bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of platelet signature on monocytes isolated from HIV infected individuals
Authors: Suwunnakorn Sumanun , Singh Meera V. , Singh Vir B. , Palshikar Mukta , Thakar Juilee , Weber Emily A. , Maggirwar Sanjay B.
Published: January 2023

Background

Higher levels of platelet-monocyte complexes (PMCs) are detected in human immunodeficiency virus (HIV)-infected individuals. Such complexes are implicated in expansion of monocytes exhibiting inflammatory phenotype, and their translocation into tissues, thus promoting development of cerebrovascular and cardiovascular disorders. Here we have recruited small sub-set of HIV-infected individuals to investigate how platelets cause epigenetic changes in monocytes of HIV-infected individuals. We posit that the formation of PMCs facilitates monocyte differentiation via exchange of biomaterial between activated platelets and monocytes during HIV-infection.

Methods

We performed ATAC-seq to measure changes in chromatin accessibility from two types of monocytes, PMC-derived or non-PMC-derived, isolated from HIV-infected persons.

Results

Using ATAC-Seq data, we were able to detect distinct changes in chromatin accessibility patterns within the genomic regions of proinflammatory mediators in PMC-derived monocytes. Chromatin accessibility of inflammatory genes was dramatically increased in PMC-derived monocytes. This suggested to us a possibility of active exchange of molecules that causes such alterations in monocytes.

Conclusions

Increases in chromatin accessibility of pro-inflammatory genes occur in the PMC-derived monocytes in persons with HIV. This change is likely due to the transfer of platelet antigens to monocytes via cell-cell interaction, suggesting a novel mechanism through which platelets trigger inflammatory response in monocytes. Thus, our initial findings pave a way that has a strong diagnostic as well as therapeutic value.
Viral vaccines improve PD-1 therapy against cancer
Authors: Penaloza-MacMaster Pablo A , Chung Young Rock , Dangi Tanushree , Penaloza-MacMaster Pablo
Published: January 2023

Programmed cell death-1 (PD-1) therapy is effective against various types of cancer. However, only 30% of cancers respond to PD-1 therapy, highlighting the need to further improve its efficacy. In this study, we interrogated whether one could exploit the immunostimulatory properties of viral vaccines to enhance PD-1 therapy in a B16 melanoma model that is poorly responsive to PD-1 therapy. Immunization of tumor-bearing mice with clinically approved viral vaccines, such as the smallpox virus vaccine and the yellow fever virus vaccine (YFV-17D), resulted in an unprecedented improvement of PD-1 therapy. This synergistic effect on PD-1 therapy was more potent upon a secondary immunization, suggesting a positive effect of recruiting pre-existent immune memory to the tumor microenvironment. Interestingly, co-administration of viral vaccines together with PD-1 therapy did not increase tumor-specific CD8 T cell responses relative to PD-1 therapy alone. Mechanistically, the adjuvant effect of viral vaccines on PD-1 therapy seems to be dependent on innate immune responses. These findings may be important for improving the efficacy of checkpoint-based immunotherapies by exploiting the immunogenicity of viral vaccines. Altogether, our data suggest that viral vaccines could be repurposed as immunotherapy adjuvants, and that the beneficial effects of viral vaccines on PD-1 therapy can be further improved by repetitive boosting of bystander immunity.

R21 AI132848-01A1 (NIAID); IRG-15-173-21 (ACS), and the Lurie Cancer Center.
STAT1-dependent tolerance of intestinal viral infection
Authors: Sharon Andrew J , Filyk Heather A , Fonseca Nicolette M , Simister Rachel L , Yuen Wallace , Hardman Blair K , Robinson Hannah G , Seo Jung Hee , Rocha-Pereira Joana , Welch Ian , Neyts Johan , Crowe Sean A , Osborne Lisa C
Published: January 2023

The diverse commensal ecosystem present in the mammalian intestine requires the host immune system to maintain a tolerogenic environment. The virome is an understudied component of the microbiota which promotes intestinal homeostasis and protection from injury, but the host mechanisms that regulate tolerance to viral commensals are poorly described. The antiviral signal transducer STAT1 has previously been shown to mediate tolerance to systemic viral pathogens by suppressing adaptive immune responses and protecting the host from immunopathology, but we sought to characterize its role in the context of a commensal intestinal viral infection.

We used a persistent strain of murine norovirus (MNV CR6) to interrogate host mechanisms of viral tolerance and commensalism, as CR6 has previously been shown to promote intestinal homeostasis. While CR6 infections of wildtype mice were asymptomatic and limited to the colon, STAT1-deficient mice exhibited virus-induced weight loss and mortality accompanied by systemic viral spread, colonic bacterial dysbiosis, CD4+ T cell dysfunction and hyperaccumulation of CD8+ T cells. However, clinical manifestation of virus-induced disease in STAT1-deficient mice was independent of T cells and the bacterial microbiota. Instead, therapeutic control of viral replication was sufficient to prevent virus-induced disease despite ongoing T cell dysregulation. Collectively, our data indicate that STAT1 maintains tolerance to a viral component of the intestinal microbiota by control of viral replication rather than immunopathology, suggesting that STAT1 uses distinct strategies to tolerate pathogenic and commensal viruses.
Zika Virus Infects Primary Human Glial Cells and Results in Neuroinflammation and Cell Death
Authors: Sun Jiaren , Liang Yuejin , Hu Haitao , Zhang Biao
Published: January 2023

Zika virus (ZIKV) infection in humans has been linked to serious birth defects, including abnormalities of the brain and eyes. Here, we used two lines of primary human glial cells from the brain and found both are susceptible to ZIKV infection. Viral RNA abundance increased from 6 to 36 h post-infection (hpi). Both cells showed cytopathic effects with cell shedding, suspension and death at 48 hpi. We found increased mRNA levels of IL-6, IL-1b, CXCL10 (IP-10), IL-8 and CCL2 in ZIKV-infected microglial cells, as well as those of TNF-a in astrocytes. Elevated levels of CXCL10 protein were also observed in microglial cells at 48 hpi. These results suggested that glial cells may directly contribute to neuroinflammation by releasing pro-inflammatory cytokines. ZIKV infection can also activate ISGs, as evidenced by increased mRNA levels of IFN-b, OAS1, Mx1 and IFITM1 in infected microglia at 48 hpi. In addition, ZIKV can induce levels of caspase-1 and caspase-3 expression in microglia and astrocytes, respectively. These findings suggest that activation of glial cells contributes to neuroinflammation and induces distinct cell death pathways in different glial cells. Finally, we examined the antiviral effect of azithromycin in infected glial cells and found a dose-dependent effect (0, 10 and 20 uM) on viral reduction. Azithromycin treatment significantly decreased gene expression of TNF-a. Moreover, the antiviral effect of azithromycin was confirmed in mouse models of ZIKV infection. Collectively, our study demonstrated that ZIKV can infect primary human glial cells, leading to inflammatory responses and cell death. Azithromycin treatment inhibits viral load in glial cells and alleviate neuroinflammation during ZIKV infection.
LncRNA HOTAIRM1 promotes the immunosuppressive functions of MDSCs through upregulating HOXA1 expression during chronic HCV infection
Authors: Yao Zhi Q , Thakuri Bal Krishan Chand , Zhang Jinyu
Published: January 2023

HOXA transcript antisense RNA myeloid-specific 1 (HOTAIRM1) is a long non-coding RNA (lncRNA) that is critical for the myeloid cell development by targeting HOXA1. We have previously shown that myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that have immunosuppressive functions. However, the role and mechanisms of HOTAIRM1 in the differentiation and function of MDSCs in the setting of viral infection remain elusive. In this study, we demonstrated that HOTAIRM1 and its target gene HOXA1 were upregulated and closely associated with the expression of immunosuppressive molecules, such as arginase 1 (Arg1), inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS), and signal transducer and activator of transcription 3 (STAT3) in myeloid cells derived from individuals with chronic hepatitis C virus (HCV) infection. We further revealed that HCV-associated exosomes could induce HATAIRM1 and HOXA1 expression via the miRNA-mediated signaling pathway. Importantly, overexpression of HOTAIRM1 or HOXA1 in healthy myeloid cells promoted MDSC differentiation and suppressive functions, evidenced by an increased frequency of MDSCs, expression of immunosuppressive molecules, and inhibition of co-cultured CD4 T cells to produce IFN-γ. Conversely, silencing HOTAIRM1 or HOXA1 expression in HCV myeloid cells partially restored the frequencies of MDSCs, the expressions of their suppressive molecule, and the IFN-γ production in co-cultured T cells. Taken together, this study reveals that HOTAIRM1-HOXA1 enhances the immunosuppressive function of MDSCs and may be a potential target for immunomodulation in conjunction with antiviral therapy during chronic viral infection.
MicroRNA-92a controls the balance between inflammatory and regulatory T cells in CNS autoimmunity
Authors: Fujiwara Mai , Raheja Radhika , Garo Lucien P , Ajay Amrendra K , Saxena Shrishti , Gandhi Roopali , Weiner Howard L , Gopal Murugaiyan
Published: January 2023

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are mediated by dysregulated autoreactive T cell responses in the central nervous system (CNS). This dysregulation consists of an imbalance between inflammatory T helper cells, such as Th17 cells, and Foxp3+ T regulatory cells (Tregs). MicroRNAs (miRNAs), a class of small non-coding RNAs, are known to play a key role in immune function, and to be dysregulated in EAE and MS. However, identifying specific miRNA pathways that directly connect clinical activity with immune mechanisms in CNS autoimmunity has been a challenge. Previous work has identified miR-92a as one of the most significantly elevated miRNAs in the sera of MS patients, which positively correlates with neurological symptoms and brain atrophy. We now report a major functional role for miR-92a in CNS autoimmunity. MiR-92a is increased during EAE, and its loss strikingly attenuates clinical disease. This attenuation is accompanied by reduced Th17 and increased Treg cells in the CNS. Mechanistically, we found that T cell-intrinsic miR-92a inhibits the development and function of Treg cells while promoting those of Th17 cells by targeting Foxo1, a key transcription factor in T helper cell biology. Preclinical administration of miR-92a inhibitor phenocopies miR-92a loss and ameliorates EAE. Analogous to mice, miR-92a is significantly elevated in MS patient T cells, and reciprocally regulates human Treg and Th17 differentiation. These findings suggest miR-92a skews the Treg/Th17 balance to promote CNS autoimmunity, and that miR-92a silencing may be of therapeutic benefit for MS patients.
Elevated levels of pentraxin 3 correlate with neutrophil counts and coronary artery dilation during acute Kawasaki disease, as a predictor of coronary artery lesions
Authors: Ching Lauren Lei , Nerurkar Vivek R , Lim Eunjung , Shohet Ralph V , Melish Marian E , Bratincsak Andras
Published: January 2023

Kawasaki disease (KD) is the leading cause of acquired heart disease in the developed world. 25–30% of untreated patients will develop irreversible coronary artery lesions (CAL). Pentraxin-3 (PTX-3), has been well studied in inflammatory diseases, in particular vascular endothelial dysfunction associated with cardiovascular disease. We hypothesized that PTX-3 played an important role in the development of KD associated CAL, and investigated circulating levels of PTX-3 in KD patients serum. Children with acute KD were followed from diagnosis through normalization of the clinical markers of inflammation. Blood draws and echocardiograms were conducted at several timepoints, acute (prior to IVIG treatment), sub-acute (5–10 days after IVIG treatment), and convalescent (1–4 months after KD diagnosis) phases. In this study we enrolled 70 children with KD, which included 26 (37%) who presented with CAL. Patients included in this study came from a diverse ethnic background. We observed a significant increase in PTX-3 levels during acute KD phase, compared to sub-acute and convalescent phases. PTX-3 levels during acute KD were significantly higher among KD patients with CAL. Further, PTX-3 levels were significantly correlated to coronary artery diameter size during acute KD and neutrophil counts throughout KD progression regardless of coronary artery diameter. We observed PTX-3 levels highly correlated to neutrophil counts, a known source of PTX-3 in acute inflammation and an important player in the development of KD vasculitis. Therefore, we propose PTX-3 as a novel factor associated with acute KD and in the development of KD associated CAL, and neutrophil derived PTX-3 as an important player of KD vascular dysfunction.
Laying Siege to the Castle: The Role of Sepsis Related Cytokines and Inflammation in Bringing Down the Barriers
Authors: Campbell Carmen Elaine , Boisvert William A
Published: January 2023

The endothelium plays a critical role in the progression from sepsis to organ failure. The endothelium lines all blood vessels and organs in the body. As such, it provides a protective barrier against pathologic micro-organisms. The endothelium is also responsible for dynamically regulating the passage of small molecules, fluid, and cells between the bloodstream and tissues. In sepsis, many factors play a role in compromising the endothelium. Dysfunctional endothelium often results in development of capillary leak, tissue hypoperfusion, hypoxia, edema, and shock. This study evaluates the individual contributions of sepsis-related immunomodulatory cytokines as well as a potential synergistic effect of cytokine combinations on vascular endothelial permeability. The Electric Cell-substrate Impedance Sensor was employed to evaluate the barrier function (Rb) and cell membrane capacitance (CMC) of endothelial cell monolayers treated with an array of cytokines including TNF-α IFN-γ, TGF-β, IL1-β, IL-6, IL-8, IL-10, and IL-12. The ECIS results revealed significant differences in the Rb of cell monolayers exposed to sepsis related cytokines compared to cytokine-free control samples. Components of intercellular tight junctions and associated cell adhesion molecules also revealed differences in the endothelial cell monolayer under immunofluorescence microscopy. Thus, cytokines as individual entities or in combinations that may be produced during episodes of sepsis significantly alter the endothelial morphology and permeability.
The role of Interleukin-6 in age-related frailty syndrome
Authors: Jergovic Mladen , Thompson-Ryder Heather L , Asghar Arveen , Nikolich-Zugich Janko
Published: January 2023

Aging is accompanied by chronic low-grade inflammation – a term which relates to increase in serum levels of cytokines such as interleukin 6 (IL-6), TNF-α and acute phase proteins such as C-reactive protein. Multiple studies in humans have shown that IL-6 most reliably increases with aging. Frailty is a geriatric syndrome characterized by decrease in physical ability and increased vulnerability to stressors. One of the most consistent findings in frail subjects is an elevation of serum IL-6 but we lack research which delineates is this a causational relationship. We developed a mouse model with inducible IL-6 expression (IL-6TET-ON/+ mice) that enables overexpression of IL-6 following stimulation with doxycycline (dox) administrated in food. IL-6 induction was dose dependent and led to increase in frailty as measured by 30-point murine clinical frailty index. Mice induced to express high levels of IL-6 quickly displayed an increase in frailty index, decrease in muscle grip strength and loss of fat. Mice induced to express 3–4 fold increase in IL-6 similar to frail humans displayed similar changes after months of induction. We measured IL-6 levels in serum and various tissues (gut, muscle, adipose, spleen) of aged (28-month-old) frail mice and adult controls as well as dox fed IL-6TET-ON/+ mice. We observed that IL-6 levels were increased in serums and spleen homogenates of aged mice and IL-6TET-ON/+ mice but not in other tissues. We determined that neutrophils were the main producers of IL-6 and their numbers were higher in spleens of aged and IL-6TET-ON/+ mice compared to adult controls. We conclude that elevated IL-6 serum levels are directly associated with age-related frailty and that spleen neutrophils are likely the main producers of IL-6.
JAK2V617F expressing macrophages stimulate IL-6 production through PKM1 mediated STAT3 activation
Authors: Ikezoe Takayuki , Li Rongqing , Sun Na , Chen Xin , Yang Jing
Published: January 2023

The JAK2V617F mutation is intimately involved in the pathogenesis of myeloproliferative neoplasms (MPNs) including polycythemia vera and essential thrombocytosis. The thrombotic events are serious complications occurring in individuals with MPNs which affect their prognosis. Interestingly, JAK2V617F is detected in some individuals who developed thrombosis without MPNs and thrombotic risk factors. These observations prompted us to hypothesize that JAK2V617F arising in myeloid cells could contribute to cardiovascular disease by promoting the production of proinflammatory cytokines. To test this, we established JAK2V617F expressing murine macrophages (JAK2V617F macrophages) and explored their function. We found that the levels of p-STAT3, one of downstream targets of JAK2, were markedly elevated in JAK2V617F macrophages in associated with an increase in the production of IL-6 at baseline and following stimulation with LPS, as measured by Western blotting and ELISA, respectively. While, inhibition of STAT3 by C188-9 significantly decreased the production of IL-6 in the culture medium harvested from JAK2V617F macrophages. In further experiments, JAK2V617F endowed macrophages with elevated glycolytic phenotype in parallel with aberrant expression of PKM1. Interestingly, silencing of PKM1 inactivated STAT3. On contrary, ectopic expression of PKM1 activated STAT3. Importantly, JAK2V617F contributed to PKM1 protein stabilization via blockade of lysosomal-dependent degradation via chaperone-mediated autophagy (CMA). All of these observations indicated that JAK2V617F could protect PKM1 from CMA-mediated degradation, leading to activation of STAT3, which promoted IL-6 production.
Nrf2 differentially regulates pro- and anti-inflammatory function of Th17 cells through RORγT, SOD3 and Ahr
Authors: Lin Xun , Tawch Suzanne , Gaudino Stephen , Roy Suyasha , Castillo Patricia , Elsegeiny Waleed Abdel Wahab , Wakabayashi Nobunao , Oury Tim , Pociask Derek , Chen Kong , Syritsyna Olga , Coyle Patricia , McLinskey Nancy , Melville Patricia , Awasthi Amit , Kolls Jay K. , Kumar Pawan
Published: January 2023

IL-17A and IL-22 derived from CD4+ T cells are significant for both inflammatory and tissue protective responses. However, it remains unclear whether IL-17A and IL-22 can be regulated differently to achieve targeted functional outcome.

Here, we showed that Nrf2 selectively regulated IL-17A and IL-22 responses in CD4+ T cells. Nrf2−/− mice challenged with Ovalbumin (Ova) + LPS displayed reduced IL-22 but enhanced IL-17A response. Interestingly, CDDO-Im, a selective Nrf2 activator, induced IL-22 but suppressed IL-17A response in CD4+ T cells polarized under Th17 cell condition. CDDO-Im-activated CD4+ T cells had lower Rorc, but increased Cybb, Sod1 and Sod3 transcript. Luciferase reporter assay showed that IL-17A promoter activity is inhibited by Nrf2 at the presence of RORγT, suggesting that inhibition of IL-17A by Nrf2 is RORγT-dependent. CDDO-Im-impaired IL-17A response was also abolished in SOD3−/−, showing that SOD3 is involved in CDDO-Im-mediated inhibition of IL-17A. Furthermore, we found that CDDO-Im induced aryl hydrocarbon receptor (Ahr) and its downstream Cyp1a1 and Cyp1b1 gene expression. Additionally, CDDO-Im induced Nqo1 expression, an Nrf2-activated gene, in WT mice but not in Ahr−/− mice. CDDO-Im-mediated induction of IL-22 production in CD4+ T cells was abrogated in Ahr−/− mice, supporting that Ahr is involved in the induction of IL-22 by Nrf2. Finally, upon the stimulation of anti-CD3 and anti-CD28, CDDO-Im also mediated the inhibition of IL-17A but induction of IL-22 production in PBMCs from relapsing-remitting multiple sclerosis patients.

Collectively, our data show that Nrf2 promotes IL-22 production via Ahr, whereas RORγT and SOD3 are involved in Nrf2-mediated inhibition of IL-17A expression in Th17 cells.
The role of IL-29 in obesity-induced inflammation and insulin resistance
Authors: HSU YU HSIANG , CHIU CHIAO JUNO , LIN TIAN YU
Published: January 2023

Adipocyte-macrophage crosstalk plays a critical role in regulating adipose tissue microenvironment and causes chronic inflammation in the pathogenesis of obesity. Interleukin-29 (IL-29), a member of type 3 interferon family, plays a role in host defenses against microbes. The macrophage is one of the cellular sources of IL-29; however, the role of IL-29 in metabolic disorders is still unclear. We explored the function of IL-29 in the pathogenesis of obesity-induced inflammation and insulin resistance. We found that serum IL-29 level was significantly higher in obese patients. IL-29 upregulated IL-1β, IL-8, and monocyte chemoattractant protein-1 (MCP-1) expression and decreased glucose uptake and insulin sensitivity in human SGBS adipocytes through reducing glucose transporter 4 (GLUT4) and AKT signals. Inhibition of IL-29 could reduce inflammatory cytokine production in the macrophage-adipocyte co-culture system, which mimics an obese microenvironment. In vivo, IL-29 reduced insulin sensitivity and increased the number of peritoneal macrophages in high-fat diet (HFD)-induced obese mice. IL-29 increased M1/M2 macrophage ratio and enhanced MCP-1 expression in adipose tissues of HFD mice. Therefore, we have identified a critical role of IL-29 in obesity-induced inflammation and insulin resistance, and we conclude that IL-29 may be a novel candidate target for inhibiting obesity-induced inflammation and insulin resistance in patients with metabolic disorders.
A Novel Viral Signaling Complex Containing Host Chemokine Receptor CXCR4, Interleukin-10 Receptor, and Human Cytomegalovirus pUS27
Authors: Spencer Juliet , Tu Carolyn
Published: January 2023

CXCR4 is a chemokine receptor that plays an essential role regulating cell movement during development and immune responses. Human cytomegalovirus (HCMV) is a widespread herpesvirus causing little disease except in immune compromised hosts, especially transplant recipients. HCMV establishes latency in myeloid cells and maintains lifelong infection by manipulating chemokine signaling, especially the CXCR4 pathway. HCMV US27 encodes a viral chemokine receptor (pUS27) that constitutively upregulates host CXCR4 expression, while UL111A encodes cmvIL-10, a homolog of cellular interleukin-10 (IL-10) that binds the IL-10 receptor (IL-10R). Downstream effects of cmvIL-10 include activating Stat3 and enhancing CXCR4 signaling responses to chemokine CXCL12. We used the proximity ligation assay (PLA) to examine the cellular distribution of host CXCR4 and IL-10R, and the results show the two receptors colocalized to discrete clusters on the cell surface. Exposure to CXCL12 caused increased receptor clustering and triggered calcium mobilization. We found that pUS27 was also closely associated with both CXCR4 and IL-10R in the same PLA clusters in transfected HEK293 cells and in HCMV-infected fibroblasts. The addition of CXCL12 and cmvIL-10 resulted in significantly higher receptor clustering, calcium flux, and cell migration of transfected and virus-infected cells. These results indicate that CXCR4, IL-10R, and pUS27 all associate to form a novel virus-host signaling complex that enhances signaling and chemotaxis to CXCL12 during HCMV infection.
CCR4 and CCR7 make distinct contributions to the spatial distribution and negative selection of different thymocyte subsets
Authors: Li Yu , Lancaster Jessica N , Nam Seo Hee , Seo Hyemin , Selden Hilary J. , Ehrlich Lauren Ilyse Richie
Published: January 2023

Central tolerance mechanisms, including clonal deletion and Treg selection, are required to remove autoreactive T cells generated during thymocyte differentiation. To ensure T cell tolerance to diverse self-antigens, the majority of the peptidome is displayed in a sparse mosaic by medullary thymic epithelial cells and dendritic cells in the medulla. Thus, thymocytes must efficiently enter the medulla and scan multiple antigen presenting cells to ensure complete tolerance. We and others have previously reported that the chemokine receptors CCR4 and CCR7 are important regulators of medullary enrichment, rapid motility, and negative selection of post-positive selection thymocytes; however, it is not known whether CCR4 and CCR7 play distinct or redundant roles in central tolerance. Here, we report that early post-positive selection thymocytes undergo chemotaxis towards CCR4 ligands, while progressively mature cells migrate towards CCR7 ligands. Synchronized positive selection experiments reveal that CCR4 is upregulated within hours of receiving a positive selection signal, while CCR7 upregulation is delayed for about 24h. 2-photon microscopy reveals that CCR4 facilitates entry of early post-positive selection cells into the thymic medulla. These findings suggest that CCR4 and CCR7 promote distinct stages of negative selection. Consistent with this possibility, flow cytometric analysis reveals that CCR4 deficiency results in a selective decline in early-stage negative selection, while CCR7 deficiency impairs only late-stage negative selection. Altogether, our data suggest a new model in which CCR4 and CCR7 mediate medullary entry of distinct thymocyte subsets and contribute differentially to central tolerance.
Reduced level of CX3CR1 positive T-cells and monocytes in children with, newly diagnosed, Type 1 diabetes
Authors: Dadfar Elham , Ghaderi Amineh , Moshfegh Ali , Barcenilla Hugo , Casas Rosaura , Samuelsson Ulf
Published: January 2023

Background

The chemokine CX3CL1 is associated with many autoimmune diseases. Type 1 diabetes (T1D) is an autoimmune disease characterized by an insufficient insulin production due to T-cells mediated destruction of pancreatic beta cells. CX3CL1/CX3CR1 plays an important role in recruitment and extravasation of T-cells and monocytes to the inflammatory site.

Aim

To investigate the role of CX3CL1 and its receptor CX3CR1 in children with T1D in acute phase (day 0), 10 days and 3 months after treatment compared to healthy subjects (HS).

Methods

Blood samples from children (range 2–18 years) with new onset type 1 diabetes (day 0 (n=14), day 10 (n=9) and 3 month (n=14)) and 24 age matched HS was collected. The concentrations of CX3CL1 in plasma (T1D day 0, day 10 and HS) was measured by ELISA. Receptor expression of CX3CR1 on monocytes and T-cells was analyzed by flow cytometry.

Results

The study showed significant higher concentrations of CX3CL1 in serum from T1D day 0 compared to HS (p<0.05) and T1D day 10 (p<0.01). No significant difference was observed between T1D at day 10 and HS. Furthermore, the level of CX3CR1 positive T-cells was significantly lower in T-cells from T1D day 0 compared to HS (p<0.003) and T1D 3 months (p<0.007). In addition, the amount CX3CR1 positive monocytes were significant lower in T1D day 0 compared to HS (p<0.003).

Conclusion

The low amount of CX3CR1 positive T-cells and monocytes in peripheral circulation in acute phase T1D, despite the high level of s-CX3CL1, indicate that the T-cells and monocytes might have transmigrated to the inflammatory site. This study indicates that CX3CL1/CX3CR1 axis could play a role in the pathogenesis of T1D.
CRISPR-based identification of factors influencing IL-22 and IL-17 expression in type III innate lymphoid cells
Authors: Brown Rachel Anne , Dangel Andrew W , Saini Ankita , Oltz Eugene M.
Published: January 2023

Cytokine regulation of immune responses is influenced by multiple factors, including the target cell type and its microenvironment. Type III innate lymphoid cells (ILC3s) rapidly produce cytokines, including interleukin (IL)-22 and IL-17, in response to pathogens or injury. Dysregulation of IL-22 or IL-17 contributes to numerous pathologies, including autoimmune diseases and malignancies. Thus, a deeper understanding of the factors that regulate productive expression of IL-17/22 by ILCs or by their type III helper T cell counterparts is warranted.

We have used a genome-wide CRISPR interference screen to elucidate genes that drive IL-22/17 expression by ILC3s. We engineered the murine ILC3-like line, MNK3, to inducibly express catalytically dead Cas9 (dCas9) linked to the transcriptional repressor KRAB. Following lentiviral transduction of a sgRNA library, cells were stimulated with IL-23 and IL-1b. Guides from cell populations sorted by levels of IL-17/22 expression were sequenced and analyzed for depletion (target may be necessary for cytokine expression, e.g., il17, jak3, and il23r) and enrichment (target’s suppression may promote cytokine expression). We are currently validating “hits” that are components of several biological pathways, including transcriptional regulation, chromatin modification, notch/wnt and cadherin signaling, and RNA splicing. Results will also be presented for a complementary CRISPR activation screen. These data will contribute new insights into the biological processes necessary for proper expression of type 3 cytokines by innate and adaptive lymphoid cells.
IL-17 promotes protective antibody during gut infection through IκB-ζ-dependent LN stromal cell activation
Authors: Wu Dongwen , Majumder Saikat , McGeachy Mandy J
Published: January 2023

Fibroblastic reticular cells (FRCs) provide critical regulation of T and B cell. Our recent study shown metabolic reprogramming through Interleukin 17 (IL-17) was critical for the FRCs expansion and autoantibody production. Although studies shown IL-17 is important for B-cell activation and induction of IgA responses, the mechanisms have been unclear. We further hypothesized IL-17 signaling in FRCs is critical to drive protective antibody responses, and prior FRCs activation through a distinct inflammatory episode could enhance this response. We employed Dextran Sulfate Sodium (DSS) induced-colitis followed by Citrobacter rodentium infection model. Mice pretreated with DSS shown increased FRCs expansion and germinal center (GC) formation and enhanced anti-Citrobacter antibody production, leading to a quicker Citrobacter clearance, less colon inflammation and better outcome. IL17a neutralization in DSS phase blocked both the expansion of FRCs and the enhanced B cell response. We further confirmed FRCs failed to expand during Citrobacter infection in FRC-restricted IL-17RA deletion mice. These mice had reduced antibody production in both serum and feces, along with higher Citrobacter burden. We previously shown IL-17 induction IκB-ζ was required for metabolic reprogramming in vitro. Now, we found FRC-intrinsic IκB-ζ expression was required for in vivo Cpt1a (a rate-limiting transporter for fatty acid oxidation) expression, GC formation and antibody responses. RNAseq data suggest GC survival factors such as IL-6 and BAFF were increased by IL-17 signaling in FRCs. In conclusion, IL-17 indirectly promotes GC formation and antibody responses by inducing IκB-ζ expression in activated LN stromal cells during gut infection.
WGCNA identification of CXCL13 and associated genes involved in the Tumor Immune Microenvironment (TIME) of lung adenocarcinoma
Authors: Young Corey D , Dammer Eric , Nunez Sha’Kayla , Griffen Tiara , Carey Kaylin , Dill Courtney Denise , Lillard James W
Published: January 2023

Lung cancer is the second most commonly diagnosed cancer, causing the most cancer-related deaths in the United States. Non-small cell lung cancer (NSCLC) accounts for over 75% of lung cancer cases of which lung adenocarcinoma (LUAD) is the most common. Chemokines and their receptors have been shown to be highly expressed in a number of cancers, presumably leading to abnormal signaling. Elevated levels of CXCL13, and its receptor, CXCR5, have been correlated with the overall features of mortality, but the exact mechanisms in which these factors act to promote NSCLC progression are not fully understood. Here, we studied the role of CXCL13, CXCR5, and associated genes expressed in NSCLC tumor immune microenvironment (TIME). The software package WGCNA was used to establish clusters of highly correlated genes related to clinical characteristics. A total of 23,329 transcripts were used to identify 37 modules (gene co-expression networks). The light-cyan module, consisting of 491 genes contained both CXCR5 and CXCL13. The light-cyan module is a hub gene heavy module with the top 300 transcripts exceeding our hub gene threshold (kMe > 0.6). The light-cyan module transcripts where negatively correlated with age. The top biological processes associated with the light-cyan module include immune system processes, regulation of lymphocyte activation, regulation of immune response and cellular defense response. Collectively, the gene clusters identified along with their clinical correlates provide a novel insight into the complex biology of NSCLC and its TIME.
Defining IL22 regulatory logic in type 3 lymphoid cells
Authors: Saini Ankita , Bando Jennifer K. , Collins Patrick L. , Cella Marina , Colonna Marco , Oltz Eugene M.
Published: January 2023

The cytokine IL-22 is a major effector expressed by type 3 immune cells (T helper 17, Th17 and type 3 innate lymphoid cells, ILC3s), and has diverse roles in maintaining intestinal homeostasis, defense against pathogens, cell proliferation, and tissue regeneration. Perturbations in the homeostatic levels of IL-22 have been associated with various inflammatory disorders and tumors. However, significant gaps remain in our understanding about the regulatory mechanisms controlling IL-22 expression by type 3 cells in health and disease.

In this regard, we generated a stable clone of Mnk3, a mouse ILC3-like cell line, which harbored a doxycycline-inducible version of catalytically dead Cas9 nuclease (dCas9) fused with KRAB repressor domain. We performed a CRISPRi screen in this dCas9-KRAB Mnk3 line by infecting it with a lentiviral library of sgRNAs targeting IL22 regulatory space. Our CRISPRi screen identified a set of candidate enhancers important for IL22 expression in type 3 cells. Chromatin profiling data suggested a differential pattern of activities for these enhancer elements in immune cell subsets. We have now deleted several enhancers from the germline of mice and will present results describing the impact of these mutations on IL-22 expression in type 3 lymphoid cells from the innate and adaptive immune systems.

Collectively, this study defines the cis-regulatory circuits controlling IL22 in type 3 immune cells and thus will open new avenues for therapeutic intervention in IL-22 pathologies.
Single-cell analysis maps the dynamic landscape of mouse iNKT cell development and identifies Cbfb as a key regulator for early commitment
Authors: Wang Jie , Adrianto Indra , Liu Tingting , Wu Xiaojun , Sant’Angelo Derek B , kronenberg mitchell , Zhou Li , Mi Qing-Sheng
Published: January 2023

Invariant natural killer T (iNKT) cells are innate-like T cell that share the characteristics of T cell and NK cell. iNKT cell acquire an effector function and differentiate into iNKT1, iNKT2 and iNKT17 sub-lineages prior to the thymic export. Although remarkable advancements in iNKT cell development have been made, the underlying molecular programs that guide their differentiation and heterogeneity remain unclear. Here, we profiled the transcriptomes of over 17,000 iNKT cells and the chromatin accessibility states of over 39,000 iNKT cells from the mouse thymus across stage 0 to stage 3 using scRNA-seq and scATAC-seq. By integrating iNKT cells transcriptome and chromatin accessibility profiles, we found that both iNKT2 and iNKT17 lineage commitment occur at stage 0, while iNKT1 cells initiate their differentiation as early as stage 1. Furthermore, iNKT1 and iNKT2 cells exhibit extensive phenotypic and functional heterogeneity, while iNKT17 cells are relative identical. Reconstruction of iNKT developmental trajectories revealed two possible pathways stemming from iNKT progenitors. More importantly, we identified Cbfβ (core-binding factor subunit beta) and known Egr2 and Slamf6 exhibit similar expression pattern in iNKT precursors. Deletion of Cbfβ in the mice blocked iNKT cell development at stage 0 and stage 1, and Cbfβ-deficient iNKT cells were failed to be differentiated into effector iNKT subsets. Bone marrow transfer experiments further indicated that the defective iNKT cell development in Cbfβ KO mice was cell-intrinsic. Overall, our findings revealed the cellular heterogeneity of iNKT cells, mapped the potential developmental trajectories, and identified Cbfβ as a key regulator for early iNKT cell commitment.
CD4 and CD8 double positive T cell progenitors in mediastinal adipose tissue and the aortic arch differentiate to CD4 T cells
Authors: Winkels Holger , Ghosheh Yanal , Kobiyama Kouji , Kiosses William B , Marcovecchio Paola , Orecchioni Marco , Wolf Dennis , Thiault Nicolas , Seumois Gregory , Vijayanand Pandurangan , Ley Klaus
Published: January 2023

Using 10× Genomics-based single-cell RNA sequencing on digested atherosclerotic mouse aortas, we discovered an unexpected immune cell population whose transcriptome was similar to thymic CD4+CD8+ cells (DPs). Flow cytometry showed DP presence in the aortic arch (AA) and mediastinal adipose tissue (MAT), but not in lymph nodes, spleen, blood or other tissues. DPs in MAT or AA did not change with atherosclerosis in Apoe−/− mice. Fluorescence microscopy of cleared whole mounts placed DPs in the AA adventitia and near microvessels in MAT. MAT and AA DPs were absent in Rag2−/− mice and in day3 and 21 thymectomized mice. Deep transcriptional profiling from sorted thymic, MAT, and AA DPs showed hundreds of differentially expressed genes. While thymic DPs were enriched for TCR signaling pathways, extra-thymic DPs were enriched for stress responses and cell cycle pathways. Pulse-chase experiments demonstrated a five-day turnover of MAT and AA DPs. Functionally, sorted MAT DPs developed successfully into conventional T cells (with a bias to CD4) in hanging drop cultures. Pharmacologic inhibition of cell migration by S1P1 blockage with FTY720 did not affect the presence of these cells, suggesting that migration of extra-thymic DPs does not require passage into blood. Transplantation of congenically marked thymi under the kidney capsule of thymectomized mice reconstituted T cells in the inguinal lymph node, but failed to reconstitute extra-thymic DPs. The discovery of two new, transcriptionally distinct subsets of extrathymic DPs reveals a novel, unexpected local escape mechanism from the thymus. Their association with blood vessels suggests possible angiotrophic functions.
THEMIS functions as a cytoplasmic adaptor and nuclear transcription factor in developing thymocytes and mature T cells
Authors: Thiault Nicolas , Kakugawa Kiyokazu , Takazawa Ikuo , Verstichel Greet , Taniuchi Ichiro , Cheroutre Hilde
Published: January 2023

THEMIS functions in the cytoplasm as an adaptor molecule which dampens TCR signaling. During thymic development, THEMIS sets the affinity threshold for activation, enabling the selection of conventional T cells in response to low affinity antigens. Although these events occur in the cytoplasm, THEMIS possess a nuclear localization signal (NLS) and a significant amount of THEMIS resides in the nucleus.

To investigate the importance of THEMIS in the nucleus, we deleted the Themis NLS to force THEMIS in the cytoplasm. Surprisingly, T cell development was impaired similar to the Themis null mice, suggesting that THEMIS may have important roles in the nucleus as well. Alternatively, we established Themis mutant mice, in which THEMIS localized exclusively in the nucleus or where THEMIS was constantly present in both compartments. Normal conventional T cells were absent in both mutant mice. Since lack of THEMIS or the constant presence of THEMIS in the cytoplasm or nucleus or both, all resulted in aberrant T cell development it indicated that the translocation of THEMIS to or/and from the nucleus is essential for its function.

To examine the role of THEMIS in T cells in the periphery, we generated mutant mice where THEMIS is expressed normal during thymic development but altered post-thymically. Adoptive transfer of naïve CD4 T cells with altered THEMIS expression into RAG deficient host induced enhanced sever colitis and furthermore, we observed that forced localization of THEMIS in the nucleus redirects the polarization of CD4 T cells toward the pathogenic TH17 helper lineage.

These findings identify a dual function for THEMIS as a cytoplasmic adaptor during TCR signaling and a nuclear factor directing gene expression in thymocytes and mature T cells.
Cellular requirements for B7 expression in thymic iNKT cell development
Authors: Watanabe Masashi , Celli Sofia , Alkhaleel Farrah , Hodes Richard J
Published: January 2023

We had previously shown that the B7-CD28 co-stimulatory pathway is important for thymic iNKT cell development. iNKT cell development is dependent on glycolipid/CD1d complex presented by CD4+ CD8+ double positive thymocytes, with no requirement for CD1d-expression on other cell types, including thymic epithelial cells (TEC), dendritic cells (DC), or B cells. However, the identity of the specific cell type(s) providing B7 co-stimulation for developing thymic iNKT is unknown. Here we have analyzed the cellular requirement for B7 expression utilizing B7 conditional KO approaches. We found that B7 expression on TEC, DC and B cell have essential but redundant roles in supporting thymic iNKT cell development. These results suggest that during thymic iNKT cell development, signal 1 (glycolipid/CD1d-TCR) and signal 2 (B7-CD28) are provided by spatially and temporally distinct cell types and may provide new insight into how co-stimulation can function in immune system development.
Loss of Dll4 expression in thymic epithelial cell monolayer culture is mediated by reduction in both burst size and burst frequency of its gene transcription
Authors: Han Jianxun , Kianizad Korosh , Zúñiga-Pflücker Juan Carlos
Published: January 2023

Commitment of thymic seeding progenitor (TSP) cells to the T-lineage depends on Notch signaling, which requires engagement of the Notch1 receptor on TSPs by the Notch ligand Dll4 on thymic epithelial cells (TECs). We previously showed that when TECs are cultured as monolayers (2-D), Dll4 expression is rapidly lost, as is their ability to support T-cell development in vitro. In the present study, we aim to examine the regulation of Dll4 expression in TECs at the levels of gene transcription and mRNA degradation in TECs from E15 embryos using a combination of multiple techniques. The half-life of Dll4 mRNA in TECs was found, by utilizing quantitative PCR (qPCR) combined with Uridine RNA analog pulse-labeling, only approximately 30 min, which makes its level very sensitive to changes in the rates of gene transcription. Nonetheless, reduction in gene transcription, rather than in mRNA stability, was determined to be the primary molecular mechanism underlying the loss of Dll4 expression in 2-D TECs. FoxN1, the transcription factor that drives thymic epithelial cell development, was found to bind to multiple sites at the Dll4 gene promoter and an enhancer region within its third intron by luciferase reporter assay. Consistent with the luciferase reporter assay result, enforced expression of FoxN1 in 2-D TECs restored Dll4 expression significantly. Further detailed analysis with PrimeFlow RNA assay revealed that FoxN1 regulates only the burst size, but not the burst frequency, of Dll4 gene transcription. We are currently working on identifying the transcription regulator(s) that could bind to the enhancer region to regulate the burst frequency of Dll4 gene transcription.
Bone marrow-derived cells contribute to the maintenance of thymic stroma including thymic epithelial cells
Authors: Chakrabarti Shami , Hoque Mohammed , Jamil Nawshin Zara , Meer Neelab , Singh Varan J , Pezzano Mark T
Published: January 2023

In paradox to critical functions for T-cell selection and self-tolerance, the thymus undergoes profound age-associated atrophy and loss of T-cell function, which are further enhanced by cancer therapies. Identification of thymic epithelial progenitor populations capable of forming functional thymic tissue will be critical in understanding thymic epithelial cell (TEC) ontogeny and designing strategies to reverse involution.

We identified a new population of progenitor cells, present in both thymus and bone marrow (BM), that co-express the hematopoietic marker CD45 and the definitive thymic epithelial marker EpCAM and maintains the capacity to form functional thymic tissue. Confocal analysis and qRT-PCR of sorted cells from both BM and thymus confirmed co-expression of CD45 and EpCAM. Grafting of C57BL/6 fetal thymi under the kidney capsule of H2BGFP transgenic mice revealed that peripheral CD45+EpCAM+GFP+ cells migrate into the developing thymus and contribute to both TECs and FSP1-expressing stromal cell. Sorted BM-derived CD45+EpCAM+ cells contribute to reaggregate thymic organ cultures (RTOCs) and differentiate into keratin and FoxN1 expressing TECs, demonstrating that BM cells can contribute to the maintenance of TEC microenvironments previously thought to be derived solely from endoderm.

BM-derived CD45+EpCAM+ cells represent a new source of progenitor cells that contribute to thymic homeostasis. Future studies will characterize the contribution of BM-derived CD45+EpCAM+ TEC progenitors to distinct functional TEC microenvironments in both the steady-state thymus and under conditions of demand. Cell therapies utilizing this population may prove useful for counteracting thymic involution in cancer patients.