bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytokine and chemokine profiles in a human respiratory disease model of Ebola virus
Authors: O’Brien Cecilia , Brannan Jennifer , Kuehne Ana , Josleyn Nicole M. , Parkhill Robert , Edwards Darin , Dye John M.
Published: January 2023

Category A agents, such as Ebola virus, pose a great risk to national security because of high rates of transmission and mortality and the gravity of impact to public health. The use of Ebola virus as a bioweapon is a significant concern, and rapid diagnostics, safe vaccines, and efficacious therapeutics are needed. The Mucosal Tissue Equivalent (MTE) component of the MIMIC™ lung mucosal model is an in vitro/ex vivo respiratory tissue model comprised of human lung epithelial cells that mimics the vascularized lower airway alveolar tissue micro-environment. The use of the MTE allows for the inclusion of human immune components, which may be a better predictor of the human immune response than animal models. In this study, Ebola virus was applied at the air-liquid interface of the differentiated human epithelium and expression of cytokines was measured. Peripheral blood mononuclear cells (PBMCs) were applied to apical layer of the MTEs prior to inoculation with Ebola virus (Kikwit). We quantified cytokines and chemokines using supernatants serially collected on days 0, 3, 6, 9, and 15 post infection. In the infected MTEs containing human PBMCs, we detected pro-inflammatory cytokines, colony stimulating factors and endothelial growth factors. This “cytokine storm” would be expected to play a role in inflammation, recruitment of cells to the site of infection, and induction of a specific immune response to acute viral infection in humans. This human lung infection model will be used to evaluate existing therapeutic antibody and vaccine candidates for Ebola virus. Going forward, this system may provide a more rapid, relevant and cost effective means to evaluate vaccine candidates than can be accomplished by using animal models.
Type I and III interferon signaling contributes to severe pathogenesis of human metapneumovirus clinical isolates
Authors: Xu Jiuyang , Zhang Yu , Tollefson Sharon J. , Manni Michelle L. , McHugh Kevin J. , Alcorn John F. , Sarkar Saumendra N. , Williams John V.
Published: January 2023

Human metapneumovirus (HMPV) is a leading cause of respiratory tract infection in infants and children. The disease outcome of HMPV infection ranges from mild infection to severe bronchiolitis and fatal pneumonia. HMPV was discovered in 2001 and its disease mechanisms are not fully understood. To gain insight into HMPV pathogenesis, we tested nine HMPV clinical isolates in an established C57BL/6 mouse model. HMPV clinical isolates induced variable disease severity ranging from mild to fatal disease. Mice infected by non-virulent strain TN/94-49 did not show significant weight loss, but mice infected with virulent isolate C2-202 showed dramatic weight loss and 40% mortality within 5 days post-infection. These findings were confirmed in other inbred mouse strains. C2-202 infected mice also suffered from impaired pulmonary function post-recovery. Lung virus replication did not correlate with disease severity, suggesting immune-mediated pathogenesis. Further investigation revealed increased type I and III interferons, pro-inflammatory cytokine production, and neutrophil infiltration in C2-202 infected mice. HMPV C2-202 infection also inhibited dendritic cell recruitment and down-regulated MHC-II surface expression on macrophages. Stat1/Stat2 double knockout mice lacking interferon signaling exhibited reduced weight loss, lessened disease severity, and increased dendritic cell recruitment after C2-202 infection. Our results indicate that severe disease caused by HMPV clinical isolates was due to exuberant interferon response leading to immuno-pathological tissue damage. These data suggest interferon signaling plays an important role in HMPV pathogenesis, and thus serves as a potential therapeutic target.
A single infection with a malaria parasite protects mice from lethal autoimmune glomerulonephritis
Authors: Bolland Silvia , Kole Hemanta K. , Scott Bethany , Amo Laura
Published: January 2023

Infections can alter the prevalence of autoimmune diseases such as SLE. Epidemiological studies have showed a relative absence SLE in West Africa (areas of endemic malaria), even though SLE is highly prevalent in populations of African descent living outside of Africa. We hypothesize that malaria infections might reduce the incidence of autoimmune lupus. In the present work, we assess the effect of the non-lethal Plasmodium yoelii 17XNL in a well-characterized animal model of SLE (FcγRIIB−/−). Mice deficient in the inhibitory Fc receptor for IgG (FcγRIIB) develop spontaneous disease driven by the production of anti-nuclear autoantibodies, a broad immune activation, splenomegaly, anemia and lethal glomerulonephritis with a pathology similar to human SLE. We infected FcγRIIB−/− mice at 2 months of age with P.yoelii and followed the progression of autoimmune disease for 5 months. Infection with P. yoelii increased the titer of a broad range of autoantibodies, with concomitant immune-complex deposition and complement fixation in the kidney. While lupus-prone mice succumbed from renal disease detected by protein in urine and serum urea nitrogen, malaria-infected lupus-prone mice did not develop proteinuria or lethal disease. Infection with the parasite did not alter tolerance mechanisms leading to lupus but instead prevented end-point tissue pathology. P. yoelii infection seems to inhibit pathogenic leukocyte infiltration without affecting the autoimmune inflammation of glomeruli. These results indicate a possible route for lupus protection that specifically targets renal function, which remains a cause of the substantial morbidity and mortality in SLE patients.
Lupus-suppressing effect of VSV-primed CD8+ T cell subsets
Authors: Kole Hemanta K. , Scott Bethany , Crampton Steve , Sule Gautam , Deane Jonathan , Bolland Silvia
Published: January 2023

Pathogenic infections are important environmental modifiers of autoimmune disease as they can alter the immune system in a way that either promotes or reduces autoimmune responses. We tested the effect of infection with vesicular stomatitis virus (VSV) on the FcγR2B−/− (R2) mouse strain that spontaneously develops lupus disease. Infecting FcγR2B−/− mice with live VSV reduces autoantibody levels and inflammatory pathology, and prolongs survival. We showed that VSV-mediated protection can be transferred in a CD8 cell adoptive transfer experiment. Legend screening for surface markers on CD8 T cells indicated that CD8 T cells from VSV infected mice express high level of markers linked to exhausted and regulatory phenotypes. After testing the transfer of various CD8 T cell subsets, we have identified CD8+CD122+NKG2D+ cells as mediator in VSV-induced Lupus amelioration. Preliminary analysis of RNA Sequencing data for gene expression profiles among various CD8 subsets, indicate that the protective cells express higher level of genes linked to memory phenotype. Our working hypothesis is that the viral infection may induce the expansion of long lived CD8 populations that regulate the humoral response.
Bacterial biofilm product Curli/eDNA induces NETs and serum anti-Curli/eDNA levels correlate with bacteriuria and lupus activity.
Authors: Pachucki Ryan J. , Corradetti Chelsea , Tursi Sarah , Kohler Lynne , Nicastro Lauren , Gallucci Stefania , Kilpatrick Laurie , Tükel Çagla , Caricchio Roberto
Published: January 2023

Infections are a major contributor to lupus disease. We have previously demonstrated that bacterial amyloid curli, produced by E.coli, can accelerate disease in mouse models of lupus. Interestingly curli incorporates extracellular DNA, which in turn can be both adjuvant and a self-antigen in lupus. Finally, uropathogenic E. coli (UPEC) is responsible for the majority of urinary tract infections in SLE. Based on our previous results, we hypothesize that exposure to UPEC triggers anti-curli/eDNA antibodies and curli/eDNA complexes can stimulate the innate immune system. We investigated 98 lupus patients who met at least 4 SLICC criteria. Results were compared to 54 age, sex and race matched healthy controls. We tested the production of anti-curli/DNA complex of both IgG and IgA subclasses. We than correlated the levels of anti-curli/DNA antibodies with clinical parameters. Finally, we stimulated human neutrophils with curli/eDNA complexes. We found that anti-curli/eDNA IgG levels were detected in both lupus and controls plasma. In lupus patients, anti-curli/eDNA levels correlated with persistent bacteriuria and disease flares (p<0.05). In addition, anti-curli/eDNA antibodies recognized dsDNA suggesting a potential molecular mimicry mechanism for curli/eDNA with self-antigens such as dsDNA. We also found that IgA anti-curli/eDNA levels were higher (p<0.01) in lupus donors compared to controls. Finally we found that curli/eDNA induced neutrophil extracellular traps (NETs) and the induction was ROS-dependent. We conclude that curli/eDNA complexes from UTIs can activate the innate immune system and IgG and IgA anti-curli/eDNA are strong biomarkers for disease activity and severity.
The REV-ERBs: Negative Regulators of Th17 Cell Development and Autoimmunity
Authors: Amir Mohammed , Chaudhari Sweena M. , Wang Ran , Campbell Sean , Kamenecka Theodore M. , Solt Laura A.
Published: January 2023

Th17 cells have been associated with the pathogenesis of several autoimmune diseases, including multiple sclerosis. While the nuclear receptor (NR) RORγt is considered the lineage determining transcription factor for Th17 cells, the REV-ERBs (REV-ERBα and REV-ERBβ), two other members of the NR superfamily, are often co-expressed in the same tissues as members of the ROR subfamily of NRs (i.e. RORγt) and bind to the same DNA response elements. In contrast to the RORs, which are transcriptional activators, the REV-ERBs are transcriptional repressors, suggesting mutual cross talk and co-regulation of their shared target genes. While the REV-ERBs are best known for their roles in the circadian rhythm and metabolic processes, their role in Th17 cell development and Th17 mediated inflammatory diseases is largely unexplored.

Our data indicates that over expression of the REV-ERBs inhibits Th17 cell differentiation whereas genetic deletion of the REV-ERBs perturbs Th17 cell differentiation. Using two commonly used mouse models of autoimmunity known to be Th17 driven [experimental autoimmune encephalomyelitis (EAE) and adoptive transfer model of colitis], we found that loss of REV-ERB expression exacerbates EAE and colitis compared to WT controls, largely as a consequence of an increased number of pro-inflammatory Th17 cells. Finally, using REV-ERB-selective small molecules that we have developed, we discovered that pharmacological modulation of REV-ERB activity significantly inhibits the development of autoimmunity. Overall, our data suggests that the REV-ERBs are potent negative regulators of Th17 differentiation and identifies the REV-ERBs as viable therapeutic targets for the treatment of Th17-mediated autoimmune diseases.
The contribution of autoantibody-inducing CD4+ T cell (aiCD4 T cell) help to the B cell maturation and possible autoantibody in Systemic Lupus Erythematosus
Authors: Sakurai Keiichi , Tsumiyama Ken , Shiozawa Shunichi
Published: January 2023

INTRODUCTION

Autoantibody-inducing CD4 T (aiCD4 T) cell seems indispensable for the self-organized criticality theory that induces Systemic Lupus Erythematosus (SLE). We previously found that the aiCD4 T cell belongs to CD45RBlo122lo CD4 T cell subpopulation. We here analyze the B cell population and CD19+ splenic B cell receptor (BCR) repertoire, and test our contention whether aiCD4 T cell helps B cell maturation.

METHODS

BALB/c mice were immunized with OVA twelve times and SLE was induced. Cell surface markers of B cell were detected with flow cytometry. CD19+ splenic B cells were sorted and BCR repertoires of these cells were analyzed on next generation sequencer.

RESULTS

After twelve times immunization of mice with OVA, follicular B (Fo B) cell and germinal center B (GC B) cell were significantly increased(P<0.05), whereas marginal zone B cell was decreased. Activation markers of the B cell were increased and in particular, CD80 was significantly increased (P<0.05). In addition, the number of clonotypes of CD19+ splenic B cells in OVA immunized mice was increased. Among these repertoires, the same pairs of V and J usage were frequently found in OVA immunized mice.

CONCLUSION

Fo B cell and GC B cell and the activation markers of B cell, as well, were significantly increased in OVA immunized mice, and the result of BCR repertoire analysis suggested that somatic hyper mutation occurred frequently in OVA-stimulated mice, which is compatible with the contention that aiCD4 T cells helps the maturation of B cells into germinal center B cells to enhance autoantibody formation thereby inducing SLE.
Stage specific requirement of TCF-1 for restraining Th17 immunity
Authors: Zhang Jing , He Zhiheng , Sun Zuoming
Published: January 2023

Besides the positive and negative selection for shaping T cell repertoire that only for attacking foreign antigens, central immune system also control the type and scale of immune responses in the periphery. T cell factor 1 (TCF-1) is highly expressed in the early thymic progenitors and has been shown to restrain Th17 responses in the periphery. However, it is not well understood which specific T cell developmental stage is critical for TCF-1 to determine the magnitude of Th17 immunity in the periphery. Here we showed that conditional deletion of TCF-1 early at hematopoietic, but not CD4+CD8+ double positive (DP) stage, resulted in severe reduction of thymic cellularity, enhanced Th17 differentiation and more aggravated experimental autoimmune encephalomyelitis (EAE). We also observed abnormal IL-17 expression in developing CD4−CD8− double negative (DN) thymocytes. Expression of wild-type TCF-1 in CD4−CD8− DN thymocytes, but not peripheral mature T cells, inhibited IL-17 expression. We also demonstrated that TCF-1 binds to IL-17 promoter region to perform its histone deacetylase (HDAC) function and hence restrain IL-17 expression in thymus. Our study first demonstrated the stage specific requirement of TCF-1 to restrain peripheral Th17 responses and thus provide an additional way to develop treatment for autoimmune diseases.
CD28 suppresses IL-23- and IL-1β-driven human Th17 development
Authors: McGeachy Mandy J. , Revu Shankar , Wu Jing , Rittenhouse Natalie , Menk Ashley , Delgoffe Greg M. , Poholek Amanda C.
Published: January 2023

Th17 cells drive autoimmune disease but also control commensal microbes. A common link between antigens from self-proteins or commensal microbiota is relatively low activation of T cell receptor and costimulation signaling. Indeed, strong TCR/CD28 stimulation suppressed Th17 cell differentiation from human naïve T cells, but not effector/memory cells. CD28 suppressed the classical Th17 transcriptional program, while inducing known Th17-regulators, and acted through an Akt-dependent mechanism. Th17 cells differentiated without CD28 were not anergic: they showed robust proliferation and maintained Th17 cytokine production following restimulation. IL-23 and IL-1β promoted glucose uptake and increased glycolysis. Although modestly increased compared to CD28 costimulation, glycolysis was necessary to support Th17 differentiation, indicating that cytokine-mediated metabolic shifts were sufficient to obviate the classical requirement for CD28 in Th17 differentiation. Together these data propose that in humans, strength of TCR/CD28/Akt activation serves as a rheostat tuning the magnitude of Th17 development driven by IL-23 and IL-1β.
Identifying novel mechanisms regulating the expression of the inflammatory chemokine IP-10
Authors: Richard Mara Lennard , Caldwell Tomika , Nowling Tamara K. , Gilkeson Gary S. , Zhang Xian
Published: January 2023

In response to the innate immune signals, mammalian cells produce inflammatory cytokines and chemokines to activate the immune system, and their expression is tightly regulated. IFN-gamma Inducible Protein (IP-10), also known as C-X-C motif chemokine 10 (CXCL10), is an inflammatory chemokine belonging to the CXC chemokine family. IP-10 is chemotactic for neutrophils, and altered expression of IP-10 is associated with many inflammatory diseases. Fli-1 belongs to the ETS transcription factor family. We have demonstrated that the Fli-1 transcription factor is a novel regulator in modulating the expression of many inflammatory mediators, including monocyte chemotactic protein 1 (MCP-1), chemokine (C-C motif) ligand 5 (CCL5) and interleukin 6 (IL-6). In this report, we found that murine endothelial cells transfected with Fli-1 specific siRNA produced significantly lower IP-10 after stimulation with Toll-like receptor 4 ligand LPS compared to the cells transfected with control siRNA. The production of IP-10 in endothelial cells with LPS stimulation is dose-dependent. We demonstrated that Fli-1 binds to the IP-10 promoter by Chromatin immunoprecipitation (ChIP) assay. Mechanisms by which Fli-1 regulates expression of IP-10 are currently being investigated. Together, the results indicate that Fli-1 is a novel, critical transcription factor in regulating the expression of the pro-inflammatory chemokine IP-10
Soluble Transforming Growth Factor Beta-1 enhances murine mast cell cytokine release
Authors: Lyons David O. , Plewes Michelle R. , Pullen Nicholas A.
Published: January 2023

TGF-β1 is traditionally known as an immunosuppressive cytokine. Previous studies have investigated its effects on FcɛRI-dependent mast cell activation, yet there is an incomplete understanding of TGF-β1 on mast cell function, for example, without sensitization to IgE. We investigated the effects of soluble TGF-β1 on IL-6 and IL-13 cytokine production in C57BL/6 bone marrow-derived mast cells in the presence and absence of FcɛRI-mediated activation. Our experiments found IL-6 production to be significantly enhanced independent of IgE sensitization or FcɛRI crosslinking; this was similar to treatment with stem cell factor (SCF), which also enhanced IL-6 production in this resting state. Additionally, IL-13 production was significantly enhanced following IgE-mediated stimulation of TGF-β1 treated mast cells; an effect independent of IgE was not observed, and treatment with SCF had no significant impact. Lastly, via confocal microscopy, we interrogated the cell-surface expression of FcɛRI, CD117 (c-Kit; SCF receptor), and TGFβRII with and without soluble TGF-β1 and/or FcɛRI crosslinking. This experiment revealed no apparent effect of TGF-β1 on FcɛRI and CD117 receptor co-localization – in both treatments we observed an equivalent decline in co-localization following IgE-mediated activation. TGFβRII was also found to weakly co-localize with those receptors, however this co-localization was unchanged across all treatments. Together, our findings provide novel insight into the stimulatory role of TGF-β1 on mast cell function both in the presence and absence of an allergic-like stimulus, suggesting alternative pathways play a pivotal role in regulating mast cell function beyond that of a canonical IgE-mediated pathway.
Proposed 3-D Structure of a Critical Protein-Protein Interaction Required for Transcription of the IL1B Gene Coding for Human Interleukin 1β
Authors: Pulugulla Sree H. , Workman Riley , Rutter Nathan , Esposito Emilio X. , Yang Zhiyong , Madura Jeffry D. , Auron Philip E.
Published: January 2023

We previously reported that expression of the human IL1B gene coding for Interleukin 1β in LPS-activated macrophages requires long-range chromatin looping mediated by a mutual DNA binding domain (DBD) interaction between two transcription factors, Spi-1/PU.1 (Spi1) at the promoter and C/EBPβ at a far-upstream enhancer. We have now combined preexisting X-ray structures and advanced computational techniques with additional biochemical and cell-based studies in order to further investigate the structure of this critical interaction. In vitro binding was used to generate a surface interaction map for the two proteins that suggested an association similar to that of a C/EBPβ·Myb X-ray structure reported by others. The distinction is that, in addition to a C/EBPβ leucine zipper interaction similar to that with the Myb DBD, there is an interaction between a carboxyl-tail extension of one of the C/EBPβ leucine zipper monomers with Spi1. This is consistent with our previous report that the carboxyl-tail of C/EBPβ is critical for association with arginine 232 within a pocket at one end of the Spi1 DNA recognition helix. L-arginine, a known anti-inflammatory, was computationally identified to bind in this interaction pocket. In vivo studies of LPS-activated THP-1 macrophages treated with L-arginine decreased IL1B transcription in parallel with a reduction in C/EBPβ association with Spi1 on the IL1B promoter. No significant change in direct binding of Spi1 and C/EBPβ to DNA was observed. These results suggest that L-arginine inhibits IL1B gene expression by competitive inhibition of C/EBPβ binding to Spi1 in the absence of any effect on direct DNA binding for either factor, and support the predicted structure of the C/EBPβ·Spi1 interaction.
The Notch signaling pathway controls basophil responses during helminth-induced type 2 inflammation
Authors: Webb Lauren M. , Peng Seth Alexander , Oyesola Oyebola , Cubitt Rebecca L. , Danko Charles , Wojno Elia Dion Tait
Published: January 2023

Type 2 inflammation is characterized by production of the cytokines IL-4, IL-5 and IL-13 and is required for clearance of gastrointestinal helminth parasites, which infect over 2 billion people worldwide. Basophils are innate immune cells that promote expulsion of the helminth Trichuris muris in mice. However, the molecular mechanisms that control basophil function and gene expression during helminth-induced Type 2 inflammation remain unclear. We show that during T. muris infection, basophils localized to the intestine and upregulated components of the Notch signaling pathway, which regulates inflammatory gene expression programs. In vitro, Notch inhibition abrogated IL-33-elicited IL-4 and IL-6 production from basophils by directly targeting Il4 and Il6. Transcriptional profiling of Notch-deficient basophils revealed that Notch directs basophil responsiveness to inflammatory cues and effector gene expression. Mice lacking basophil-intrinsic Notch signaling had impaired worm clearance and decreased intestinal Type 2 effector mechanisms following infection. These findings demonstrate that Notch regulates basophil gene expression and effector function during helminth-induced Type 2 inflammation. We are now actively investigating how basophils interact with other cells in the intestine to orchestrate optimal Type 2 immune activation, and the role Notch plays in this process. These findings increase our understanding of the molecular signals that control innate immunity during Type 2 inflammation and may have far-reaching impact on the development of therapeutics aimed at modulating Type 2 immune responses.
Langerhans cells are required for the priming and induction of an optimal immune response against an acute DNA virus infection
Authors: Wong Eric , Xu Ren-Huan , Tang Lingjuan , Meng Jennie , Sigal Luis J.
Published: January 2023

Resistance to ectromelia (ECTV) virus infection is dependent on the induction of a strong type I interferon (T1-IFN) response, which our lab has recently determined is primarily mediated by inflammatory monocytes (iMo) that are recruited to the draining lymph node (dLN). However, the mechanism behind how iMo are recruited is unclear. Accumulation of iMo is dependent on the production of CCL2 and CCL7 by CD11c+MHC-IIhi migratory dendritic cells (skin-mDCs) that originate from the skin of the footpad (initial site of infection), specifically CD103+CD207+ double-positive dermal DCs (DP dermal DCs), CD103−CD207− double-negative DCs (DN dermal DCs) and Langerhans cells. The transport of skin-mDCs to the dLN is dependent on the intrinsic utilization of the TLR9-MyD88-IRF7 axis, highlighting a key role for DNA sensing mechanisms in the skin. Analysis of infected and uninfected skin-mDC subsets reveal differential effector roles between the groups, with DP dermal DCs and Langerhans cells exhibiting high expression of pro-inflammatory cytokines and chemokines. Ablation of skin-mDC recruitment to the dLN with pertussis toxin (Ptx) resulted in a reduction in iMo accumulation and subsequent T1-IFN/pro-inflammatory expression with significantly higher mortality in infected B6 mice (which are normally 100% resistant to ECTV-related mortality). Finally, depletion of Langerhans cells, but not DP dermal DCs, resulted in a significant reduction in iMo accumulation in the dLN and consequently high mortality rates, therefore indicating that Langerhans cells may play an integral role in the priming and breadth of the host immune response against acute viral infections.
Oxidative stress enhances mitochondrial DNA-dependent type I interferon responses.
Authors: Lei Yuanjiu , Martinez Camila Guerra , West Phillip
Published: January 2023

It is increasingly well-appreciated that mitochondrial DNA (mtDNA) can engage the cGAS-STING cytosolic DNA sensing machinery to trigger type I interferon (IFN-I) responses. However, the exact immunostimulatory features of mtDNA and whether other nucleic acid sensors participate in triggering IFN-I remain poorly defined. We have discovered that mitochondrial reactive oxygen species potentiate mtDNA-driven IFN-I responses by enhancing the immunostimulatory properties of this DNA toward the cGAS-STING pathway. Moreover, we report that elevated oxidative stress results in the accumulation of mtDNA species with unique secondary structures that enhance IFN-I production. Finally, we report that Zbp1/DAI cooperates with cGAS to sustain IFN-I responses triggered by mtDNA. These results provide new insight into the molecular mechanisms of mtDNA recognition by the cytosolic DNA sensing machinery, with broad implications for understanding human diseases with mtDNA as a pathogenic inflammatory driver.
Type I and II IFNs differentially regulate IFNGR1 to tune IFNγ responsiveness in myeloid cells
Authors: Crisler William J. , Eshleman Emily M. , Lenz Laurel L.
Published: January 2023

Interferons (IFNs) act on macrophages to shape immune responses against microbial infections and tumors. Type I and II IFNs signal to induce transcription of IFN-stimulated genes (ISGs). Binding of the type II IFN (IFNγ) to its receptor, IFNGR, induces ISGs that enhance macrophage inflammatory and anti-microbial activity critical for bacterial infection. Conversely, when type I IFNs bind their receptor (IFNAR), they reduce expression of myeloid cell surface IFNGR and dampen macrophage responsiveness to IFNγ. Macrophages stimulated with IFNγ also reduce cell surface IFNGR, but this was previously attributed to ligand-induced receptor internalization. Here, we confirm the ability of IFNγ stimulation to rapidly reduce cell surface IFNGR in cultured mouse and human macrophages. However, we find this ligand-induced reduction in IFNGR is associated with reduced ifngr1 transcript abundance. Unlike IFNβ, IFNγ did not impede de novo transcription from an ifngr1 promoter-driven reporter construct, suggesting IFNγ reduces ifngr1 transcript abundance via a distinct mechanism. BMDMs pulsed with IFNγ transiently became refractory to a secondary IFNγ treatment as measured by impaired STAT1 phosphorylation. As shown previously, type I IFNs cause a reduction in myeloid cell surface IFNGR1 seen 72 hpi with systemic L. monocytogenes (Lm), and this reduction was abrogated by myeloid cell expression of a transgenic IFNGR1 in fGR1 mice. In contrast, IFNγ was responsible for a reduction in IFNGR on myeloid cells early after systemic Lm infection (24 hip), and this was not abrogated in fGR1 mice. Together, our results demonstrate that type I and II IFNs act via distinct mechanisms to prevent hyper-activation of myeloid cells following exposure to IFNγ.
Cholinergic signaling in the dorsal motor nucleus regulates systemic inflammatory responses via the vagus nerve
Authors: Kressel Adam M. , Tsaava Téa , Pavlov Valentin A. , Chavan Sangeeta S. , Tracey Kevin J.
Published: January 2023

The autonomic nervous system regulates organ homeostasis via neural reflex circuits. The inflammatory reflex is the prototypical neural circuit composed of afferent and efferent fibers that travel via the vagus nerve to regulate TNF production. Previous studies have demonstrated that stimulation of the cervical vagus nerve attenuates serum TNF levels and cytokine production in the spleen, but efferent vagus nerve neurons originate both in the dorsal motor nucleus of the vagus (DMV) and in the nucleus ambiguus (NA) in the brainstem. The origin of the specific fibers that regulate splenic TNF production was previously unknown. Here, we selectively stimulated cholinergic neurons in the DMV and measured TNF in endotoxemic mice. A fiber-optic cannula was inserted under stereotactic guidance into the DMV in transgenic mice expressing channelrhodopsin under the choline acetyltransferase promoter. Animals were subjected to either optogenetic stimulation (n=15) or no light (sham control, n=15) for five minutes (473nm laser, 20Hz, 25% duty cycle). After 24 hours, animals were challenged with intraperitoneal lipopolysaccharide (0.25mg/kg), and serum collected after 90 min. Optogenetic stimulation but not sham stimulation of the cholinergic neurons in the DMV significantly attenuated endotoxin-induced serum TNF levels. Additionally, direct neural recording of splenic nerve activity during optogenetic stimulation of the DMV revealed that signals originating in the brainstem travel via the vagus nerve to the spleen. Together these studies provide new insights into the identity and central origin of the efferent vagus nerve fibers regulating TNF production during endotoxemia.
The histone acetyltransferase MOF promotes macrophage inflammation in diabetic wounds.
Authors: den Dekker Aaron D. , Joshi Amrita , Davis Frank M. , Kimball Andrew , Boniakowski Anna , Schaller Matthew , Kunkel Steven L. , Moore Bethany B. , Gallagher Katherine
Published: January 2023

Macrophage (Mϕ) plasticity is essential for the repair and remodeling of wounds. However, under pathologic conditions such as type 2 diabetes (T2D), Mϕs exist in a chronic inflammatory state that drives continued tissue destruction and results in impaired wound healing. We have previously shown that Mϕs isolated from wounds in a murine model that mimics physiologic development of T2D (diet-induced obese mice; DIO) display increased levels of inflammatory cytokines at both a gene expression and protein level. Although the underlying mechanisms remain unclear, we and others have shown that Mϕ phenotype can be controlled by epigenetic modifications that alter gene expression. Our recent work provides evidence that the diabetic milieu alters gene expression and sets an “epigenetic signature” in myeloid cells that result in an exaggerated inflammatory phenotype and delayed wound healing in a murine model. Further, our recent work has found that the histone acetyl transferase, Males absent on the first (MOF), responsible for acetylation of lysine 16 on histone H4 (H4K16), was critical for expression of genes involved in fatty acid metabolism. In the present study, we find that wound Mϕs from DIO mice have increased expression of MOF at day 5, when Mϕs shift from a pro-inflammatory to an anti-inflammatory state within wound tissue. This change in MOF was accompanied by increased expression of components of MOF regulatory complexes, MSL1/2 and KANSL1. Our findings reveal a previously unreported role for MOF in promoting a pro-inflammatory state in innate immune cells. Moreover, our data suggest that the diabetic milieu promotes increased expression of MOF which, in turn, may contribute to maintaining the persistent inflammatory state in wound Mϕs.
Regulatory circuits governing identity and function of human type 1 ILCs
Authors: Collins Patrick , Cella Marina , Porter Sofia , Mccullen Matthew , Colonna Marco , Oltz Eugene M.
Published: January 2023

Innate lymphocytes develop from common progenitors but diverge functionally into a broad range of subsets that comprise a rapid response system to pathogens. In mice, type 1 innate lymphoid cells (ILCs), characterized by IFN-g expression, segregate into two major groups: cytotoxic natural killer (NK) cells and cytokine-producing, helper like ILC1s. In humans, this functional dichotomy is complicated further by heterogenous populations of circulating or tissue-resident NK cells, which exhibit distinct potentials for cytotoxicity or cytokine expression. Although developmental and functional kinships among NK cells have been studied extensively in mice, little is known about the molecular programs governing NK populations in humans. Here, we present integrated “-omics” analysis of human type 1 ILCs from blood and mucosal tissues, which identifies the key factors and biologic pathways establishing cell-type and functional identifies, as well as their underlying gene regulatory programs.
An essential role of transcription factors PU.1 and IRF8 in follicular B cell development and the germinal center response
Authors: Wang Hongsheng , Jain Shweta , Sun Jiafang , Kovalchuk Alexander L. , Morse Herbert C.
Published: January 2023

The transcription factors PU.1 and IRF8 regulate an unknown number of gene programs for differentiation of many hematopoietic cells including B cells, dendritic cells and myeloid cells. Their roles in B cell development were previously studied using B cell-specific conditional deletion mouse models, such as PU.1flox/flox-CD19Cre, IRF8flox/flox-CD19Cre, IRF8−/−PU.1flox/flox-CD19Cre, or IRF8flox/flox-PU.1flox/flox-CD19Cre mice. While PU.1-deficient B cells are phenotypically normal, deletion of IRF8 in B cells caused a moderate expansion of marginal zone B cells. Double deletion of PU.1 and IRF8 caused moderate expansion of plasma cells (PCs) in vitro. In this report, we investigated IRF8 and PU.1 double deletion mice using Mb1-Cre - termed DKO mice. FACS analysis revealed normal levels of early stage B cells in the bone marrow and transitional B cells in the spleens of DKO mice. However, follicular B cells were markedly reduced and the MZ B cell compartment was modestly expanded in DKO mice. The peritoneal CD5+ B-1a cells, which are a major source of circulating IgM, were completely absent in DKO mice. Surprisingly, the serum levels of IgM were higher and the levels of IgG3 and IgA were slightly lower in DKO than control mice. While the levels of serum IgG1 were comparable between DKO and control mice, DKO mice had no serum IgG2b or IgG2c antibodies. More intriguingly, following immunization with NP-KLH/alum, although the DKO mice produced more IgM-secreting PCs early on, they failed to generate germinal centers and produced markedly reduced levels of NP-specific switched IgG antibodies. Taken together, these data revealed a critical role of IRF8 and PU.1 in differentiation of follicular and germinal center B cells.
Progressive upregulation of oxidative metabolism facilitates plasmablast differentiation to a T-independent antigen
Authors: Price Madeline J. , Patterson Dillon , Scharer Christopher D. , Boss Jeremy M.
Published: January 2023

Transitioning from a metabolically quiescent naïve B cell to an antibody-secreting plasmablast requires division-dependent cellular differentiation. Though cellular processes such as cell division and antibody secretion demand significant ATP and metabolites, the metabolic processes used to generate ATP during plasmablast formation are not well described. Here, the metabolic machinery requirements for plasmablast formation were determined. Following T-independent stimulation with lipopolysaccharide, B cells increased expression of the oxidative phosphorylation machinery in a step-wise manner. Such activated B cells have increased capacity to perform oxidative phosphorylation, but showed additional dependency on glycolytic metabolism. Plasmablasts displayed higher oxidative metabolism to support antibody secretion, as inhibiting oxidative ATP production with oligomycin resulted in decreased antibody titres. Differentiation orchestrated by Blimp1 was required for this increase in oxidative metabolism as Blimp1-deficient cells proliferate normally, but neither differentiate to plasmablasts nor upregulate oxidative phosphorylation. Therefore, Blimp1 expression is required for full metabolic activity, and proliferation alone is insufficient to induce full oxidative phosphorylation. Together, these findings identify a shift in metabolic pathways as B cells differentiate, as well as the requirement for increased metabolic potential to support antibody production.
The histone demethylase LSD1 regulates B cell proliferation and plasmablast differentiation
Authors: Haines Robert Richard , Barwick Benjamin G. , Majumder Parimal , Scharer Christopher D. , Boss Jeremy M.
Published: January 2023

In order to generate humoral immunity, B cells must differentiate into antibody-secreting cells in response to antigen. LSD1 is a histone demethylase that regulates multiple cellular differentiation pathways and has been implicated to function during B cell differentiation through interaction with Blimp-1. Here we show that LSD1 is necessary for maximal proliferation and differentiation of naïve B cells into LPS-induced plasmablasts. LSD1-deficient B cell differentiation results in the targeted upregulation of hundreds of genes, a subset being Blimp-1 repressed genes, as well as the targeted downregulation of cell cycle genes. Plasmablasts lacking LSD1 fail to close chromatin at naïve B cell active enhancers in addition to PU.1 and IRF4 binding sites. LSD1 directly regulates the Blimp-1 target gene Sell through H3K4me1 demethylation at its Blimp-1 target binding sites. Our data defines LSD1 as a critical transcriptional and epigenetic repressor in plasmablasts.
Defining the mechanism of BATF specificity in Th cell lineages
Authors: Fu Yongyao , Kaplan Mark H.
Published: January 2023

IL-9 is a pleiotropic cytokine produced by Th9 cells and inappropriate expression is associated with immune disorders. The basic leucine zipper transcription factor (BATF) is expressed in multiple Th subsets and cooperates with other factors to regulate gene transcription. BATF-deficiency results in diminished IL-9 in Th9 cells and decreased IL-17 in Th17 cells. However, ectopic expression of BATF activates only the lineage-specific cytokine; IL-9 in Th9 cells and IL-17 in Th17 cells. The mechanism for this restricted activity is unclear. In this study, we demonstrate a lineage specific mechanism of how BATF activates Il9 in Th9 and Th17 cells. Using ChIP and chromatin accessibility assays we found that BATF binds to the Il9 gene locus at a greater level in Th9 cells compared to Th17 cells. This observation may be because the DNA in Th17 cells is in a heterochromatic state. Increasing the chromatin accessibility of Il9 gene locus in Th17 cells followed by BATF overexpression leads to increased IL-9 production. BATF forms a heterodimer with Jun family members to induce gene expression. The expression level of c-Jun and JunB in Th17 cells is lower compared to Th9 cells. In testing the role of these factors, we observed that the loss of c-Jun or JunB impairs IL-9 production in Th9 cells, indicating that Jun family members promote IL-9 production. Ectopic expression of BATF with either c-Jun or JunB significantly increases IL-9 production in Th17 cells, suggesting the low expression of Jun family members limits the ability of BATF to induce IL-9 in Th17 cells. Taken together, our results reveal a mechanism for how BATF achieves specificity of cytokine induction in Th subsets.
The plasma kallikrein-kininogen pathway is critical in the pathogenesis of colitis in mice
Authors: Wu Yi
Published: January 2023

The kallikrein-kinin system (KKS) consists of two serine proteases, prekallikrein (pKal) and factor XII (FXII), and a cofactor, high-molecular-weight kininogen (HK). Although the KKS is activated in humans and animals with inflammatory bowel disease (IBD), its role in the pathogenesis of IBS has not been characterized. Here we determined the role of the KKS in the pathogenesis of IBD using mice that lack proteins involved in the KKS. In two colitis models, induced by dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS), mice deficient in HK, pKal, or bradykinin receptors displayed attenuated phenotypes including body weight loss, disease activity index, colon length shortening, reduced histological scoring, and increased colonic production of cytokines. Infiltration of neutrophils and inflammatory monocytes in the colonic lamina propria was reduced in HK-deficient mice. Reconstitution of HK-deficient mice through intravenous injection of HK recovered their susceptibility to DSS-induced colitis, increased IL-1β levels in the colon tissue, and bradykinin concentrations in plasma. In contrast to the phenotypes of other mice lacking other proteins involved in the KSS, mice lacking FXII had comparable colonic inflammation to that observed in wild-type mice. The concentration of bradykinin was significantly increased in the plasma of wild-type mice after DSS-induced colitis. In vitro DSS-induced pKal activation, HK cleavage, and bradykinin plasma release were prevented by the absence of pKal or the inhibition of Kal. Unlike DSS, TNBS-induced colitis did not trigger HK cleavage. Collectively, our data strongly suggests that Kal contributes to experimental colitis by promoting bradykinin release from HK.
Enterohepatic Helicobacter species modulate severity of DSS-induced colitis but do not prevent amelioration by indole-3-carbinol
Authors: Alkarkoushi Rasha Raheem , Singh Udai P. , Chatzistamou Ioulia , Bam Marpe , Nagarkatti Prakash S. , Nagarkatti Mitzi , Testerman Traci L.
Published: January 2023

Enterohepatic Helicobacter (EHH) species are bacteria that colonize the colons and biliary ducts of humans and other mammals. Virtually nothing is known about the mechanisms of EHH-induced colitis in wild-type mice or humans. Indole-3-carbinol (I3C), a chemical extracted from cruciferous vegetables, ameliorates Dextran sulfate sodium (DSS)-induced colitis. We hypothesized that specific EHH species would alter the course of DSS-induced colitis and possibly the responses to I3C treatment. We infected C57BL/6 mice with human- and rodent-associated EHH species and measured the effects on DSS-induced colitis and response to I3C treatment. We found that H. muridarum(H.m) and H. cinaedi exacerbate DSS-induced colitis and delay recovery. Furthermore, I3C ameliorated colitis and shifted the Treg/Th17 balance in H.m-infected mice. Moreover, the microRNAs expression pattern was altered in H.m-infected mice when compared to that of uninfected mice. Interestingly, enhanced colitis, as well as increased Th17 cells in spleens and mesenteric lymph nodes, were observed in H.m-infected DSS-treated mice(DHM). I3C treatment of DSS + H.m-infected mice (DMI) decreased the expression of pro-inflammatory IL17 and RORC as well as increased anti-inflammatory Foxp3 when compared to DHM group. These immunological changes correlated with the microRNAs expression. This demonstrates that the presence of EHH species alters susceptibility to DSS-induced colitis. Importantly, mice with exacerbated colitis respond to I3C. In the future, we plan to study the mechanisms underlying the effects of EHH on colon cancer development and the efficacy of I3C in both the colitis and colon cancer models.
Increased levels of Th1-inducing cytokines are detected in cervical secretions of women with Chlamydia trachomatis infection limited to their cervix
Authors: Lee De’Ashia E. , Zhong WuJuan , Dong Li , Russell Ali Russell , O’Connell Catherine , Wiesenfeld Harold , Hillier Sharon , Sempowski Gregory D. , Zheng Xiaojing , Darville Toni
Published: January 2023

Background

Chlamydia trachomatis (CT) is the leading bacterial sexually transmitted infection worldwide. CT can ascend to the upper reproductive tract in women, and cause tissue damage resulting in reproductive sequalae. Data from animal and human studies indicate a role for the Th1 cytokine, IFNγ, in protection from infection and disease.

Methods

We recruited 246 women at high risk for CT infection. Cervical swabs and endometrial biopsies were collected for detection of sexually transmitted pathogens, including CT, by nucleic acid amplification test (NAAT). Cervical secretions collected using cervical sponges were screened for 57 cytokines using multiplex bead arrays. Data were analyzed by logistic regression.

Results

92 (37%) women were NAAT+ for CT in only their cervix and 69 (28%) women had CT infection detected at both their cervix and endometrium. Compared to women with endometrial infection, women with only cervical infection had higher levels of CX3CL1 (p<0.05), a potent chemoattractant of T cells and monocytes; CXCL14 (p<0.01), a chemoattractant and activator of dendritic cells, monocytes, and NK cells; and the Th1 inducing cytokine IL-12p70 (p=0.07). Conversely, type I IFN-inducible proteins CXCL9 (p<0.05) and CXCL10 (p<0.01) were significantly increased in women with endometrial infection, compared to women with cervical infection only.

Conclusions

Th1-associated cytokines in cervical secretions are associated with limitation of infection to the cervix, whereas type I IFN-associated chemokines are associated with endometrial CT infection. These proteins may be used as biomarkers to identify women at increased risk for upper reproductive tract pathology and those who could benefit from enhanced screening.
Intestine-specific CCR10+ plasma cells regulate migration of intestinal regulatory T cells
Authors: Zhao Luming , Hu Shaomin , Davila Micha L. , Xiong Na
Published: January 2023

In intestines, B cells are activated in gut-associated lymphoid tissue (GALT) to differentiate predominantly to IgA antibody-secreting cells (IgA-ASCs), which acquire a CCR10+ specific gut-homing property and migrate into intestinal lamina propria where they secrete IgA antibodies specifically acting on commensal bacteria and food-borne antigens for homeostatic regulation. While IgA antibodies were extensively investigated, differentiation of B cells and their functional mechanisms in establishment of intestinal homeostasis are still incompletely understood. We herein report a novel process in which CCR10+ differentiated B cells regulate intestinal homeostasis. We found that CCR10+ IgA-ASCs preferentially interact with activated regulatory T (Treg) cells in GALT and lamina propria to help their localization in colons. In CCR10-knockout mice, defective intestinal migration of IgA-ASCs resulted in reduced Treg cells and spontaneous development of inflammatory symptoms in colons. In addition, in IgA-deficient mice, there was preferential compensatory generation of CCR10+ IgG-ASCs that substituted CCR10+ IgA-ASCs to promote T cell homeostasis and regulate commensal bacteria in colons. The preferential compensatory generation of CCR10+ IgG-ASCs was also observed in IgA-deficient patients. These findings aid our understanding of B cell differentiation and functional mechanisms in intestinal homeostatic regulation.