bluuebunny/crossref_metadata_2025 huggingface dataset:
Functions and mechanisms of chondroitin disaccharide-induced miRNAs in the regulation of CD44 expression involved in the development of experimental autoimmune encephalomyelitis
Authors: Zhou Juhua , Ma Xinting , Li Yanmin , Zhong Yin , Nagarkatti Prakash S , Nagarkatti Mitzi
Published: January 2023

Multiple sclerosis (MS) is a chronic demyelinating neurodegenerative disease of the central nervous system (CNS). In this study, we investigated the effects of chondroitin disaccharide and its induced miR-29b and miR-199b on CD44 expression in mouse lymphocytes, as well as the effects of miR-29b and miR-199b on the development and progression of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model of MS. The effects of chondroitin sulfate disaccharide on the changes in cellular immunity were investigated in EAE induced in mice by MOG and PTX. Real-time PCR and flow cytometry were used to investigate the effects of the anti-miRs and mimics of miR-29b and miR-199b on CD44 expression in mouse splenocytes after electroporation. After the transduction of antai-miRs and mimics of miR-29b and miR-199b into splenocytes, they were adoptively transferred into mice intravenously and the changes in immune cells and cytokines were analyzed by flow cytometry and ELISA. Our studies revealed that chondroitin disaccharide significantly up-regulated several specific miRNAs such as miR-29b and miR-199b, and thus dramatically inhibited EAE in mice. In vitro and in vivo studies demonstrated that miR-29b and miR-199b could down-regulate the expression of CD44 and modulate the expression of IL-17 and IL-4 in the lymphocytes from EAE mice. Therefore, miR-29b and miR-199b could inhibit the expression of CD44, modulate the expression of IL-4 and IL-17, and thus suppress lymphocyte proliferation and migration, leading to significant inhibition of EAE development. Thus, miR-29b and miR-199b could also be used as therapeutic targets for EAE and MS.
Induced lymphatic circulation decreases IL-17 expression in the lymph nodes of rats in adjuvant-induced arthritis
Authors: Volin Michael V , Zanotti Brian , Hager Denver , Lai LinhChi , Gober Calley , Pulpulaan Paul Anthony , Incrocci Ryan , Swanson-Mungerson Michelle Anne
Published: January 2023

Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic inflammation mediated by Th-17 cells. Rat adjuvant-induced arthritis (AIA) is a Th-17-dependent RA model with dysfunctional lymphatic circulation. We hypothesized that induction of lymphatic circulation would attenuate arthritic inflammation. We utilized Lymphatic Pump Treatment (LPT), an osteopathic lymphatic manipulation, to determine if inducing lymphatic flow would alleviate arthritic inflammation. LPT was accomplished on AIA rats by gradually increasing compression on the chest wall for 5 seconds followed by a rapid release. This technique was performed on the rats for 1 minute 3X/day over 7 consecutive days following the onset of measurable inflammation on day 14 post adjuvant injection. Ankle caliper measurements, articular index scoring, popliteal lymph nodes, peripheral blood, and spleens were collected. The LPT-treated rats fell into two groups. LPT responders and non-responders. LPT responders had significantly reduced inflammation (as determined by both in ankle circumference and index scoring). These therapeutically treated animals also had significantly decreased IL-17 expression in their draining popliteal lymph nodes. These results along with our previous findings indicate that LPT can reduce arthritis, alter lymphocyte trafficking and differentiation in secondary lymphoid organs during inflammation. These data suggest that induced lymphatic circulation using osteopathic manipulation may serve as a therapy for chronic inflammation in autoimmune disorders and may also be an enhancement to current treatments.
Androgens may mediate protection through an effect on immunoregulation in lupus-prone mice
Authors: Ma Jing , Harder James W , Alard Pascale , Kosiewicz Michele M
Published: January 2023

Lupus is much more prevalent in females. Depleting androgens via castration increases disease and mortality in male NZBxNZWF1 (BWF1) mice, suggesting androgens are protective. We have found that treatment of male BWF1 mice with CD103-neutralizing antibody induces disease and increases mortality suggesting CD103+ cells are important for disease protection in males. Mesenteric lymph node (MLN) CD103+ dendritic cells (CD103DC) from castrated male mice exhibit decreased ability to induce conversion of CD4+Foxp3+ cells (Tregs) in vitro and decreased expression of RALDH2, an enzyme involved in retinoic acid synthesis (hallmark of CD103DC function) vs control mice. Also, castrated BWF1 male mice have significantly decreased circulating and peripheral LN Tregs vs control mice, suggesting androgens may affect Tregs via an effect on CD103DC. We have found CD103DC express androgen receptor (AR) mRNA levels that are lower than splenic macrophages (positive controls), but higher than splenic myeloid DC (negative controls) suggesting that androgens could have a direct effect on CD103DC. To determine whether signaling through the AR is required for androgen-mediated protection and CD103DC function, male BWF1 mice were treated with flutamide (AR inhibitor). Male mice treated with flutamide exhibited increased disease incidence and severity, and decreased CD103DC function including a decreased ability to induce conversion of Tregs in vitro and decreased expression of RALDH2 mRNA. Taken together, these data suggest that androgens may influence immunoregulation via a direct effect on CD103DC function and may, at least in part, mediate protection from lupus through this mechanism.
LPS elevates ceramide levels through regulating ORMDL3 expression in macrophages and acute respiratory distress syndrome model
Authors: Ma Jason , Ting Nai-Chun , Kuo Ming-Ling
Published: January 2023

Acute respiratory distress syndrome (ARDS) is a respiratory system failure defined by the acute onset of noncardiogenic pulmonary edema and hypoxemia. After triggered by diverse pulmonary toxicants, macrophages accumulate in lung and generate cytotoxic and proinflammatory mediators that induce pathogenic responses, including cytokines, chemokines, proteases and bioactive lipids. Ceramide is a lipid lies within cell membrane bilayer and functions as a signaling molecule, causing production of inflammatory cytokines and cell apoptosis. The generation of ceramide is controlled by serine palmitoyltransferase (SPT), which forms complex with orosomucoid-like protein 3 (ORMDL3) to regulate ceramide levels. Since the level of ceramide is highly associated with lung injury, we aim to investigate how LPS induces ceramide level. PMA-differentiated THP-1 cells were activated by LPS and the ceramide levels were analyzed by using UPLC-TOF-MS system. Proteins regulating ceramide synthesis and relative transcription factors were further determined. We found that ORMDL expressions were down-regulated and ceramides were increased in THP-1 cells upon LPS activation. ORMDL3 transcription factors, p300 and STAT6, were down-regulated but not Ets-1, CREB nor ubiquitin ligase Cbl-b. We established a LPS-induced B6 model for ARDS and analyzed BALF cells, cytokine and chemokine levels, and ceramide composition. ORMDL3 is also down-regulated in ARDS model, which may result in the increase of ceramide levels. Taken together, LPS may increase ceramide levels by regulating ORMDL3 expression through p300-STAT6 axis. These results may provide new therapeutic strategies for ARDS patients by targeting ORMDL3 to control ceramide levels.
Macrophage-restricted tyrosine phosphatase SHP2 is a novel anti-atherosclerotic regulator
Authors: wu chenxia , Xu Chen , Ma Lan , Mao Wei , Cheng Hongqiang , Ke Yuehai
Published: January 2023

Atherosclerosis is a main pathological basis for cardiovascular diseases. Macrophages, featured in phenotypic and functional plasticity, play critical roles in initiation, progression, and regression of atherosclerosis. Our previously studies have shown that protein tyrosine phosphatase SHP2 regulates macrophage polarization, the immune homeostasis mediated by the interactions between macrophages and epithelial cells in mouse lung and intestine, and a tumor-favorable immune microenvironment. However, a potential role of SHP2 in atherosclerotic macrophages remains unknown. The expression of SHP2 is increased in macrophages within human atherosclerotic plaques. SHP2 allosteric inhibitor and macrophage-specific SHP2 deletion worsened atherosclerosis in ApoE null mice. Total cholesterol and low-density lipoprotein were increased in SHP2 inefficient mice. SHP2 inefficiency promoted inflammatory M1 polarization and inhibited anti-inflammatory M2 polarization, which was ApoE-null-dependent. Less cholesterol accumulation was observed in SHP2 inefficient macrophages and cholesterol transporter CD 36 was downregulated. To further explore the underlying mechanism, RNA sequencing was performed using SHP2 deleted macrophages with or without oxLDL treatment. Taken together, our studies using SHP2 knockout mice and specific inhibitor suggest that SHP2 is a novel anti-atherosclerotic regulator in promoting inflammatory macrophage phenotype.
The effect of N-butyl benzyl phthalate, a common plasticizer, on endometriosis development
Authors: Lee Joyu , Sharma Pooja , Suen Jau-Ling
Published: January 2023

Endometriosis is an estrogen-dependent and chronic inflammatory disease, which retrograded menstrual endometrial cells enter the pelvis and abnormally grow outside the uterine cavity. Severe symptoms such as infertility and chronic pelvic pain are typically observed in 6%–10% of reproductive-aged women. Previous study demonstrates that phthalates are capable to disrupt the endocrine system, especially by mimicking estrogen functions. Among them, n-butyl benzyl phthalate (BBP), a plasticizer for vinyl foams and artificial leather, is able to work as a selective agonist through the binding of ERα, and subsequently interrupt the endocrine regulation. This study aimed to explore the effect of BBP on endometriosis. We established a surgery-induced murine model and exposed the mice at physiologically relevant dose of BBP mimicking human exposure. The result showed that chronic exposure to BBP did not promote the growth of endometrial lesions, however, the lesion survival rate in BBP-treated mice was significantly increased compared to vehicle-treated mice. Our data showed that BBP exposure promoted the infiltration of monocytes (Ly6c+CD11b+F4/80-) and plasmacytoid dendritic cells (pDCs; CD11c+/lowPDCA-1+) in the lesions. In addition, the percentages of CD44+ cells in the infiltrated monocytes and pDCs in the BBP-treated group were significantly higher than those in the control. Also, BBP exposure promoted ICAM-1 expression in CD45− endometriotic lesion cells. The results suggest that BBP exposure may enhance endometrial cell adhesion through altering innate cell subset migration. The detail mechanism of BBP effect on endometriosis development awaits further investigation.
NFkB mediated sustained inflammation promotes hepatobiliary injury in Sickle Cell Disease
Authors: Pradhan-Sundd Tirthadipa , Vats Ravi , Sundd Prithu
Published: January 2023

Hepatic crisis is an emergent complication affecting sickle cell disease (SCD) patients, however, the molecular mechanism of sickle cell hepatobiliary injury remains poorly understood. Using the knock-in humanized mouse model of SCD and SCD patient blood, we sought to mechanistically characterize SCD-associated hepatopathophysiology applying our recently developed quantitative liver intravital imaging, RNA sequence analysis, and biochemical approaches. SCD mice manifested sinusoidal ischemia, progressive hepatomegaly, progressive inflammation, hyperbilirubinemia, and increased ductular reaction under basal condition. NFkB activation in the SCD mice liver inhibited FXR signaling and its downstream targets, leading to loss of canallicular bile transport, sustained inflammation and altered bile acid pool. Intravital imaging revealed impaired bile secretion into the bile canaliculi, which was secondary to loss of canallicular bile transport and bile acid metabolism, leading to intrahepatic bile accumulation in SCD mice liver. These findings are the first to identify that NFkB-FXR dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury and liver inflammation in SCD. Improved understanding of these processes could potentially benefit the development of new therapies to treat sickle cell hepatic crisis.
Longitudinal Immune and Genomic Monitoring Reveals Signatures of Immune-related Adverse Events in Cancer Patients Receiving Checkpoint Inhibitor Therapy
Authors: Khan Shaheen , Moses Angela , Imam Marjaan , Wakeland Edward K , Arana Carlos , Fattah Farjana , Li Quan-Zhen , von Itzstein Mitch , Hsieh David , Popat Vinitha , Barnes Spencer , Xie Yang , Khan Saad , Gerber David
Published: January 2023

Despite the remarkable success of immune checkpoint inhibitor (ICI) therapy, a significant number of patients develop severe and unpredictable immune-related adverse events (irAEs) affecting a wide variety of organs. Concerns over irAE have led to the exclusion of patients with autoimmune disease from ICI clinical trials. Role of host genetic and immune factors in mediating irAEs remain unclear and it is not clear if the manifestations of irAEs is associated with response to therapy. Here, we use multi-faceted approach to characterize changes in host immune system in 200 patients receiving ICI therapy at baseline and post immunotherapy. In addition, we assessed genetic predisposition to autoimmunity using the Illumina SNP array and via targeted resequencing of over 150 immunoregulatory loci including the HLA region. In this meeting, we will present our initial genetic data, serum cytokine and autoantibody profiles and RNA sequencing on peripheral blood mononuclear cells (PBMCs) at baseline and post immunotherapy in patients with and without irAEs. Our preliminary findings suggest that patients who developed irAEs have lower baseline levels and greater post-treatment increases in key interferon gamma inducible cytokine/chemokine levels. Our autoantibody profiling data reveals unique sets of autoantibodies associated with specific irAEs. We will present a comprehensive analysis of immune and genetic correlates of irAEs and response to therapy. We hope our study can help gain insight in to the mechanisms underlying irAE and to identify biomarker signatures predictive of irAEs and/or response. These findings may ultimately help identify high-risk patients, customize therapy, expand use of immunotherapy and prevent toxicities.
Cervical Cancer induces NLRP3 inflammasome activation in human peripheral blood monocytes
Authors: Fernandes Fernanda Pereira , Leal Vinícius Nunes Cordeiro , Pontillo Alessandra
Published: January 2023

Background

Chronic inflammation plays an important role in tumorigenesis of HPV-associated cervical cancer (CC). However the role of inflammasome on CC has not been yet elucidated.

Aim

To investigate the interplay between inflammasome and CC to determine the contribution of inflammasome activation in the progression of CC.

Methods

Inflammasome activation was analyzed in SiHa, Caski, C33 cells lines in monolayer and spheroids cultures representing the CC model. The cell lines was also co-cultured with human monocytes isolated from Healthy donors (HD). Peripheral blood mononuclear cells (PBMC) of patients with CC or precursor lesions (CIN) were also stimulated to evaluate inflammasome activation.

Results

The co-culture of CC cell lines and monocytes induced an increased production of IL-1ß in spheroids model. The culture with the specific NLRP3 inhibitor (MCC-950) showed that the IL-1ß is derived from monocytes through NLRP3 receptor activation. These results indicated that CC is able to activate NLRP3 inflammasome and IL-1ß production in monocytes, however how it happens it still remains unclear. CC conditioned medium was not able to activate monocytes suggesting that possibly a physical interaction between cancer cells and monocytes is needed. Moreover whether IL-1ß production is beneficial or not for the tumor control is still debated. IL-1ß production is higher in LPS-induced CC PBMC than in CIN PBMC.

Conclusion

Our finds suggests that CC is able to activate NLRP3 inflammasome and IL-1ß production in monocytes, and IL-1ß production is higher in patients with CC.
Noncanonical NF-κB signaling is suppressed through the negative regulation of the essential kinase NIK and attenuation is associated with eosinophilic esophagitis pathogenesis
Authors: Allen Irving C , Morrison Holly , Eden Kristin , Brown Stephan , Hart Michael , McGowan Emily
Published: January 2023

Eosinophilic esophagitis (EoE) is an allergic disease of the esophagus driven by T cell and eosinophil responses to dietary allergens, resulting in chronic mucosal inflammation. NF-κB signaling is a master regulator of gene transcription and drives diverse biological processes of relevance to EoE. NF-κB activation occurs through either canonical or noncanonical pathways. In general, there is a relative paucity of knowledge regarding noncanonical NF-κB regulation and signaling. Previously, we have shown that mice with hyperactive noncanonical NF-κB signaling are more susceptible to Th1/Th17 mediated experimental colitis and cancer. Conversely, mice with attenuated noncanonical NF-κB signaling spontaneously develop EoE. The objective of our current study is to translate these findings from mouse models to human patients. Clinical specimens were collected from a cohort of 16 EoE patients and representative controls. Gene expression and protein analysis revealed a significant decrease in p52, RelB, and chemokines specifically associated with noncanonical NF-κB signaling correlated with EoE. However, we also identified a significant change in several mediators that should increase noncanonical NF-κB signaling upstream of the essential kinase NIK. Thus, our data suggest a disconnect between pathway activation and p52/RelB mediated gene transcription, with NIK functioning as a key regulatory target. Further analysis identified 6 regulatory proteins that target NIK in EoE patient specimens and robustly attenuate downstream noncanonical NF-κB signaling. Together, these data demonstrate the significance of this understudied pathway in human EoE and identify potential targets for future therapeutic strategies.
Absence of SIRPα signaling drives TLR9-induced hemophagocytic lymphohistiocytosis-like disease independent of IFN-γ in mice
Authors: Kidder Koby , Bian Zhen , Shi Lei , Liu Yuan
Published: January 2023

The non-physiological phenomenon whereby macrophages phagocytose healthy host cells is a hallmark of severe inflammatory disorders such as hemophagocytic lymphohistiocytosis (HLH). The mechanisms underlying the nascence of hemophagocytic macrophages, however, remain largely unknown. Under healthy conditions, macrophages are non-hemophagocytic and therefore do not eat healthy host cells, owing to the inhibitory myeloid receptor SIRPα. However, we previously showed that, unless inflammatory stimulation via TLR agonists or certain pro-inflammatory cytokines is also provided, the mere absence of SIRPα does not drive macrophages to become hemophagocytic. Here we show that hemophagocytic macrophages may arise under inflammatory conditions given an absence of SIRPα signaling and thereby lead to a severe inflammatory state reminiscent of HLH. SIRPα-knockout (Sirpα−/−) mice injected with the TLR9 agonist CpG rapidly developed characteristics of HLH, e.g., hemophagocytosis, anemia, thrombocytopenia, leukopenia, hepatosplenomegaly, hyperferritinemia, hypertriglyceridemia, and hypercytokinemia, with much greater severity than wild-type mice. Interestingly, IFNγ, a putative driver of HLH, was dispensable in the immunopathology of TLR9-driven HLH in Sirpα−/− mice, as IFNγ neutralization did not ameliorate any symptoms of HLH. As with CpG, other TLR agonists (Poly I:C, LPS, Zymosan) or cytokines (TNFα, IL-6, IL-17A) induced hemophagocytic macrophages that incurred severe HLH in Sirpα−/− mice. Together, these findings implicate that a pre-disposing loss of SIRPα signaling under inflammatory conditions may give rise to hemophagocytic macrophages that drive an HLH-like disease independent of IFNγ.
Repeated exposures to the environmental allergen methylisothiazolinone reduce viability of fibroblasts derived from allergic sites in response to allergen challenge in vitro
Authors: Chatterjea Devavani , Blum Melissa , Koehler Jennifer
Published: January 2023

Chronic, unexplained vulvar pain or vulvodynia affects 8% of women of reproductive age, and is more common with histories of yeast infections and allergies. We have established mouse models of localized, provoked genital pain driven by repeated labiar and vaginal exposures to allergens such as oxazolone, dinitrofluorobenzene and the common chemical preservative methylisothiazolinone (MI). Pain sensitivity is concomitant with tissue mast cell increases and reduced when mast cells are reduced by chemical degranulation or topical phytocannabinoid treatment. Vulvar fibroblasts from patients show altered inflammatory responses in vitro.to repeated topical application of the allergenic cosmetic preservative methylisothiazolinone (MI) to the murine vaginal canal produces long-lasting contact hypersensitivity. Here, we have optimized methods to grow skin and vaginal canal fibroblasts from outbred Swiss mice that are responsive to lipopolysaccharide and zymosan treatment in vitro. We also show that in vitro MI treatment reduces the viability of derived fibroblasts in a dose-dependent manner, with a more robust effect on cell lines derived from MI-treated skin, indicating that previous exposure to MI in the skin primes fibroblasts to undergo cell death upon future exposures. Our data suggest that fibroblasts may become more sensitive to allergens as a result of previous exposures, which could contribute to the development of chronic allergic and pain sensitivity after repeated low dose chemical exposures.
Microarray analysis of human abdominal aortic aneurysm (AAA) detected altered gene expression profiles including H19
Authors: Lu Song , Li Liping , White John V , Zhang Xiaoying , Nwaneshiudu Ifeyinwa , Ntaoula Nectaria , Lin Wanlu , Solomides Charalambos , Gaughan John , Monos Dimitri , Oleszak Emilia , Platsoucas Chris D
Published: January 2023

AAA is often a life-threatening disease with poorly understood pathogenesis. We examined gene expression profiles between AAA and normal aorta specimens by cDNA microarray analysis. AAA wall specimens from 5 patients with AAA and grossly normal aortic walls from 3 transplant donors were employed in this study. Total RNA was extracted from each specimen, RT into cDNA, and labeled with phosphorus α-32P. cDNA probes were then hybridized onto human cardiovascular microarray and analyzed by densitometry. Quantitative differences of some genes were further explored by SYBR green-mediated qRT-PCR analysis. Pairwise comparisons of both microarray and qRT-PCR results were made by Student t-test. Most of the 588 genes studied had similar expression among AAA specimens and control aortas, however, 25 genes were upregulated and 26 downregulated. Expression of MMP-9, TIMP3, collagen 1A1 (COL1A1), COL6A3, COL15A1, decorin, MCP 1, Apo E, Apo D, H19 and others was increased. Expression of COL4A4, biglycan, caveolin 3, galectin 1, LDL receptor-related protein 1, extracellular SOD 3, ANP receptor A precursor and others was decreased. Most qRT-PCR results agreed well with those from microarray analysis, with few exceptions. A number of genes that have been previously postulated to participate in AAA pathogenesis, have been upregulated or their expression has been altered. We also revealed some genes that have not been yet studied in human AAA. Of note, H19, one of the most highly up-regulated transcripts in two mouse aneurysm models, was also significantly upregulated in human AAA. These results indicate that many genes involved in different cellular functions might contribute to the pathogenesis of AAA.

Supported in part by grant RO1 HL64340 from NIH.
Investigating the role of B cell signaling in atherosclerosis
Authors: Ma Shelby , Moriarty Alina , Keeter W. Coles , Waseem Tayab , Gauronskas Phillip Joseph , Galkina Elena
Published: January 2023

INTRODUCTION

Atherosclerosis is an inflammatory disease of the large and medium size arteries. While specific antigens are not well characterized, strong data demonstrate that the immune response is involved in atherogenesis. The role of B cells in atherosclerosis is B cell specific: B2 cells are proatherogenic and B1 and MZ B cells serve as protective. B cell activation is dependent on B cell receptor signaling along with a secondary signal, such as CD45. To date, it is unknown how CD45 signaling regulates activation and functions of B cells in atherosclerosis.

METHODS

We took advantage of a previously characterized transgenic mice that express low levels of CD45 (CD45L/L). We performed adoptive transfer of CD45L/L or WT B cells into B cell-deficient mice injected with AAV-PSCK-9 to induce atherosclerosis. After 16 wks of a western diet feeding aortas were collected and stained with Oil Red O. Spleens from the CD45L/L and WT recipients were analyzed by FACS and isolated splenic B cells were tested in BCR-induced Ca2+ flux, proliferation, and survival assays.

RESULTS and CONCLUSIONS

Our preliminary data demonstrate that low CD45 expression by B cells increases atherosclerosis in the CD45L/L vs WT recipients. Unexpectedly, elevated aortic plaque burden was accompanied by decrease in pathological Tfh cells and a significant elevation in protective B1 B cells in the CD45L/L compared to WT recipients. Future analysis will determine levels of proliferation and activation of B cells in both groups. These results suggest that CD45-dependent activation of B cells impacts the distribution of B cell subsets and highlight a potential role of BCR-related signaling in the regulation of immunity in atherosclerosis.
Defining mechanisms of semaphorin 4A stimulatory action on human Treg cells
Authors: Chapoval Svetlana P , Qi Xiulan , Neuwald Andrew F , Keegan Achsah Dorsey
Published: January 2023

We had shown previously that semaphorin 4A (Sema4A) suppresses allergic inflammation in mice (Mucosal Immunol., 2012, 5:409-19), regulates human Treg cell stability, and induces the generation of Treg cells in PBMC cultures acting through its Plexin B1 receptor (Immunohorizons, 2019, 3:78–87). Plexin B1 forms functional homodimers or heterodimers with other Plexin B molecules and co-precipitates with Neuropilins (NRPs) in transfected 293T cells. Furthermore, NRPs were shown to stabilize Sema/Plexin interactions. In this study, we sought to identify potential partner(s) for Plexin B1 in mediating Sema4A action. We examined surface expression of Plexin B2, B3, and NRPs 1, 2 in human Treg cells by flow cytometry, and observed that they express NRP-2 but not NRP-1, Plexin B2, or B3. Moreover, culture of PBMC with recombinant human Sema4A-Fc (rhSema4A) in vitro resulted in the downregulation of NRP-2 expression on human Treg cells suggesting a specific ligand-receptor interaction. We propose that Sema4A action on human Treg cells may be mediated by a Plexin B1-NRP-2 complex. Our ongoing research is focused on cryoEM studies of ligand-receptor and receptor-receptor interactions in conjunction with sequence/structure statistical analyses using the DARC (Deep Analysis of Residue Constraints) program (Scientific Reports, in press) with the goal of developing Sema4A-based therapies for allergic disease.
IQ motif-containing GTPase-activating protein 1 is essential for the optimal maintenance of lung ILC2s
Authors: Tayama Shunichi , OKUYAMA YUKO , PHUNG HAI THE , ASAO ATSUKO , KOBAYASHI SHUHEI , KAWABE TAKESHI , TAKANORI SO , ISII NAOTO
Published: January 2023

Group 2 innate lymphoid cells (ILC2s) play critical roles in type 2 immunity and are crucial for pathogenesis of various types of inflammatory disease. IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a ubiquitously expressed scaffold protein that is involved in multiple cellular functions such as cell survival and trafficking. While the roles for IQGAP1 in T and B lymphocytes have been uncovered, the physiological significance of IQGAP1 in innate lymphocytes remains to be elucidated. In the current study, we demonstrate that using bone marrow chimeras, the deficiency of IQGAP1 caused an impaired survival of lung ILC2s in a cell-intrinsic manner and that Iqgap1−/− mice displayed decreased accumulation of ILC2s after administration of papain and thereby reduced pathology of the disease. Moreover, Iqgap1−/− ILC2s showed a significantly enhanced apoptosis as compared to wild-type ILC2s under both steady-state and inflammatory conditions. Together these results identify for the first time that IQGAP1 is essential for homeostasis of ILC2s in the lung.
The Role of Tyrosine Kinase Itk in T helper 17 and T regulatory cells in Hypersensitivity Pneumonitis
Authors: Elmore Jessica , Carter Chavez , Koylass Nicholas , Bennett Amelia , Mead Max , Kim Alexia , Huang Weishan , August Avery
Published: January 2023

The balance of inflammatory and suppressive cytokines is critical in controlling inflammatory responses, and the pro- and anti-inflammatory cytokines IL17A and IL10 has been implicated in numerous pulmonary inflammatory diseases. The tyrosine kinase, Itk, plays a critical role in T cell activation. Itk is required for the development of Th17 cells and their production of IL17A in allergic lung inflammation. Furthermore, Type I regulatory and Foxp3+ T regulatory (Tregs) cells, producers of IL10, are positively and negatively regulated by Itk respectively. Farmer’s lung, a subset of hypersensitivity pneumonitis, develops due to repeated exposure to the bacteria Saccharopolyspora rectivirgula (SR) and is dependent on IL17A and regulated by IL10. Surprisingly, exposure to SR drives robust CD4+ T cell IL17A response even in the absence of Itk, with pulmonary inflammation. Transcriptomic analysis of sort purified WT and Itk−/− IL17A producing CD4+ T cells from SR-exposed mice revealed an enrichment of Notch signaling pathway in the absence of Itk. SR also induced the Itk independent development of a population of IL17A producing Foxp3+ Tregs cells, and a significant decrease in IL10 producing Tr1 cells. These data suggest that Itk regulates the expression of IL10, and pathogenic Th17 cells via Notch signaling. These studies suggest that TCR signaling through Itk differentially regulates the development of inflammatory Th17 cells and suppressive Tregs and Tr1 cells in response to SR exposure. Understanding how Itk modulates the development of Th17/Treg cytokine responses will allow us to better understand the precise role of Itk in the regulating the balance of pro- and anti-inflammatory cytokine production during airway inflammation.
Potential role of thioredoxin-interacting protein in non-IgE mast cell degranulation
Authors: Mendoza Ryan P , Anderson Colin C , Roede James R , Brown Jared M
Published: January 2023

Non-IgE mediated pathways of mast cell degranulation result in similar pathological outcomes as IgE mediated degranulation without prior IgE sensitization thereby making it difficult to identify causative agents and develop therapeutics for patients. We have demonstrated that 20 nm silver nanoparticles (AgNP) induce non-IgE mast cell degranulation through an unknown mechanism. However, we have established that this non-IgE response is strain dependent with C57BL/6 bone marrow derived mast cells (BMMCs) being high responders and LP/J BMMCs being low responders. Further, RNA sequencing of high-responder and low-responder BMMCs demonstrated a significant increase in thioredoxin-interacting protein (txnip) in the low-responders after AgNP mediated degranulation. We explored this protein to determine its regulatory potential in non-IgE mast cell degranulation. At 1hr following exposure to AgNP, txnip mRNA levels increased in LP/J but not C57BL/6 which confirmed RNAseq results. Txnip protein levels were similarly decreased in BMMCs from both strains at 1hr; however, there was an increase in the low-responder BMMCs at six hours post-exposure that did not occur in the high-responder BMMCs. siRNA knockdown of txnip resulted in increased BMMC degranulation by AgNP. Txnip is strongly involved in cellular metabolism. Seahorse XfP results demonstrated a lower energy and glycolytically focused phenotype in low-responders compared to high-responders. C57BL/6 have significantly lowered glycolytic capacity after AgNP-mediated degranulation not present in LP/J. Our data suggests that txnip plays a role in non-IgE mediated mast cell degranulation and may modulate cellular metabolism.
M2-like, dermal macrophages are maintained via IL-4/CCL24 mediated cooperative interaction with eosinophils in cutaneous leishmaniasis
Authors: Lee Sang Hun , Chaves Mariana M. , Kamenyeva Olena , Gazzinelli-Guimaraes Pedro H. , Kang Byung Hyun , Pessenda Gabriela , Kaiuska Passelli , Tacchini-Cottier Fabienne M , Kabat Juraj , Jacobsen Elizabeth A , Nutman Thomas B , Sacks David
Published: January 2023

Tissue-resident macrophages (TRMs) maintain tissue homeostasis, but they can also provide a replicative niche for intracellular pathogens such as Leishmania. How dermal TRMs proliferate and maintain their M2 properties even in the strong TH1 environment of the L. major infected dermis is not clear. Here, we show that in infected mice lacking IL-4/IL-13 from eosinophils, dermal TRMs shifted to a pro-inflammatory state, their numbers declined, and disease was attenuated. Intravital microscopy revealed a rapid infiltration of eosinophils followed by their tight interaction with dermal TRMs. IL-4-stimulated dermal TRMs, in concert with IL-10, produced a large amount of CCL24 which functioned to amplify eosinophil influx and their interaction with dermal TRMs. An intraperitoneal helminth infection model also demonstrated a requirement for eosinophil-derived IL-4 to maintain tissue macrophages through a CCL24-mediated amplification loop. CCL24 secretion was confined to resident macrophages in other tissues, implicating eosinophil-TRM cooperative interactions in diverse inflammatory settings.
Suppression of Inflammation of the Cytokine Storm Following Francisella Tularensis Infection is Mediated by Natural Killer T Cell Subsets
Authors: Setzu Nicole R , Spencer Charles T
Published: January 2023

Francisella tularensis is intracellular bacteria which is the causative agent of the disease tularemia. Highly virulent in both humans and animals, it takes only as few as ten microorganisms to cause a lethal infection. The bacteria can enter via direct or indirect routes causing the activations of the host innate inflammatory response to ensue. The function of the bacteria once its entered the host cell is to be opsonized by phagocytic cell, survive and continue to proliferate in the host causing pathogenicity.

The bacteria invade host dendritic cells, neutrophils, and predominately macrophages. This causes a mass inflammatory response resulting in the cytokine storm. Activation of natural killer T cells has been shown to suppress inflammation in in vitro functional assay studies. We know that select populations of NKT cells influence the cytokine storm at the onset of infection. Our hypothesis seeks to determine whether type one versus type two natural killer T cells are inhibiting inflammation. Based on our preliminary results we hypothesize that the type one NKT cells will show cell suppression.
CONTRIBUTIONS OF FEVER TO THE PROMOTION OF CELLULAR ANTIMICROBIAL DEFENSES
Authors: Barreda Daniel R
Published: January 2023

Thermal changes impact molecular and cellular immune responses. Herein we take advantage of evolutionarily conserved mechanisms for the control of fever to define its contributions to host immune antimicrobial defenses. Our work demonstrates that discrete thermoregulatory programs provide a broader range of immunological benefits than previously anticipated. Complementing prior findings on the modulation of immune gene expression, high resolution quantitative monitoring of cell function showed predictable changes to leukocyte recruitment and pathogen killing capacity. Assessment of kinetics of leukocyte infiltration to a challenge site and changes to the distribution of cellular subsets showed changes in the efficiency of the teleost acute inflammatory response. Ex vivo characterization of cell function as well as in vivo evaluation of host-pathogen interactions pointed to enhancements in immune defenses and pathogen clearance. These have obvious positive implications for health, setting the stage for valuable applications.
Activation-induced cell protrusions and vesicle secretion of human lung macrophages revealed by super-resolution microscopy
Authors: Dechantsreiter Susanne , Ambrose Ashley , Hessel Edith M , Beinke Soren , Tannahill Gillian , Davis Daniel M
Published: January 2023

Recent studies using super-resolution microscopy have established that the organisation of immune cell receptors impacts signal integration and cellular activation. Understanding the nano-scale dynamics of surface receptors on tissue specific macrophages is especially important, as they are phenotypically diverse and possess a large repertoire of receptors. However, tissue macrophages are highly auto-fluorescent, severely limiting the utility of light microscopy. Here, we report a novel correction technique which utilises a moving median filter to remove auto-fluorescent noise from stochastic optical reconstruction microscopy datasets. Using this, we visualised lung macrophages (LMs) activated through Fc receptors by IgG-coated glass slides, representing a 2D model of the phagocytic synapse. This unexpectedly revealed the formation of protrusions, at the surface of LMs but not blood-derived macrophages. Class I MHC protein accumulated at the tips, consistent with a role for macrophage protrusions in antigen presentation. Additionally, staining for the exosome marker CD81 revealed the secretion of extracellular vesicles. Classically, cell-derived vesicles are studied after bulk isolation, which is harsh and comes with many caveats. Imaging them directly upon secretion allows analysis of their properties on a cell-by-cell basis in a near-native state. We discovered that LM vesicles appeared distinct from those secreted from blood-derived macrophages in that their average diameter was much smaller (80 nm ± 19 nm vs. 159 nm ± 78 nm). Thus, our correction method for super-resolution microscopy revealed novel cell biology – protrusion formation and vesicle secretion – triggered upon activation of human LMs.
Temporal transcriptional, metabolic, and functional re-programming of THP-1 macrophages by the Lactobacillus rhamnosus R0011 secretome
Authors: Jeffrey Michael P , MacPherson Chad W , Tompkins Thomas A , Green-Johnson Julia May
Published: January 2023

Macrophage responses to activation are fluid and dynamic and their ability to respond appropriately to subsequent challenge is integral to host defence. Recent evidence suggests that bacteria influence macrophage differentiation and subsequent polarization into pro-inflammatory (M1) and immunoregulatory (M2) phenotypes through direct interactions. However, many questions surround indirect communication mechanisms mediated through secretomes derived from gut bacteria, such as lactobacilli. We examined the effects of secretome-mediated conditioning on macrophage phenotype, focusing on the ability of the Lactobacillus rhamnosus R0011 secretome (LrS) to drive macrophage polarization and to prime responses to subsequent challenge with lipopolysaccharide (LPS). Transcriptional profiling revealed increased M2-associated gene transcription (IL10R, CD36, CD163, TLR1, and TLR8) in response to LrS conditioning. Cytokine and chemokine profiling confirmed these results, indicating increased M2-associated chemokine and cytokine production (IL-10, CCL1, 17, 20, CXCL1 and 2). Metabolite utilization assays indicated diminished reliance on glycolysis for energy generation, coupled with increased phagocytic capacity, characteristics of functional M2 activity. LPS challenge of LrS-conditioned THP-1s revealed heightened responsiveness, indicative of innate immune priming. Overall, the LrS conditions THP-1s into M2 macrophages and primes responses to subsequent LPS challenge. Secretome conditioning of macrophages to respond robustly to inflammatory challenge within an M2 phenotype is an uncharacterized and potentially important route through which lactobacilli can train innate immunity.
Deletion of fetal-specific maternal CD4+ T cells during human pregnancy
Authors: Krupp Katharine , MacDougall Julia , Porrett Paige
Published: January 2023

Maternal T cell responses to fetal alloantigens are thought to play a critical role in facilitating successful pregnancy, but mechanisms of maternal-fetal tolerance remain largely unclear. Studies in murine models suggest that deletion of fetus-specific CD4+ T cells may contribute to tolerance; however, the role of deletion in human pregnancy is unknown. Here we use bulk TCR sequencing to assess changes in the maternal CD4+ T cell repertoire over the course of human pregnancy.

Peripheral blood was collected from four primigravid women during the first and third trimesters, and a single peripheral blood sample was taken from their respective partners. Maternal CD4+ T cells were isolated using FACS, and TCR sequencing was performed on genomic DNA using the ImmunoSeqTM assay. The maternal alloreactive repertoire was defined using mixed lymphocyte reactions (MLRs) in which irradiated paternal PBMCs were used to stimulate maternal blood.

An average of 32,077 (SD=9,609) unique CD4+ T cell clones/subject were identified at each timepoint. Within the small cohort examined in this study, we observed a notable increase in repertoire clonality in two of four women (average change in clonality=0.0045 vs. 0.0003). The two subjects that had an increase in CD4 clonality had a significant reduction in the number of alloreactive CD4 clones detected in the third trimester relative to the first whereas this number was effectively unchanged in the other two subjects (first:third=1:0.1±0.02 vs. 1:1.0±0.1). These findings suggest that substantial deletion of CD4+ alloreactive clones occurs in some women over the course of gestation. Deletion of alloreactive T cell clones may thus represent one mechanism of maternal-fetal tolerance in human pregnancy.
Cyclic GMP-AMP synthase Regulates the Development of experimental autoimmune encephalomyelitis
Authors: Mittal Monisha , Tu Sha , Guo Hui , Zhang Jian
Published: January 2023

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that activates type 1 IFN response via cGAS/cGAMP/STING-dependent pathway to defend against viral and bacterial infections. The role of c-GAS in adaptive immunity is completely unknown. In this study, we attempted to determine whether cGAS also regulates T cell responses using mice deficient for cGAS. We found that cGas−/− mice develop ameliorated experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, which correlates with defective Th1/Th17 responses. This reduced disease activity in cGas−/− mice is unlikely due to the expression cGAS in T cells because Rag1−/− recipients receiving Wild-type and cGas−/− CD4+ T cells develop a comparable degree of EAE. Therefore, our data suggest that cGAS expression in innate immune cells may indirectly regulate pathogenic Th1/Th17 responses during EAE induction.
IRF4 regulates the proliferative capacity of activated B cells during B cell differentiation
Authors: Patterson Dillon , Scharer Christopher D. , Mi Tian , Hicks Sakeenah L. , Zhang Qiang , Boss Jeremy M
Published: January 2023

Cell division is essential for B cell differentiation to a plasma cell (PC). While the cell division coupled changes in the expression of transcription factors that coordinate the PC program have been described, little is known regarding how these factors coordinate the proliferative program. To address this, we exploited an adoptive transfer system and monitored the cell division/differentiation kinetics in response to lipopolysaccharide (LPS). In this system, wildtype (WT) B cells undergo at least 8 cell divisions before differentiating into PC, and the same requirement is also observed following NP-ficoll or influenza immune challenge. Modeling division rates over time identified a proliferative burst between 48 and 60 hours following LPS injection. In contrast, Interferon Regulatory Factor 4-deficient (IRF4−/−) B cells divided but failed to undergo the proliferative burst, stalling at divisions 2–6. To assess the scope of IRF4-dependent reprogramming, WT and IRF4−/− B cells at divisions 0, 1, and 3–6 were sorted for ATAC- and RNA-seq. RNA-seq data revealed hundreds of differentially expressed genes (DEGs) when IRF4 was deleted, a subset of which included MYC target genes. Indeed, IRF4−/− cells failed to upregulate MYC compared to WT cells and consequently, we observed little increase in cell size following immune challenge. Furthermore, IRF4−/− B cells exhibited aberrant cell cycle distribution when compared to WT cells at the same timepoint and division. ATAC-seq data also exposed hundreds of differentially accessible regions, the majority of which contained a known IRF4 binding motif and mapped to a corresponding DEG. Together, these data reveal a critical role for IRF4 in maintaining the proliferative response.
SHARPIN constrains IL-21-induced apoptosis in CD40-activated B cells to mediate the germinal center reaction and T-dependent antibody response
Authors: wang jingwei , Wu Shuai , Kaiser William , Yan Hui , Xu Zhenming
Published: January 2023

SHARPIN is an obligatory component of the fully functional LUBAC complex, which is implicated in the antibody response. Here, we have addressed the B cell-intrinsic function of SHARPIN by generating mice in which only B cells expressed non-functional SHARPIN, as encoded by the missense Cpdm mutation. These mice were normal in B cell development and homeostatic survival, but severely impaired in mounting specific IgM and IgG responses to the T-dependent antigen NP-CGG, concomitant with reduced generation of class-switched B cells and plasma cells, likely resulting from the virtual absence of germinal centers. Consistent with their failure in the germinal center development in vivo, as dependent on CD154 and IL-21 produced by T follicular helper cells, Sharpin mutant B cells activated by CD40 engagement in vitro displayed normal proliferation, but significantly elevated apoptosis upon stimulation with IL-21. This cytokine, while critical for CD40-induced plasma cell differentiation, up-regulated expression of pro-apoptotic Bim and downregulated anti-apoptotic Bcl-2 and Bcl2l1, thereby activating the mitochondria-dependent intrinsic apoptosis pathway. Indeed, Sharpin mutant B cells, upon stimulation by CD154 and IL-21, expressed the apoptosis gene signature at a high level and showed increased Caspase 3 activation. These, together the rescue of the survival of Sharpin mutant B cells and their production of class-switched IgG by apoptosis inhibitor Z-VAD, show that SHARPIN plays an important role in protecting germinal center B cells from apoptosis, thereby mediating the antibody response.