Meng Hu , Okumura Mariko , May Rebecca , Robertson Tanner , Pai Ruth-Anne , Nace Arben , Hopkins Christian , Jacobsen Elizabeth , Haldar Malay , FitzGerald Garret , Behrens Edward , Minn Andy , Seale Patrick , Cotsarelis George , Kim Brian , Seykora John , Arany Zoltan , Kambayashi Taku
Published: February 2023

Obesity is a serious public health concern. Emerging studies indicate that the immune system can regulate weight and energy homeostasis. Here we show that the cytokine Thymic Stromal Lymphopoietin (TSLP) stimulates T cells to induce selective white adipose loss, which protects against obesity, improves glucose metabolism, and mitigates nonalcoholic steatohepatitis. Surprisingly, adipose loss was not caused by alterations in food intake, absorption, or energy expenditure. Rather, it was caused by the excessive loss of lipids through the skin as sebum, causing mice to develop a striking “greasy hair” phenotype. Inhibition of sebum secretion or depletion of T cells prevented TSLP-driven adipose loss. TSLP-driven white fat loss and sebum hypersecretion were both mediated via the direct activation of CD4+ or CD8+ T cells by TSLP in an antigen-independent manner, and these T cells could be seen infiltrating skin sebaceous glands. Furthermore, we also found that TSLP and T cells regulated sebum release and sebum-associated anti-microbial peptide expression in the skin at steady-state. In human skin, TSLP expression also correlated directly with sebum-associated gene expression. Together, our study establishes a paradigm in which adipose loss can be achieved by sebum hypersecretion and reveals a previously unknown role of adaptive immunity in skin barrier function through the regulation of sebum secretion.
Microbial metabolite Urolithin A induces expansion of Regulatory T Cells in aryl hydrocarbon receptor (AhR)-dependent manner
Authors: Ghosh Sweta , Haribabu Bodduluri , Jala Venkatakrishna R
Published: February 2023

Intestinal inflammation and loss of gut barrier integrity contribute to severity of inflammatory bowel diseases (IBD). Gut microbiota and their metabolites play an important role in maintenance of immunological balance within the gut. Previously, our group demonstrated that treatment with Urolithin A enhanced gut barrier function by inducing junctional proteins and blocked LPS-induced inflammation. Moreover, treatment with this microbial metabolite mitigated colitis in pre-clinical models. In the current study, we identified novel function for Urolithin A, where treatment with Urolithin A significantly expanded FoxP3+ T regulatory cells (Tregs) both ex vivo and in vivo models. Urolithin A failed to induce Treg expansion in AhR−/− mice suggesting Urolithin A mediates its activities through AhR. Moreover, treatment with Urolithin A mitigated murine model of dextran sulfate sodium (DSS)-colitis and enhanced Treg cell population compared to vehicle treatment. These results suggest that Urolithin A offers multi-pronged beneficial activities on gut epithelial layer (enhanced barrier function), macrophages (down regulation of inflammatory cytokines) and expansion of Treg cells to protect against colitis.
T-bet+ B cells promote adipose tissue inflammation and metabolic disorder during obesity.
Authors: Vanz Carlo , Hagglof Thomas , Dudley Elizabeth , Leadbetter Elizabeth
Published: February 2023

Obesity is a public health crisis affecting a large and ever-growing portion of the global population. Obesity is associated with an increased risk of death and comorbidities, resulting in lower quality of life for obese patients and increased economic costs on healthcare systems. Obesity is linked with a low-grade and chronic inflammation in the adipose tissue, which further exacerbates metabolic dysfunctions. Understanding the mechanisms and immune cell types promoting this inflammation would allow for the development of new therapies to restore immune regulation in obese patients. Our lab has identified a subset of B lymphocytes which is expanded in the adipose tissue of humans and mice during obesity. This subset is characterized by the co-expression of T-bet and CD11c, production of IgG2c, and its expansion is dependent on the presence of iNKT cells. In vivo studies with mice fed a high fat diet reveal that these Tbet+ B cells promote inflammation. Serum transfer experiments and flow cytometry analysis revealed that this effect is mediated through the production of CXCL10 and antibodies. The absence of T-bet+ B cells does not appear to affect adipose CD4+ T cells or NK cells, but it reduces macrophage polarization towards a more proinflammatory phenotype. Transgenic mice engineered to lack T-bet+ B cells not only have reduced adipose tissue inflammation, but present improved metabolic function as well, gaining less weight on a high-fat diet than littermate controls and presenting improved glucose tolerance. Based on these results, T-bet+ B cells represent an interesting potential target to reduce inflammation in obese patients and improve their metabolic functions.
Milk-derived osteopontin shapes the development of the intestinal microbiota
Authors: McClanahan Kathleen , Greer Michael , Olivares-Villagómez Danyvid
Published: February 2023

Breast-feeding is important for the growth and development of infants, delivering nutritional support as well as a wealth of immunological factors that provide protection while the infant immune system matures. Formula-fed infants have an increased risk of negative health outcomes, including obesity, autoimmunity, and infections, and display dysbiosis of the intestinal microbiota compared to breast-fed infants. Of the many components of milk, bioactive proteins provide a potential mechanism by which breast milk can impact the infant microbiota. One protein of interest is osteopontin (OPN), a highly glycosylated phosphoprotein involved in a range of physiological processes including bone mineralization, inflammation, wound healing, and homeostasis of lymphoid cells in the intestinal epithelium. Osteopontin constitutes about 2% of the protein component of milk; however, the impacts of milk-derived osteopontin on the development of the infant microbiota are not yet well understood. We propose that milk-derived osteopontin plays a critical role in the development of the neonatal gut microbiota, and that lack of osteopontin intake during the post-natal period leads to gut dysbiosis.
Peroxisome proliferator-activated receptor gamma activation reverses adipose tissue regulatory T cell dysfunction.
Authors: Smith Alan J , Shantaram Dharti , Liu Joey , Wright Valerie P. , Bradley David , Hsueh Willa A.
Published: February 2023

Adipose tissue (AT) regulatory T Cells (Tregs) play an essential role in regulating inflammatory and metabolic responses. Obesity is associated with a decrease in AT Tregs and a corresponding increase in tissue inflammation and systemic insulin resistance in both humans and mice. In human samples, we have found Tregs isolated from visceral AT (VAT) display a dysfunctional phenotype characterized by decreased viability, decreased effector function, and increased expression of inhibitory co-receptors (PD-1 and OX40). VAT Tregs cultured with peripheral blood mononuclear cells (PBMCs) display reduced ability to suppress proliferation, while peripheral blood Tregs suppress proliferation normally. As peroxisome proliferator-activated receptor (PPAR)-gamma has been shown to be a regulator of AT Tregs, we hypothesize that the Treg dysfunction can be reversed by addition of the PPAR-gamma agonist Pioglitazone. We isolated Tregs from VAT and peripheral blood and added cells to CFSE-labelled PBMCs in the presence and absence of Pioglitazone. After 4 days culture, Pioglitazone was found to significantly improve the ability of VAT Tregs to suppress proliferation. This effect was specific to AT Treg populations as Pioglitazone alone did not affect PBMC proliferation. These data suggest that Treg dysfunction contributes to AT inflammation and activation of PPAR-gamma promotes VAT-specific Treg activity providing therapeutic implications for PPAR-gamma agonists. (Partially supported by the OSU Cancer Center Support Grant P30CA016058).
The impact of type I interferon driven cytokines on the plasmablast response to viral pathogens
Authors: Steffen Tara L , Hassert Mariah , Brien James D
Published: February 2023

The plasmablast B cell (PB) response to viruses provides the first wave of functional virus specific antibodies. PBs may undergo immunoglobulin class switch recombination (CSR), giving these antibodies a diverse array of properties and functions that contribute to their mechanisms of viral clearance. Dendritic cells (DCs) can prime a PB response through native antigen production, cell surface interactions, and soluble mediators including cytokines. Additionally, we know that type I interferon (IFN) has dynamic regulatory functions by inducing expression of interferon stimulated genes (ISGs) in addition to having potent anti-proliferative and anti-viral effects. We hypothesize that signaling of type I IFN inhibits the PB response induced by virial infection by impacting the cytokine milieu, magnitude and diversity of the PB response, and mechanisms of antibody mediated protection. As DCs are potent producers of type I IFN and mediate direct interactions with B cells, we employed a DC specific knockout of Ifnar1 from the type I IFN receptor, CD11c Cre X Ifnarfl/fl, to challenge our hypothesis. Overall, we show that ablation of type I IFN signaling followed by infection with Zika virus (ZIKV) results in an increase in PB frequency in lymphoid tissue, increase in CSR, and potent increase in the neutralization capacity of polyclonal sera. The results of these studies can be leveraged to increase immunogenicity of mRNA and attenuated vaccines while targeting for a quicker antibody response.
Characterizing infection of B lymphocytes with vaccine and wild-type strains of measles virus
Authors: Melot Logan , Coughlin Melissa , Rota Paul A , Bankamp Bettina
Published: February 2023

Wild-type (w.t.) measles virus (MeV) infection can result in immunosuppression that does not occur following vaccination. A rhesus macaque model indicated that B cells are a target of infection. The role of B cell infection in MeV-induced immune suppression is not fully understood. We characterize the differences in viral replication in human B cells infected with vaccine (Edmonston Zagreb [EZ]) or w.t. virus.

B cells from healthy donor blood were infected (MOI 1.0) with a GFP-expressing EZ virus or a w.t. virus to evaluate viral replication after 24 and 48 hours. The number of GFP+ cells (EZ infected) or hemagglutinin (H)+ cells (w.t. infected) was measured via flow cytometry. Transcription of the viral nucleoprotein (N) gene was measured by mRNA-specific cDNA generation and N gene specific qPCR. Production of infectious viral particles was measured via endpoint dilution.

In B cells infected with EZ, 30% of cells expressed GFP at 24 hours and 45% of cells expressed GFP at 48 hours. There was a 4.4-fold increase in N gene transcription from 0 to 48 hours. There was no detectable increase in viral titer over input after 48 hours. In B cells infected with w.t. virus, 10% of cells expressed viral H at 24 hours with no increase in H expression at 48 hours. The w.t. virus had a 2.4-fold increase in viral transcription from 0 to 48 hours. There was no detectable increase in viral titer over input after 48 hours.

MeV-infected B cells show viral protein production and N gene transcription at 24 and 48 hours. However, w.t. virus replication may be less efficient as shown by lower percentages of H+ cells at 24 and 48 hours and lower levels of N gene transcription at 48 hours. There is no evidence of infectious virus production by either strain of MeV despite evidence of replication.
Type I Interferon Shapes Humoral Immunity to SARS-CoV-2
Authors: Goldman-Israelow Benjamin , Mao Tianyang , Song Eric , Lu Peiwen , Wong Patrick , Lucas Carolina , Klein Jon , Iwasaki Akiko
Published: February 2023

The role of type I interferon (IFN-I) in SARS-CoV-2 infection has remained controversial. Elevated IFN-I levels are associated with increased COVID-19 morbidity and mortality; however, impaired IFN-I signaling, either genetic or via autoantibodies, is also associated with worse outcomes. While these studies have focused on the role of IFN-I in COVID-19 pathogenesis, its role in modulating adaptive immunity to SARS-CoV-2 remains unknown. Here we investigate the role of IFN-I in shaping the humoral immune response to SARS-CoV-2 infection in both COVID-19 patients and mouse models of SARS-CoV-2 infection. We find that patients with COVID-19 who have higher levels of plasma IFN-I develop lower anti-spike (S1) and anti-receptor binding domain (RBD) IgG levels. Similarly, we find that mice lacking the interferon-α/β receptor (IFNAR) or treated with IFNAR blocking antibodies develop higher levels of anti-SARS-CoV-2 S1 and RBD IgG, as well as higher levels of anti-S1 IgA, IgG1, and IgG2. Furthermore, we find that IFNAR deficiency promotes the development of follicular helper T cells, germinal center B cells, and antibody-secreting cells in the draining lymph node. Accordingly, patients with higher serum IFN-I levels have reduced circulating follicular helper T cells. Despite higher levels of anti-SARS-CoV-2 IgG, sera from IFN-I sufficient or deficient mice harbored comparable neutralizing activities, suggesting reduced antibody potency (IC50/IgG) in IFNAR-deficient mice. Similarly, patients with higher serum IFN-I levels produced higher-potency anti-SARS-CoV-2 antibodies. These findings highlight a beneficial role for IFN-I in modulating cellular responses critical for promoting a potent antibody response to SARS-CoV-2.
Live attenuated viral vectors based on the rubella vaccine strain RA27/3
Authors: Berkower Ira , Zhou Wenshuo , Perry-Anderson Jasper , Felber Barbara , Virnik Konstantin
Published: February 2023

We have developed a series of live viral vectors based on the live attenuated rubella vaccine strain RA27/3. These vectors can express a variety of heterologous vaccine antigens without interfering with vector growth or stable antigen expression. The vaccine inserts were expressed for at least 5 to 10 passages in Vero cells. Proteins as large as the ectodomain of HIV gp140 could be expressed (90 kDa), and they retained the native conformation. Recently expressed vaccine inserts include: P27 Gag protein of SIV, the NANP repeat sequence of malaria circumsporozoite protein, and the receptor binding domain of Sars CoV-2 S1 spike protein.

We have studied these vectors for immunogenicity in a rhesus macaque model. The rubella vectors are based on the rubella vaccine strain, for which one or two doses can elicit lifelong immunity. The vectors are a little different: The best immune response was observed when the macaques were primed with a DNA vaccine, followed by rubella vectors, or when they were primed with rubella vector and then boosted with the native protein. In both cases, an immunization strategy with only one exposure to rubella antigens was better than giving it twice.

This could occur if the secondary response to rubella antigens inhibited the response to the vaccine insert. Alternatively, the strong anamnestic response to a boost of protein antigens suggests that the level of protein expression by the vector was the limiting factor. In future studies, we will test whether vectors with better growth characteristics and stronger antigen expression levels can improve antibody titers, elicit neutralizing antibodies, and protect against a viral challenge.
A CD8 T cell signature in ALS4.
Authors: Campisi Laura , Gromova Anastasia , Arnold Frederick , Mei Xueyan , Beharry Cindy , Fstkchyan Yesai , La Spada Albert , Marazzi Ivan
Published: February 2023

Motor neuron disorders (MND) cause disability and death globally. Despite an increasing knowledge of the genetic defects of MND, a clear understanding of the mechanisms of disease initiation and progression is lacking. Recent advance in the field has highlighted that several genes linked to MND are essential for maintaining immune homeostasis, suggesting that dysfunctions in the immune system could influence the development of the disease or aggravate its pathology.

Amyotrophic Lateral Sclerosis 4 (ALS4) is a rare, juvenile form of MND. It is caused by mutations in the gene SENATAXIN (SETX), which encodes for a nuclear helicase ubiquitously expressed. Our group identified SETX as a transcriptional regulator of inflammatory responses.

Since SETX is involved in both MND and the control of the immune response, we hypothesized that underlying defects in the immune system could be associated to ALS4 progression.

We took advantage of the Setx-L389S+/− knock-in (KI) mouse carrying the most common causative variant of ALS4 in humans. Surprisingly, we found that SetxL389S needs to be expressed in both neuronal and immune cells to trigger disease. In parallel, we discovered a pathologic signature consisting of high frequencies of activated CD8 T cells in the central nervous system (CNS) and blood of KI animals. Further analyses showed clonal expansion of aberrant activated CD8 T cells. Dysfunctions in the CD8 T cell compartment have been confirmed in the peripheral blood of ALS4 patients, as compared to age-matched controls.

Collectively, our results suggest that ALS4 is characterized by aberrant CD8 T cell responses of possible autoimmune origin. Our findings have the potential to pave the way for immune-based diagnosis and therapy for patients.
Artemisinin selectively induces ferroptosis and reverses the immunosuppressive state of MDSCs in T cell lymphoma
Authors: Yan De Hong , Zhang Mengqi , Wan Xiaochun
Published: February 2023

The malaria drug artemisinin has previously been identified as a potent anti-lymphoma drug to treat B-cell lymphoma and myeloid leukemia, but treatment of T lymphoma is rarely reported. In this study, we found that artemisinin can not only cause ferroptosis by up-regulating ROS levels and increasing misfolded protein in T cell lymphoma via down-regulating ERK pathway, but also reduce the accumulation of MDSCs, relieve the immunosuppressive effect of MDSCs on effector T cells, induce anti-tumor immune reactivation, and inhibit tumor growth in tumor bearing mice.

This work was supported by the National Key R&D Program of China (2019YFA0906100), National Natural Science Foundation of China (Grants 82071772, 81501356 and 81373112), Key-Area Research and Development Program of Guangdong Province (2019B020201014), the Shenzhen Basic Science Research Project(Grants JCYJ20190807161419228, JCYJ20170818155135838, JCYJ20170818164619194, and JCYJ201 70413153158716), China Postdoctoral Science Foundation (2019M660220), Basic and Applied Basic Research Foundation of Guangdong Province (2019A1515110359), Nanshan pilot team project (LHTD20160004), Start-up funding (CYZZ20180307154657923).