bluuebunny/crossref_metadata_2025 huggingface dataset:
Ureaplasma parvum induces preterm birth by triggering immune responses in the fetus and at the maternal-fetal interface
Authors: Motomura Kenichiro , Romero Roberto , Theis Kevin , Winters Andrew , Xu Yi , Garcia-Flores Valeria , Zou Chengrui , Para Robert , Ahmad Madison , Slutsky Rebecca , Gomez-Lopez Nardhy
Published: January 2023

Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. One of every four preterm births is due to intra-amniotic infection, most commonly associated with Ureaplasma species. However, a causal link between Ureaplasma species and adverse pregnancy outcomes, as well as the triggered host immune responses, has not been investigated. Molecular microbiological surveys first revealed that Ureaplasma parvum is the most common bacterium in women with intra-amniotic infection. Ultrasound-guided intra-amniotic injection of U. parvum in pregnant mice resulted in preterm birth and neonatal death. Specific qPCR revealed that U. parvum invaded specific fetal (lung, intestine, spleen, fetal membranes, and placenta) and maternal (decidua, uterus, and cervix) compartments. Consistently, U. parvum induced a strong inflammatory response in the amniotic cavity, fetal membranes, placenta, and fetal lung and spleen, as well as at the maternal-fetal interface. However, U. parvum neither invaded the non-reproductive maternal organs nor induced a maternal systemic inflammatory response. An ex vivo model of intra-amniotic infection showed that U. parvum elicits pro-inflammatory responses in human amnion epithelial cells. Lastly, the treatment of U. parvum-injected mice with clarithromycin, a clinically relevant macrolide, prevented preterm birth and neonatal death. Collectively, this investigation provides a causal link between U. parvum and adverse pregnancy outcomes, and yields insights into the host immune responses triggered by this genital mycoplasma. Importantly, these data support the use of clarithromycin in the treatment of Ureaplasma-associated intra-amniotic infection leading to preterm birth.
Immune checkpoint dysregulation and immune activation in HIV-exposed uninfected children
Authors: Li Wei , McHenry Megan , Oyungu Eren , Yang Jing , Xia Ying , Syed Fahim , Yu Qigui
Published: January 2023

Children who are HIV-exposed uninfected (CHEU) are a growing population worldwide. CHEU suffer greater morbidity and mortality from infectious diseases, likely due to their impaired cell-mediated immunity (CMI) and hyper-inflammatory responses. As CMI and inflammatory responses are regulated by immune checkpoints (ICPs) that play a key role in maintaining immune homeostasis, we determined whether CHEU had altered expression of the ICP axes. We used multiplex immunoassays to quantify plasma levels of 16 soluble ICPs (sBTLA, sCD27, sCD28, sCD40, sCD80, sCTLA-4, sGITR, sGITRL, sHVEM, sICOS, sLAG-3, sLIGHT, sPD-1, sPD-L1, sTIM-3, and sTLR-2) from 46 CHEU and 37 children who are HIV-unexposed uninfected (CHUU) from AMPATH in western Kenya. We demonstrated that all ICPS except GITR were detected in all samples. Compared to CHUU, CHEU had significantly higher levels of 2 sICPs: soluble CD40 (sCD40) and soluble TIM-3 (sTIM-3). As CD40 plays an essential role in antigen-presenting cell functions, while TIM-3 is a key member of immunosuppressive ICPs that inactivate T cell activity, the dysregulated sCD40 and sTIM-3 could play a role in the impaired CMI in CHEU and can be explored as biomarkers for adverse health outcomes in CHEU. A previous study showed that the percentage of activated CD8 T cells (CD8+CD38high) was increased in Canadian CHEU. Therefore, we also studied the activation of T cells in PBMCs from Kenya and found that CHEU had higher percentages of activated CD4 (CD4+CD38+HLA-DR+) and CD8 (CD8+CD38+HLA-DR+) T cells in the peripheral blood compared to CHUU. Our study demonstrated that the ICP axes were dysregulated in CHEU, and we provided additional support to the notion that the immune system in CHEU is altered.
Expression of Pattern Recognition Receptor mRNA in Pediatric Patients with Otitis Media with Effusion
Authors: Yeo Seung Geun , Park Dong Choon
Published: January 2023

Importance

Otitis media with effusion (OME) in children often requires surgical treatment. Pattern recognition receptors (PRRs), which are involved in innate immunity in the middle ear, are believed to play an important role in relapse and chronicity of OME.

Objective

To investigated the expression of PPRs in children with OME.

Participants

Total 126 patients under 10 years of age who were diagnosed with OME and underwent unilateral or bilateral ventilation tube insertion (VTI).

Main outcomes and measures

Expression levels were compared in children with and without bacteria in the middle ear cavity, with and without concomitant diseases, by characteristics of middle ear fluid, and in children who were and were not otitis prone.

Results

Mean [SD] expression level of PRR mRNAs did not differ significantly in children with and without bacteria in the middle ear, with and without concomitant diseases, or by characteristics of middle ear fluid. However, mean [SD] expression level of TLR2 (0.037 [0.079] vs 0.093 [0.124]; MD, 0.056; 95% CI, 0.018 to 0.094), TLR4 (0.015 [0.054] vs 0.036 [0.047]; MD, 0.021; 95% CI, 0.003 to 0.039), TLR6 (0.038 [0.072] vs 0.107 [0.170]; MD, 0.069; 95% CI, 0.020 to 0.118), TLR9 (0.229 [0.256] vs 0.611 [0.840]; MD, 0.382; 95% CI, 0.146 to 0.618), NOD-1 (0.004 [0.002] vs 0.008 [0.014]; MD, 0.004; 95% CI, 0.001 to 0.008), and RIG-I (0.034 [0.086] vs 0.092 [0.191]; MD, 0.058; 95% CI, 0.002 to 0.114) mRNAs were lower in the otitis prone group than in the non-otitis prone group.

Conclusions and relevance

These findings suggest that PRRs are involved in the pathophysiology of OME. OME is frequent and chronic in children with low levels of TLR, NLR, and RIG-I mRNAs.
Direct effects of ethanol at relevant concentrations on conformation and ligand binding of Toll-like Receptor 3 (TLR3)
Authors: Pruett Stephen B , Lewis Edwin , Gwaltney Steven , Nanduri Bindu
Published: January 2023

The purpose of this study was to seek direct physical evidence that ethanol at concentrations relevant to binge drinking alters the conformation of TLR3 and decreases ligand binding. We and others have reported that TLR3-mediated responses are significantly suppressed by concentrations of ethanol that occur in binge drinkers (J. Immunol., 2004, 173: 2715–2724). This suppression can be detected at all levels of signaling that have been tested (from MAP kinases to NF-kB), but it remains possible that ethanol acts directly on TLR3 and that its other effects are secondary to altered TLR3 conformation. Purified ectodomains of TLR3 were used in this study, because they comprise most of the TLR3 molecule, and dimerization of TLR3 ectodomains is necessary for signal transduction and activation of TLR3-mediated innate immunity. Using circular dichroism (CD) to measure changes in protein conformation, we demonstrated that ethanol concentrations from 20–100 mM substantially altered the conformation of human and mouse TLR3 ectodomains complexed with a defined dsRNA ligand. Binding of poly I:C, a dsRNA analog, was significantly inhibited by ethanol. Modeling with Autodock 4 software indicated that mouse and human TLR3 contain a putative ethanol binding site with a dissociation constant consistent with the concentration dependence of the conformational changes. These results support the hypothesis that ethanol inhibits TLR3-mediated functions by directly altering the conformation of TLR3. This work was supported by NIH R01AA09505 and SBP was supported by NIH grant P20GM103646.
Targeting the TLR7 signalosome assembly by TLR7-derived decoy peptides
Authors: Toshchakov Vladimir Y , Javmen Artur
Published: January 2023

Toll-like receptors (TLRs) sense microbial molecules and initiate antimicrobial immune response. The members of the TLR family use cytoplasmic Toll/Interleukin-1R homology (TIR) domain to initiate intracellular signaling. Activated TLRs dimerize their TIRs and recruit adapter proteins to the dimer, through multiple interactions of receptor and adapter TIR domains. Although TLRs play an essential role in innate immunity, the aberrant TLR signaling may cause pathogenic inflammation. This study has screened a library of cell-permeable decoy peptides (CPDPs) derived from the TLR7 TIR for interference with TLR7 signaling and identified several CPDPs that inhibit TLR7 signaling. Peptides 7R1, 7R6, 7R9 and 7R11 inhibited the TLR7-induced signaling in murine and human macrophages. The most potent inhibitory peptide of the four, 7R11, significantly reduced the systemic cytokine levels elicited by administration of a TLR7 agonist, R-848, to mice. TLR7 TIR surface regions that correspond to inhibitory peptides generally corresponded to four TIR sites that mediate signalosome assembly for other TLRs. The cell-based Förster resonance energy transfer/fluorescence lifetime imaging confirmed that 7R9 and 7R11 interact with adapter TIRs. These findings clarify the molecular mechanisms that trigger the adapter recruitment to activated TLR7 and suggest that 7R9 and 7R11 have a significant translational potential as candidate or lead therapeutics for treatment of TLR7-mediated inflammation.
TLR3 co-localizes with LAMP1-labeled endosomes from the first minute following poly(I:C) stimulation of murine astrocytes
Authors: Mielcarska Matylda Barbara , Gregorczyk-Zboroch Karolina Paulina , Bossowska-Nowicka Magdalena , Szulc-Dąbrowska Lidia , Toka Felix Ngosa
Published: January 2023

Toll-like receptor 3 (TLR3) senses dsRNA in the acidic environment of endosomes and plays a pivotal role during infection of the brain with viruses, including herpes simplex virus type 1 (HSV-1). Studies on various cell lines corroborate TLR3 presence in LAMP1-labeled endosomes following the receptor stimulation. However, co-localization times, as well as the subjection of co-localization on the TLR3 stimulation, remain divergent depending on the cell type. In this work, we examined TLR3 residence in the LAMP1-marked endosomes in nine different time intervals following poly(I:C) stimulation of C8-D1A murine astrocytes. Interestingly, TLR3 did not localize in the lysosomal compartment in resting cells, however, the presence of the receptor was observed in LAMP1-labeled endosomes from the first minute after the addition of poly(I:C). Furthermore, 24 h following stimulation, we observed numerous lysosomes co-localizing with TLR3, which were present in all astrocytes. Our results indicate LAMP1-labeled endosomes as important organelles which may serve as the TLR3 ligand recognition sites, however, further studies on the migration of dsRNA in astrocytes are essential.
Novel Anti-bacterial and Anti-inflammatory Roles for the Macrophage Galactose-type Lectin in Pulmonary Immunity to Mycobacterium tuberculosis
Authors: Naqvi Kubra Fatima , Huante Matthew B , Endsley Mark , Gelman Benjamin , Endsley Janice J
Published: January 2023

Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is characterized by inflammatory pathology that is regulated by poorly understood mechanisms of innate immunity. Pattern recognition receptors (PRR) expressed on the surface of macrophages regulate the balance of inflammatory and antimicrobial functions that determine disease outcome. Our work identifies a novel role for the C-type lectin receptor Macrophage Galactose-type Lectin (MGL) PRR in a murine model of experimental tuberculosis. Bone marrow derived macrophages (BMDM) upregulate MGL-1, but not MGL-2, following in vitro exposure to both heat killed and live Mtb. Macrophages from MGL-1-deficient mice display greater lipid accumulation, and increased production of pro-inflammatory cytokines (IL-1b, IL-6 and IFN-g). Mtb growth is also increased in MGL−/− BMDM, independent of the inflammatory outcomes and production of reactive nitrogen species. TB in mice lacking MGL-1 results in increased pro-inflammatory cytokines, more diffuse granulomatous inflammation, and increased accumulation of foamy macrophages. Assessment of disease kinetics suggests that MGL has an important role for controlling mycobacterial burden during the establishment stages of TB disease, and has less influence once the chronic disease stage is established. Macrophages in murine and human TB granulomas contain abundant MGL associated with inflammatory foci. These results suggest that MGL is a potential new therapeutic target against TB.
Can Effector Molecules of Innate Immunity Target Microbial Motility?
Authors: Akahoshi Doug T , Natwick Dean E , Collins Sean R , Bevins Charles L
Published: January 2023

Antimicrobial peptides are key effector molecules throughout nature responsible in part for the defense against microbial pathogens and influencing the composition of colonizing microbes. Defensins are the major antimicrobial peptides produced by Paneth cells in the mammalian small intestine and are essential for maintaining homeostasis between host and microbe. While mature defensin peptides typically have microbicidal activity, human alpha defensin 6 (HD6) lacks such activity yet provides protection from invasion by bacterial pathogens. Using a transgenic mouse model expressing HD6 in Paneth cells, we reported the importance of HD6 in preventing the translocation of pathogenic Salmonella Typhimurium (STM) across the small intestinal epithelium (doi:10.1126/science.1218831); however, the mechanism underlying the inhibition of invasion is not fully understood. We hypothesize HD6 binds to extracellular bacterial protein structures, such as flagella, and inhibits swimming motility through a process of direct binding and self-assembly. Using an agar based motility assay, we show STM radially swim at a slower rate when in the presence of HD6. In ongoing studies using live-microscopy to visualize GFP-expressing STM in the presence of HD6, we observe a decrease in STM motility in a concentration dependent manner with some STM appearing to be completely immobilized, but without changes in bacterial viability. Unexpectedly, it appears the immobilization observed is influenced more by the expression of fimbriae than by the expression of flagella. With these results in mind, we plan to expand our investigation to elucidate key molecular determinants and include other microbes to test the broader relevance of this activity of HD6.
A LIVE ATTENUATED Brucella VACCINE POTENTIATES MACROPHAGE ALARMINS
Authors: Ella Bhagyaraj , Akgul Ali , Hoffman Carol , Goodwin Zakia I , Yang Xinghong , Pascual David W
Published: January 2023

Wild-type (wt) brucellae subvert host immune detection leading to their replication in the endoplasmic reticulum (ER) resulting in their persistence. Protection against Brucella infection requires a strong Th1-type immunity, characterized by the production of IFN-γ. Mucosal vaccination with a live attenuated B. abortus double mutant expressing lacZ (znBAZ) provides protection against wt B. abortus challenge via the stimulation of activated T cells. Such finding implicates rendering macrophage activation. To test the hypothesis that znBAZ diminishes subversion to enable host innate immune detection, intracellular survival of wt B. abortus, B. abortus strain 19 (S19) vaccine, and znBAZ in bone marrow-derived macrophages (BMDMs) was measured. The percentage of brucellae in LAMP1+ phagolysosomes was significantly greater in znBAZ-infected BMDMs than S19 or wt B. abortus, and znBAZ induced the alarmins, s100a8, Saa1, and Saa3. These alarmins are responsible for stimulating strong inflammatory responses by interacting with pattern recognition receptors (PRRs). Consequently, IL-1β, IL-6, IL-12, and TNFα were significantly elevated following in vitro znBAZ infection of macrophages. CD80, CD86, CD40, and MHCII expression by znBAZ-infected BMDMs was significantly increased. Analysis of lung macrophages two days after pulmonary infection reveals that znBAZ significantly increases the expression of CD86 and MHCII on interstitial and alveolar macrophages, and increased intracellular IL-1β and IL-6 by interstitial macrophages. These findings show that znBAZ enhances macrophage activation, which in turn leads to the induction of potent T cell immunity. Work is supported by NIH AI123244 & AI125516.
LncRNAs modulate immune response in myeloid cells challenged with periodontal pathogens
Authors: Naqvi Afsar , Ahmad Imran
Published: January 2023

Objective

Long noncoding RNAs (lncRNA) are an emerging class of regulatory RNAs. LncRNAs have been implicated in differentiation and regulation of various immune cells. However, the role of lncRNA in myeloid cell regulation, in the context of periodontal inflammation remains poorly studied. Here we examined lncRNA-mediated regulation of myeloid cells upon challenge with periopathic bacteria.

Method

CD14+ monocytes were purified from PBMCs and were differentiated into macrophage (Mϕ) or dendritic cells (DC). Monocytes were differentiated to M1, M2 Mϕ, or DC in presence of GM-CSF, M-CSF or GM-CSF+IL-4 respectively for 7 days. Mϕ and DC were challenged A. actinomycetemcomitans (Aa), P. gingivalis (Pg) or treated with LPS from Aa and Pg. Total RNA isolated from periopathogen treated cells was used for qPCR. LncRNA knockdown was achieved by siRNA transfection using Lipofectamine 2000 and cell surface markers, cytokine analysis, antigen uptake/processing and phagocytosis assays were performed.

Result

DC challenged with Pg LPS exhibit differential expression of various lncRNAs (19 were up, 2 were down). Expression of selected lncRNAs in periopathogen challenged Mϕ/DC show that lncRNA are sensitive to periopathogen challenge. Cells transfected with lncRNA targeting siRNA showed skewing of M1 or M2 Mϕ polarization markers and altered secretion of pro- or anti-inflammatory cytokines upon periopathogen challenge. Finally, lncRNA knockdown resulted in functional impairment of Mϕ and DC function as reflected by decreased antigen presentation and phagocytosis.

Conclusion

We have functionally characterized lncRNAs in myeloid cells challenged with periopathogens and demonstrated their regulatory role in shaping immune response.
Hypusination regulates the antibacterial response of macrophages
Authors: Gobert Alain P , Latour Yvonne L , Finley Jordan K , Asim Mohammad , barry Daniel P , Allaman Margaret M , Lindsey-Rose Kristie K , Piazuelo Maria B , Sierra Johanna C , Wilson Keith T
Published: January 2023

Introduction

Hypusine is an amino acid found only in the eukaryotic translation initiation factor 5A (EIF5A). Hypusine is synthesized by the enzyme deoxyhypusine synthase (DHPS) that transfers the N-moiety of spermidine to a lysine residue in EIF5A. This post-translational modification (hypusination) is essential to binding specific mRNAs. The EIF5AHyp/mRNA complex is translocated to the cytoplasm, reaches ribosomes, and allows for translation. We analyzed the role of hypusination in the innate response of macrophages.

Results

The gastric pathogen Helicobacter pylori stimulated DHPS expression and hypusination in murine bone marrow-derived macrophages (BMmacs). We generated myeloid-specific Dhps knockout mice (DhpsΔmye) by crossing Dhpsfl/fl mice to Lyz2-cre mice. By iTRAQ proteomics, 29 of the proteins induced by H. pylori in BMmacs from Dhpsfl/fl mice were significantly less expressed in infected DhpsΔmye mice. These included antibacterial effectors (IRG1, NOS2), cytokines (TNF-α, IL-1β), and mediators of autophagy (SQSTM, IRGM1). These findings were confirmed by Western blot. DhpsΔmye mice infected with H. pylori exhibited increased colonization (p<0.01) and gastritis (p<0.05) compared to Dhpsfl/fl mice. DhpsΔmye mice were also more colonized by the intestinal pathogen Citrobacter rodentium than Dhpsfl/fl mice (p<0.05). EIF5AHyp and NOS2 were more expressed in gastric and colonic CD68+ macrophages from H. pylori- and C. rodentium-infected Dhpsfl/fl mice versus uninfected animals, respectively. The staining for EIF5AHyp and NOS2 was markedly reduced in infected DhpsΔmye mice.

Conclusions

Hypusination in myeloid cells is critical for host innate immunity to limit the infectivity of pathogenic bacteria.
Inflammasome signalling is dynamically modulated during the course of a chronic infection by Salmonella Typhimurium
Authors: Cai David , Sad Subash
Published: January 2023

Salmonella is a bacterial pathogen that typically causes acute infections, however, typhoid strains may chronically persist in an asymptomatic state after the acute infection has been resolved. Inflammatory cell death plays a key role in controlling bacterial infections and inflammasomes are a key innate signalling complex that respond to Salmonella infection. Inflammasomes may be triggered by the recognition of pathogen associated molecular patterns such as virulence factors, including the flagella and type III secretion systems of Salmonella. Inflammasome activation results in caspase-1 mediated inflammation and inflammatory cell death. As a consequence, pathogens may regulate their expression of virulence factors to avoid immune recognition and encourage chronicity.

In this study, we sought to assess whether Salmonella-induced inflammasome signalling is modulated throughout the stages of infection to promote its chronic persistence. Through the use of in vivo models of chronic infection, we show that Salmonella virulence is dynamically modulated over the course of a chronic infection to circumvent inflammasome activation. Bacteria isolated during acute phases of infection exhibited an increased ability to induce cell death and inflammation whereas bacteria isolated at later time intervals displayed a progressive decrease in their ability to induce inflammasome signalling. The inflammation and inflammatory cell death observed was primarily dependent on the activation of the NLRP3 and NLRC4 inflammasomes. Together, these results demonstrate that Salmonella dynamically modulates the expression of its virulence factors over the course of an infection to evade immune recognition and promote chronic carriage.
Less may be more for Brucella bacteriophage therapy due to immune suppressive effects
Authors: Hedges Jodi F , Robison Amanda , Snyder Deann Teresa , Plewa Jack , Jutila Mark A
Published: January 2023

We are studying the applicability of Brucella-specific phages to counter brucellosis. With antibiotic resistance on the rise and new realizations concerning the detrimental effects of long-term antibiotic use, phage therapy may become a useful approach against bacterial infection. However, phages are frequently less effective at countering bacterial infection in vivo than is expected considering their strong anti-bacterial effects in vitro. Furthermore, the effects of phage on mammalian cells are largely unknown. We have discovered that Brucella-specific phages are capable of activating some immune components, while suppressing others. Specifically, Brucella phages can activate bovine γδ T cells directly in vitro as measured by upregulation of IL-2R, but dampen the expression of TNF-α induced by E. coli LPS. A similar effect can be measured on human THP-1 macrophages. Also, in an in vitro model of intracellular Brucella infection, low phage MOI was capable of protecting cells from infection, but, unexpectedly, use of high phage MOI diminished protection. In vivo delivery of low phage MOI to the lung was more effective at diminishing Brucella burden than higher doses of phage. Finally, combining low dose phage with innate immune stimulation was more effective at reducing Brucella bacterial burden than either treatment alone. We hypothesize that some phage preparations induce an immune suppressive response that may counteract their anti-bacterial effects. Potentially combinatorial approaches may overcome this suppressive response. Our discovery of immune suppressive effects of Brucella bacteriophages is an important consideration for using phage as a treatment. This work is supported by NIH R21 AI144496-01.
LAG-3 expression on Foxp3+ regulatory T cells is critical for controlling homeostasis and autoimmune neuroinflammation
Authors: Lee Juyeun , Min Booki
Published: January 2023

Lymphocyte activation gene-3 (LAG-3) is increasingly recognized as an important immune checkpoint molecule that controls T cell activation. However, the role of LAG-3 on Foxp3+ regulatory T cell (Treg) function remains largely unknown. To test if LAG-3 expression is critical for Treg function in vivo, we generated a novel Treg-specific LAG-3 knock out mouse model (TregΔLag3). In steady state conditions, the lack of LAG-3 in Tregs significantly elevated activated memory-like phenotypes (CD62LlowCD44hi) and higher cytokine production of peripheral T cells. Following induction of experimental autoimmune encephalomyelitis (EAE), a murine model of autoimmune inflammation in the central nervous system, TregΔLag3 mice developed severe EAE compared to that of littermate controls. Flow cytometric analyses showed that TregΔLag3 mice had increased Foxp3+ Tregs in the CNS but that these Tregs expressed lower level of CD44, CD25, ICOS, and GITR, and higher inflammatory cytokines, suggesting their functions may be defective. Unlike WT Tregs, LAG-3-deficient Tregs adoptively transferred into Treg-depleted recipients were unable to rescue the mice from lethal disease, further corroborating the findings. Importantly, Tregs expressing mutant LAG-3 deficient in the cytoplasmic domain of the LAG-3 also failed to control EAE development in vivo, suggesting the key role of LAG-3 intracellular signaling on Treg function. Taken together, our study uncovers a novel Treg-intrinsic role of LAG-3 in regulating homeostasis and inflammation.
The liver controls regulatory T cell populations by enclysis, a CD4+ T cell engulfment process
Authors: Stamataki Zania , Davies Scott , Reynolds Gary , Li Xiaoyan , Grove Joe , Barnes Nicholas , Adams David , Huang Yuehua , Qureshi Omar
Published: January 2023

Background and Aims

Regulatory T cells (Treg) dampen inflammation and they are increased in the liver compared to blood, contributing to a tolerising environment. The swift neutralisation of Treg cells is important to enable immune effector functions to combat infection, but the mode of this is not understood. We noted that hepatocytes preferentially engulfed live Treg cells, and we set out to characterise the mechanism and outcome of this new phenomenon we termed “enclysis”.

Method

We measured enclysis, the enclosure and lysis of Treg cells, in vitro and ex vivo using perfused liver wedges, in the presence of inhibitors of known cell-in-cell processes. Reporter dyes for viability, mitochondrial activity and acidification were used to determine enclysis outcome. Cell-in-cell structures were documented by light, confocal, electron and multiphoton microscopy. FoxP3+ Treg and T-bet+ T helper cells were enumerated in donor and in end-stage disease livers of autoimmune and viral diseases.

Results

Hepatocytes preferentially engulfed live Treg cells in a process that differed in mechanism and kinetic to previously described cell-in-cell structures (efferocytosis, suicidal emperipolesis, entosis). Enclysis reflects the enclosure and specific lysis of live Treg cells inside hepatocytes, and beta-catenin-positive enclytic vesicles were connected to the endocytic pathway. Enclysis was increased in patients with autoimmune compared to viral hepatitis (p=0.0011), offering an explanation for the breach of tolerance in the liver.

Conclusion

Enclysis is the preferential engulfment and deletion of live Treg cells by hepatocytes and can be modulated pharmacologically, offering a new therapeutic target for liver immune regulation.
Interferons in Treg development and function
Authors: Sjaastad Lucy E , Owen David L , LaRue Rebecca S , Farrar Michael A
Published: January 2023

Thymus-derived regulatory T-cells (Tregs) are critical for controlling immune responses and preventing autoimmunity. In the setting of viral infection, Tregs are important modulators of anti-viral immune responses and help resolve inflammation. The subset of Tregs responsible for responding to viral infections and the types of antigens they recognize are not well understood. Using single-cell RNA sequencing we identified a unique subset of thymic Treg progenitors and mature Tregs that express a strong interferon stimulated gene (ISG) signature, as well as a comparable population in the spleen. Importantly, the ISG signature is much stronger than that expressed by thymocytes receiving tonic IFN stimulation in the final stages of maturation prior to thymic egress. Using flow cytometry we established that APCs in the thymus, including mTECs, as well as SIRPα+ and CD8α+ dendritic cells, require IFNα/β or IFNλ, acting via STAT1 signaling, for the expression of the canonical ISG BST2. TCR specificity was important for selection into this subset as SM1 TCR transgenic mice, which express a TCR that recognizes a non-ISG encoded antigen, have a reduced compartment of ISG-expressing CD4SP T-cells in the thymus. We are currently generating mice that specifically lack the ISG-signature Treg subset to test their function in vivo. Based on our current findings, we propose that thymic ISG antigens serve as TCR ligands for inducing the differentiation of ISG-signature Tregs that are in close physical proximity to the ISG-producing APCs. Furthermore, we hypothesize that ISG-signature Tregs are specialized for responding to ISGs induced during antiviral immune responses and play a key role in controlling IFN-driven inflammation.
Transcriptomic and Epigenomic Regulation of Leukocytes by the Polyamine Pathway
Authors: Wang Chao , Wagner Allon , Yosef Nir , Kuchroo Vijay
Published: January 2023

Cellular metabolism can orchestrate immune cell function. We previously demonstrated that lipid biosynthesis represents one such gatekeeper to Th17 cell functional state, promoting autoimmune responses in the experimental autoimmune encephalomyelitis model. Utilizing a transcriptome-based insilico fluxomics tool, we investigated ancillary metabolic pathways associated with Th17 cell function and studied in detail the relevance of the polyamine pathway as modulators of autoimmune inflammation. We investigated this pathway using carbon-tracing fluxomics, transcriptomics, epigenomics (ATACseq) and functionally validated their relevance in autoimmune disease models using small molecule inhibitors and knockout animals.
Drak2 contributes to autoimmunity by negatively regulating IL-2 signaling and altering regulatory T cell development
Authors: Mandarano Alexandra H , Harris Tarsha L , Creasy Blaine M , Wehenkel Marie , McGargill Maureen A
Published: January 2023

Drak2, a serine/threonine kinase expressed highest in B and T cells, negatively regulates T cell activation. Previously, we showed that Drak2-deficient mice were resistant to disease in models of multiple sclerosis and type I diabetes. Interestingly, these mice were not more susceptible to infection compared to wildtype mice. While this makes Drak2 an ideal protein to target to treat autoimmune disorders without compromising immunity to infectious pathogens, it is still not clear how Drak2 functions to specifically inhibit autoreactive T cells. Here, we report a novel mechanism by which Drak2 impacts autoimmunity. We found that the resistance to type 1 diabetes was due to the absence of Drak2 in the T cells rather than the pancreatic beta cells or other immune cells. Moreover, resistance to disease was dependent on the presence of regulatory T cells (Tregs). Unexpectedly, we found that the development of thymic Tregs was increased in Drak2-deficient mice due to augmented IL-2 signaling, which led to increased proportions of peripheral Tregs compared to wildtype mice. Drak2 limits IL-2 signaling in developing T cells via interaction with Stat5. These data provide important insight into the etiology of autoimmunity by demonstrating that Drak2 not only regulates T cell activation, but also impacts the development of Tregs by regulating IL-2 signaling.
Self-originating CTL peptides induce the cross-activation of Treg cells to suppress the proliferation of self-reactive CD8+ T cells
Authors: Han Seongeun , Park Bomi , Han Chungyong , Choi Beom K.
Published: January 2023

Most of self-reactive T cells are eliminated by the central tolerance in thymus, but some of T cells with low TCR affinity survived from the central tolerance are controlled by peripheral tolerance. Although multiple factors are involved in the peripheral tolerance, the balance between Treg and effector T cells seems to be a key player in maintaining the peripheral tolerance. Here we found that cytotoxic CD8+ T cell-restricted (CTL) peptides originating from self-tumor antigens including hTERT, WT-1 and Malan-A not only induced the proliferation of CD8+ T cells, but also induced the activation and division of Foxp3+ Treg cells with a delayed kinetics. Proliferation of self-reactive CD8+ T cells were swiftly induced by the CTL peptides during the initial phase of culture, but suppressed by the expanded Treg cells in the latter phase. Cross-activation of Tregs by CTL peptides originating self Ags is not found when the CD8+ T cells specific to cytomegalovirus (CMV) antigen. These finding indicate that CTL peptides originating self Ags have a potential to activate both CD8+ T cells and Treg cells and thus, limit the T cell-mediated autoimmune responses.
Novel role for T-helper 17 signature cytokine, IL-17, in inducing CD4 effector resistance to immune suppression
Authors: Crawford Michael Patrick , Sinha Sushmita , Renavikar Pranav S , Borcherding Nicholas , Brate Ashley , Karandikar Nitin
Published: January 2023

Important immune regulatory mechanisms mediated by CD4+ and CD8+ T-cells keep destructive CD4+ T-cell responses in check. CD4+ T-helper 17 (Th17) cells, characterized by IL-17 production, play critical roles in the body’s response to infections and cancer and in the pathogenesis of autoimmune diseases such as multiple sclerosis, psoriasis, arthritis, IBD, among others. Here, we demonstrate that human CD4+ T-cells exposed to a Th17-differentiating milieu are highly resistant to immune suppression by CD8+ T-cells, compared to control Th0 cells. This resistance is mediated, in part, through the action of IL-17A, IL-17F and IL-17AF heterodimer through their receptors (IL-17RA and IL-17RC) on CD4+ T-cells themselves, but not through their action on CD8+ T-cells or APC. We further show that IL-17 can directly act on non-Th17 effector CD4+ T-cells to induce suppressive resistance through the induction of IL-1b and IL-6/STAT3 pathways, indicating a novel feedback loop. These studies reveal a novel function for IL-17 cytokines in a CD4-intrinsic mechanism of immune resistance. The pathways induced in this process may serve as a critical target for intensive investigation and therapeutic intervention.
Lactoferrin modulates BMDCs to be tolerogenic leading to enhancement of Foxp3+iTreg differentiation as well as suppression of T cell proliferation
Authors: Jang Young-Saeng , Park Hui-Won , Park Sun-Hee , Jo Hyeon-Ju , Kim Tae-Gyu , Kim Pyeung-Hyeun
Published: January 2023

DCs are professional APCs for initiating both immune response and immune tolerance. Tolerogenic DCs are generally known as regulatory DCs that suppress immune responses through regulation of T cells such as induction of T cell anergy/apoptosis and Treg. Since we have demonstrated that lactoferrin (LF) induces functional Tregs and has a protective effect against IBD, we explored the tolerogenic effect of LF on mouse bone marrow-derived DC (BMDC) and its underlying mechanisms. LF enabled BMDCs to suppress the proliferation of allogenic CD4+ T cells, and elevated levels of Arg-1, IDO-1, and CD39/73 (known as key T cell suppressor molecules). Consistently, the suppressive activity of LF-treated DCs (LF-DCs) was disappeared by inhibitors of Arg-1 and CD39. Finally, LF-DCs enhanced TGFβ1-induced OVA-specific Foxp3+ T cell population.

Collectively, these results suggest that lactoferrin effectively causes DCs to be tolerogenic by both direct suppression of T cell proliferation and indirect enhancement of Treg differentiation.
A specific microbial metabolite influences the development of peripheral intraepithelial lymphocytes
Authors: Soderholm Amelia T , Beresford-Jones Benjamin , Clare Simon , Pedicord Virginia Ahn
Published: January 2023

Peripheral CD4+CD8αα+TCRαβ+ intraepithelial lymphocytes (pIELs) are a specialised T cell population that are reduced in germ-free (GF) C57BL/6 mice and vary in frequency between SPF mice from different sources, indicating their development is dependent on the presence of intestinal microbes. The microbes and factors required for pIEL development, however, are not fully characterised. pIELs are closely associated with intestinal epithelial cells (IECs) that line the gastrointestinal tract, however the contribution of IECs in shaping pIEL development is not well defined. We investigated if microbial metabolites influence pIEL development, and whether this is mediated by microbe-IEC-pIEL interactions.

In this study we used flow cytometry to characterise pIEL frequencies in GF mice administered different microbial metabolites, and in SPF mice from different colonies and animal facilities. qPCR of IEC RNA isolated from these mice and of metabolite-treated enteroids and MODE-K cells was performed to identify candidate genes expressed by IECs that may influence pIEL development.

We identified a common bacterial metabolite that partially restores pIEL development in GF mice. pIEL frequency of SPF mice varied between different sources, and qPCR identified IEC expression of several T-cell-influencing cytokines that correlated with pIEL frequency in vivo.

Our results indicate that a common microbial metabolite contributes to pIEL development, corresponding to metabolite-induced IEC expression of T-cell-influencing cytokines. Our work further elucidates the mechanisms of commensal microbe modulation of host tolerance, which may enable better strategies to prevent and treat intestinal infections and inflammation.
IRF8 regulated interferon beta production drives Gram-negative bacteria-mediated NLRP3 inflammasome activation
Authors: Sharma Bhesh Raj , Karki Rajendra , Kanneganti Thirumala-Devi
Published: January 2023

Inflammasomes are multimeric protein complexes that orchestrate into the cytoplasm to exhibit different immune outcomes in response to microbial pathogens. Lipopolysaccharides (LPS) present in Gram-negative bacteria induces non-canonical NLRP3 inflammasome activation. We have recently found that interferon regulatory factor 8 (IRF8) was not required for caspase-11–mediated NLRP3 inflammasome activation during LPS transfection. Nonetheless, the role of IRF8 in Gram-negative bacteria mediated NLRP3 inflammasome activation has been unknown. In this study, we discovered that IRF8 promotes NLRP3 inflammasome activation in bone marrow-derived macrophages (BMDMs) infected with Gram-negative bacteria such as E. coli and C. rodentium. Furthermore, IRF8 deficient BMDMS showed reduced caspase-11 activation and gasdermin-D cleavage. Mechanistically, IRF8-mediates the activation of IRF3 for the transcription of interferon beta—which is required for caspase-11–dependent NLRP3 inflammasome activation in the infected BMDMs. Overall, we found that IRF8 regulated interferon beta production in response to Gram-negative bacteria is a driving factor for the activation of NLRP3 inflammasome.
Differential dynamics of the Jersey ruminal microbiome and immune response in the heat stress environment
Authors: Kim Euntae , Kim Myunghoo , Son Junkyu , Lee Jihwan , Kim Donghyeon , Ha Seungmin
Published: January 2023

The rumen microbiome and immune cell play important roles in metabolism and immune responses of dairy cows. Accumulation data suggesting that different species of dairy cow have differential heat stress resistance, however underlying mechanism by which non-host factors contribute heat stress responses. Thus, our study was designed to understand the changes of rumen microbiome and immune cell function to heat stress response in Holstein and Jersey cows. Respiration rate and rectal temperature were measured and rumen fluid and blood were collected for microbiome analysis and immune cell analysis under normal or heat-stressed environment from eight Holstein and Jersey cows. The rumen bacteria diversity and microbial gene enrichment was analyzed by using the Illumina HiSeq™ 4000 platform. The blood of dairy cows was collected for peripheral blood mononuclear cells (PBMCs) isolation and PBMCs were analyzed by RNA sequencing (Marcrogen, Korea) under the same environmental condition. Holstein had significantly higher respiration rate compared to that of Jersey in heat stress environment. We also find that gene transcription patterns associated with functional activities in PBMCs are differ between Holstein and Jersey. Distinct and more prominent changes of rumen bacteria taxa and functional gene abundance in Jersey cows may be associated with better adaption ability to heat stress. These data suggest that different species of dairy cows had different metabolic and immunological system to adapt heat stress responses.
Molecular Mechanisms of Immune Dysregulation during Acute Orientia tsutsugamushi Infection in Mice
Authors: Soong Lynn , Trent Brandon , Liang Yuejin , Fisher James , Card Galen E. , Walker David H. , Sun Jiaren
Published: January 2023

Orientia tsutsugamushi is a Gram- and LPS-negative bacterium that can cause severe scrub typhus in the Asia-Pacific region. While bacteria replicate freely in endothelial cell (EC) and phagocyte cytosol, little is known regarding pathogen recognition and cellular responses. Our newly developed O. tsutsugamushi infection mouse models are valuable for mechanistic studies. Here, we show that a hallmark of lethal infection is Th1-skewed, but Th2-impaired, immune responses, accompanied by severe endothelial damage and acute tissue injury. While tissue bacteria peak at disease onset (D6), neutrophil and CD8 T cell influx/activation reach peaks around D10 (prior to mouse death). We further show the pathological characteristics of vascular activation and tissue damage: 1) a significant increase of ICAM-1, VEGFR2, and angiopoietin-2 proteins in infected tissues and lung-derived ECs, 2) a loss of endothelial quiescent/junction proteins (angiopoietin-1, VE-cadherin), and 3) a functional loss of Tie2 receptor. Pulmonary macrophages predominantly display an M1-like (CD80+CD64+CD11b+) phenotype at D6 and D9, which correlated with the significant loss of CD31+CD45− ECs and M2-like (CD206+CD64+CD11b+−) cells. NanoString analyses of infected lung and spleen tissues and in vitro studies suggested a role for Clec4e in pathogen recognition and the IL-12/IL-27 axis in driving proinflammatory immune responses and cellular injury. This is the first detailed investigation of lung cellular immune responses during acute O. tsutsugamushi infection. It uncovers specific biomarkers for vascular dysfunction and M1-skewed inflammatory responses, highlighting future therapeutic research for the control of this neglected tropical disease.
Antibiotic resistant Pseudomonas aeruginosa upregulate iron piracy genes during macrophage infection and evade killing
Authors: Jorth Peter Allan , Vaillancourt Mylene , Nadeau Samantha , Caldera Juan Raphael
Published: January 2023

Antibiotic resistant bacterial lung infections are the primary cause of morbidity and mortality in people with cystic fibrosis (CF). Recently, we showed that aztreonam resistant P. aeruginosa with a mutated nalD gene were more virulent than wild type (WT), even in the absence of antibiotic treatment. Yet, the mechanisms underlying the increased virulence were unknown. Because nalD− mutants increased antibiotic efflux pump expression, we hypothesized that their hypervirulence was dependent on the efflux pump. Surprisingly, deletion of the efflux pump did not affect nalD− virulence in an acute lung infection. To determine other potential mechanisms underlying the enhanced virulence, we performed RNA-seq on WT and nalD− grown in vitro and found that nalD− increased efflux pump expression relative to WT, as expected, but also differentially regulated hundreds of genes. Exopolysaccharide genes involved in biofilm formation were among the most highly upregulated genes. Consistent with this differential gene expression, the nalD− mutant exhibited increased biofilm formation, reduced C3 complement opsonization, and also replicated better than WT in the presence of macrophages. Subsequent RNA-seq analyses of infected macrophages revealed that nalD− upregulated iron siderophore systems relative to WT. We predict that this increased iron piracy facilitates subversion of nutritional immunity during infection. Because individuals with CF frequently experience anemia, antibiotic resistance mutations that increase iron piracy could promote bacterial growth during these periods of iron limitation.
Sensory afferent neurons coordinate in host immune responses to Citrobacter rodentium
Authors: Reardon Colin , Ramirez Valerie T , Sladek Jessica
Published: January 2023

Background

Neuro-immune communication is a vital component of regulating host immune responses to pathogens. Specialized sensory neurons have recently been described to detect bacterial pathogens in the skin, lung, and small intestine, inducing either maladaptive or host-protective responses. Here we assessed the role of sensory nociceptive neurons in the host response to the enteric pathogen Citrobacter rodentium.

Methods

Selective ablation of nociceptive neurons was accomplished using the Trpv1 agonist Resiniferatoxin (RTX) 10 days prior to C. rodentium infection of mice (C57BL/6).

Results

RTX-treated mice exhibited significantly increased bacterial burden 10 days post infection and delayed pathogen clearance compared to vehicle control mice. This reduced host-protective immune response was due to significantly reduced expression of IL-22, and recruitment of CD3+ T-cells into the colonic mucosa. This process appears to be TRPV1 dependent as TRPV1 KO mice experienced a similar delay in T-cell recruitment. Expression of pro-inflammatory cytokines in RTX-treated mice was significantly enhanced, and was associated with increased numbers of colonic T-cells 29 days p.i.. As previous studies identified the sensory neuropeptide CGRP as a regulator of host immune responses, we assessed the role of CGRP receptor signaling during C. rodentium infection. Antagonism of the CGRP receptor reduced expression of Il6, and Ifng, while bacterial burden and Il22 expression was not reduced compared to C. rodentium-infected vehicle-treated control mice.

Conclusions

Our data indicate that sensory nociceptive neurons exert a significant host protective role during C. rodentium infection that is independent of CGRP.