bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibitory receptor signatures on gamma delta T cells predict ART-suppressed HIV infection, are synergistically altered by HIV and aging, and co-vary with inflammatory plasma analytes.
Authors: Snyder-Cappione Jennifer E. , Belkina Anna C. , Starchenko Alina , Drake Katherine , Proctor Elizabeth , Lauffenburger Douglas A. , Browning Jeffrey L. , Olson Alex , Lin Nina H.
Published: January 2023

Virologically suppressed HIV+ individuals have an increased risk and earlier onset of age-associated diseases such as cardiovascular atherosclerosis, cancer, and osteoporosis, referred to as HIV-Associated Non-AIDS conditions (HANAs). Whether HIV drives HANAs via same mechanisms as normal aging or other processes is unclear. We analyzed PBMC from 45 ART-suppressed HIV+ subjects (20 ≤35yo ‘younger’, 25 ≥50yo ‘older’) and 39 uninfected controls (20 younger, 19 older) with a 16-color flow cytometry panel that measures the inhibitory receptors (IRs) PD-1, TIGIT, CD160, TIM-3, and LAG-3 on CD4+ T cells, CD8+ T cells, NK cells, iNKT cells, and gd T cells. Bioinformatic (CITRUS) analysis revealed that gd T cell IR expression exclusively differentiated HIV+ subjects from controls. Also, a synergistic effect of HIV and aging on TIGIT+ and ≥ 2 IR gd T cell percentages was found. gd T cell functional profiling revealed a positive association between TIGIT expression and spontaneous release of granzymes A and B from HIV+ subjects but not controls. Using supervised multivariate modeling (Partial Least Squares discriminant analysis, PLS) of gd T IR signatures, gd T cell cytokine secretion, and inflammatory plasma analytes, all four subject groups (HIV+ and uninfected, ≤35 and ≥50yo) were effectively distinguished. Also, a statistically significant covariance between gd T cell IR signatures and levels of inflammatory plasma analytes, including IL-6, IL-1b and fibrinogen was found. Our data implicate gd T cells as a key inflammatory driver in ART-suppressed HIV infection and our PLS results suggest a divergence of ART-suppressed HIV inflammation and healthy aging processes, revealing differential “inflammaging” with and without HIV infection.
5-Fluorouracil depletion of Myeloid Derived Suppressor Cells in mice infected with LP-BM5 retrovirus.
Authors: Green Kathy A. , Green William R.
Published: January 2023

Tumor associated MDSC inhibit tumor-directed T-cell responses. We have shown the induction of monocytic MDSCs (M-MDSC) during infection of B6 mice by LP-BM5 immunodeficiency inducing retrovirus. These M-MDSCs suppressed proliferation and function of T cells, ~100% via iNOS/NO; and that of B cells, ~50% via iNOS/NO along with the negative checkpoint regulator VISTA, NOS, ROS, and other soluble mediators. Because of overlapping cell-surface phenotypes, our attempts to selectively deplete M-MDSCs in vivo have been challenging. However, 5-Fluorouracil (5-FU), a pyrimidine analog widely used in tumor chemotherapy, has recently been reported to selectively eliminate MDSCs by apoptosis (Biomed. J. 2015:38:111–116). Here, we treated LP-BM5 infected mice with an extended course of 5-FU: at 6–7 weeks post infection achieving ~50% depletion of splenic M-MDSCs, and spleen cells from depleted mice exhibited greatly decreased suppression in ex vivo assays. LP-BM5 infected mice with this level of M-MDSC depletion, had significantly less disease, including: splenomegaly and immunodeficiency – i.e. impaired B and T-cell ex vivo polyclonal responses. Also, regarding other disease parameters, the loss of LP-BM5 induced splenic: CD4 Thy1.2 and B-cell CD23 expression was greatly reduced in M-MDSC depleted mice. As compared to sham treated mice, via flow cytometry, the splenic CD8+ T cell subset from LP-BM5 infected/M-MDSC depleted mice was greatly increased in cell number and MFI. Future directions include extending these initial experiments to further characterize the effects on B and T-cells resulting from in-vivo 5-FU-mediated M-MDSC depletion during LP-BM5 retrovirus-induced immunodeficiency.
The human B cell response to infection with dengue virus serotype 2 broadly engages the plasmablast repertoire and maintains a high degree of type-specificity in memory.
Authors: Diehl Sean A. , Tu Huy , Nivarthi Usha K. , McElvany Benjamin D. , Emerling Daniel E. , de Silva Aravinda M.
Published: January 2023

The complexity of the serological response to the four serotypes of the dengue virus (DENV1-4) is a key factor confounding rational vaccine development for dengue. To refine our understanding of the basis of the humoral response to DENV, we used high-coverage approaches to unravel the early and late B cell response at the clonal level to a single controlled infection with DENV2 in humans. In peripheral blood specimens we identified the peak DENV2-induced early plasmablast response. By analyzing paired immunoglobulin heavy (IGH) and light (IGL) chains derived from peak plasmablasts we found broad evidence of lineage expansion, somatic hypermutation, and variable gene usage. We selected 92 IGH/IGL pairs from expanded lineages and found that 54 (59%) of these monoclonal antibodies (mAb) bound DENV2, five of which also neutralized virus. In these same subjects we assessed the memory B cell (MBC) repertoire at six months post infection by screening IgG from MBCs immortalized by genetic reprogramming to a germinal center-like state. Compared to the early plasmablast response, we found a drastic reduction in the DENV-specific population in the memory phase of the B cell response. There was a unexpectedly high degree of DENV2 type-specificity in the convalescent MBC pool, though heterotypic clones were also observed. MBC-derived mAbs mainly reacted to epitopes present on whole virions. Our results suggest that controlled DENV2 infection elicits a broad and complex early B cell response to DENV that contracts to yield a DENV-specific MBC compartment. These results provide insights into the basis for long-term serotype-specific humoral immunity and provide additional metrics by which to assess DENV vaccines.
Targeting TNF-α against dengue virus-induced neurotoxicity and acute viral encephalitis-like symptoms
Authors: Lin Chiou-Feng
Published: January 2023

Pro-inflammatory tumor necrosis factor α (TNF-α) facilitates dengue virus (DENV) infection in endovascular dysfunction and neurotoxicity. The introduction of TNF-α blocking therapy with antibodies and antagonists is performed for testing its therapeutic effects in this study. In DENV-infected mice, TNF-α was produced in the brains accompanied by the progression of neurotoxicity and encephalitis. DENV infection caused loss in hippocampal neurons with TNF-α expression around the damaged regions. Additionally, immunostaining showed the induction of apoptosis in hippocampal neurons. TNF-α was expressed in active microglia as well as astrocytes of DENV-infected mice. TNF-α facilitated DENV-induced neurotoxicity in vitro in murine Neuro-2a cells. By using a currently established encephalitic mice model that DENV infection caused progressive hunchback posture, limbic seizures, limbic weakness, paralysis, and lethality 7 days post-infection, TNF-α transgenic mice represented the progressive disease development while administration of neutralizing TNF-α antibody reduced dengue encephalitis and mortality. These results demonstrate an immunopathogenesis of TNF-α for mediating DENV-induced encephalitis-associated neurotoxicity and targeting TNF-α can be used as a strategy against dengue encephalitis.
Not just showboating: Shp1 may keep B cells afloat during gammaherpesvirus infection
Authors: Johnson Kaitlin E. , Mboko Wadzanai P. , Lange Philip T. , Tarakanova Vera L.
Published: January 2023

Gammaherpesviruses (GHVs) are ubiquitous pathogens that establish lifelong infections. Importantly, GHVs manipulate B cell differentiation to establish latency, and are associated with several types of B cell lymphomas. Specifically, GHV infection stimulates an abnormal germinal center expansion, and germinal center B cells are thought to be the target of malignant transformation. Thus, defining host factors that affect the germinal center response during GHV infection may offer insights into the biology of viral latency and lymphomagenesis. Shp1 is a tyrosine phosphatase that acts as a negative regulator of immune cell activation, including B cells. Therefore, we hypothesized that Shp1 opposes GHV-driven germinal center expansion. Surprisingly, our studies utilizing a mouse model of B cell-specific Shp1 deficiency have discovered that Shp1 may be required to sustain the abnormal germinal center expansion characteristic of GHV infection.

In mice with B cell-specific Shp1 deficiency we observed a significant reduction in germinal center B cells along with decreased viral loads during early stages of chronic GHV infection. Further evidence suggests that total loss of Shp1 in B cells disrupts the delicate balance of proliferation and apoptosis during germinal center expansion, resulting in an increased level of apoptosis. Based on these data, we hypothesize that Shp1 prevents hyper-activation, and subsequent cell death, of germinal center B cells during GHV infection. Ongoing studies are examining the mechanism by which Shp1 mediates B cell survival.
TBE vaccine and post TBE disease Abs drive antibody dependent enhancement of Zika infection
Authors: Demina Anna Vladimirovna , Edri Avishay , Fridman Lilach , Rouvinski Alex , Lobel Leslie , Hertz Tomer
Published: January 2023

Tick-borne encephalitis (TBE) virus belongs to the Flaviviruses, such as Zika, Dengue, Yellow fever viruses (YFV), etc. Antibody dependent enhancement (ADE) was first described between Dengue 1–4 viruses. Recently the ADE phenomenon was extended to include additional Flaviviruses that are phylogenetically close to Dengue: Zika and YFV. All arthropod borne Flaviviruses are antigenically related, however, the existence and extent of ADE between mosquito-borne and tick-borne Flaviviruses were never addressed. In order to explore ADE associated pathogenesis of sera cross-reactivity, we studied human sera from patients with acute TBE (n=30), vaccinated against TBE (n=76) and a control group without antibodies (Abs) to Flaviviruses (n=30). ELISA assays were used to profile the IgG and IgM serological repertoires to TBE and cross-reactivity to Zika antigen (Ag). Viral plaque assays (VPA) were used to test if TBE Abs cause ADE during infection with Zika virus. Flow cytometry (FACS) was used to measure ADE of Zika infection in U937 cells in the presence of the sera with TBE Abs. We found that 25% of TBE vaccinated subjects and 47% of acute TBE subjects had cross-reactive Abs to Zika Ag in ELISA. Furthermore, serum from 37/40 (92%) TBE positive subjects caused ADE in the VPA assay and 58/60 (96%) caused ADE using the FACS assay. The magnitude of Zika ADE correlated with the titer of TBE Abs. Thereby we demonstrate, that previous vaccination or infection with TBE significantly increases the risk of ADE upon a secondary infection with Zika virus. These results highlight that presence of cross-reactive Abs induced by previous exposure even to a very distantly related Flaviviruses by vaccination or infection dictates the risk of ADE upon secondary infection.
Plasmacytoid dendritic cells prime anti-viral T cells during acute HIV-1 infection but deplete and impair anti-viral T cells during chronic HIV-1 infection
Authors: Li Guangming , Ma Jianping , Cheng Liang , Zhang Liguo , Su Lishan
Published: January 2023

The role of plasmacytoid dendritic cells (pDCs) in human immunodeficiency virus type 1 (HIV-1) infection and pathogenesis is likely important but remains unclear. We have developed a monoclonal antibody that specifically depletes human pDCs in humanized mice (hu-mice) in vivo. When pDCs were depleted before/during acute HIV infection, the expression of type I interferons (IFN-I) and anti-viral interferon-stimulated genes (ISGs) was severely diminished, accompanied with a significantly up-regulated HIV-1 replication. Interestingly, we found that the anti-viral T cell response is completely abolished in the absence of pDC. We also observed an accelerated decrease of CD4/CD8 T cell ratio. However, when pDCs ablation occurred during chronic HIV infection, numbers of total human leukocytes and T cells were increased and T cell immune function was partially rescued in spite of a higher level of viral replication. We conclude that pDCs suppress HIV infection and replication as well as priming anti-viral T cell response in primary HIV infection. However, during chronic infection, pDCs promotes HIV-1 disease progression, including T cell depletion and immune exhaustion. These findings suggest that pDCs, in addition to suppressing HIV-1 infection, play distinct roles during acute and chronic phases of HIV-1 infection.
Reciprocal regulation of lung resident alveolar macrophage function and repopulation by β-catenin and PPAR-γ dictates host morbidity and tissue recovery from respiratory viral infection
Authors: Zhu Bibo , Huang Su , Jiang Li , Zhang Ruixuan , Sun Jie
Published: January 2023

Acute respiratory viral infections, such as influenza and respiratory syncytial virus infections, cause severe morbidity and mortality in children and the elderly worldwide. Lung resident alveolar macrophages (AM), residing in the alveolar space, have been recently shown as a critical regulator of host antiviral defense and the maintenance of normal lung function during acute respiratory viral infection. However, relatively less is known about the molecular regulation of AM function and repopulation in situ, and the roles of AM in the restoration of lung homeostasis at the recovery stage following pathogen clearance. Here we show that two reciprocally regulated transcription factors in AM, β-catenin and PPAR-γ, oppositely modulate host diseases and recovery following respiratory viral infection. AM β-catenin ablation results in reduced morbidity, accelerated inflammation resolution and recovery; while AM PPAR-γ deletion or constitutive β-catenin activation leads to enhanced diseases and impaired recovery following influenza and respiratory syncytial virus (RSV) infection. Mechanistically, β-catenin and PPAR-γ oppositely regulate AM antiviral, inflammatory, reparative, and immunological gene programs. Further, β-catenin and PPAR-γ distinctly modulate AM proliferation and repopulation, thereby regulating tissue recovery following infection. Strikingly, combined deficiency reveals that the main functions of β-catenin and PPAR-γ are based on mutual antagonism. Our data have revealed the critical roles of a transcriptional circuit in balancing rMΦ function and regeneration for the proper restoration of normal tissue homeostasis following infection.
Atypical Xist RNA Localization to the Inactive X in a Female-biased Murine Model of Systemic Lupus Erythematosus
Authors: Martin Anna R. , Syrett Camille M. , Myles Arpita , Atchison Michael L. , Anguera Montserrat C.
Published: January 2023

Systemic lupus erythematosus (SLE) is a severe autoimmune disease that affects women nine times more than men. The genetic basis for this bias is the X-chromosome, where the greatest concentration of immunity related genes on any chromosome can be found. Females have two X chromosomes (XX), and through a process, known as X-chromosome inactivation (XCI), silence one of their X-chromosomes randomly to have a similar level of X-linked gene expression as males (XY). In XCI, XIST RNA, a long non-coding RNA, is expressed from the future inactive X (Xi) and is bound to it in cis by the transcription factor YY1. As XIST coats the Xi, it recruits heterochromatin modifiers to condense and silence it. Previous research has shown that human SLE patient B cells exhibit altered localization of XIST RNA, thus indicating that they have partial reactivation of the Xi. To explore this relationship, we worked with NZB/W F1mice, which are a well-characterized murine model of SLE that also displays a female bias. The hypothesis of our study is that due to reduced expression of YY1, NZB/W F1 mice have altered Xist RNA localization leading to increased expression of X-linked genes in splenic B cells. Preliminary results using qPCR indicate that the concentration of YY1 is reduced across all stages of disease in NZB/W F1 when compared to age-matched wild type (WT) mice. Using Xist RNA FISH, we have observed that the activated B cells of late stage disease NZB/W F1 mice have decreased localization of Xist RNA to the Xi when compared to WT. In addition, we observed that the expression of TLR7 and CXCR3, two x-linked genes, are increased in diseased NZB/W F1 when compared to age-matched WT.
A comprehensive phenotyping program for the MRL-lpr mouse lupus model
Authors: Schile Andrew , Petrillo Melissa , Vovk Andrey , French Richard , Leighton Krystal , Dragos Zala , Hagarman James , Strobel Marjorie
Published: January 2023

MRL/MpJ-Faslpr/J (MRL-lpr) mice develop an autoimmune disease resembling systemic lupus erythematosus, a disease affecting > 1.5 million Americans. The causative mutation, Faslpr, promotes survival of self-reactive lymphocytes, leading to immune proliferation, lymphadenopathy, emergence of anti-DNA antibodies, and fatal immune complex glomerulonephrosis. Like all spontaneous autoimmunity models, MRL-lpr colonies are susceptible to inadvertent selective pressure when mice with absent or delayed phenotypes are bred. We will explain a program for promoting genetic and phenotypic integrity within MRL-lpr breeding colonies. In addition to confirmation of genetic background and Faslpr genotype, breeder phenotypes were assessed at the conclusion of the breeding period. White blood cell counts (WBC) were used to measure lymphoproliferation: at 17 weeks of age, median WBC (103 cells/μL) was 14.2 for females (range = 3.4 – 97.3; N=155) and 10.5 for males (range = 3.7 – 51.4; N=125). Urinalysis to characterize renal damage at 18 weeks of age showed a median albumin/creatinine ratio of 623 for females (range = 1 – 22806; N=163) and 95 for males (range = 1 – 6013; N=123). Median spleen weights (grams) at 18 weeks of age were 0.5464 in females (range = 0.1112 – 2.1348; N=177) and 0.4410 in males (range = 0.2078 – 1.5516; N=135). We will describe a scoring system for clinical signs present at gross necropsy, including appearance of fur/skin, lymph nodes, and organs. We will show that phenotypic integrity can be ensured over time by breeding mice from lineages showing optimal disease development criteria. This program has value beyond breeder assessment, as it can be applied to MRL-lpr mice on study and at conclusion.
BCAP promotes Lupus-like disease and regulates IFNα production in pDC
Authors: Chu Talyn , Waterman Hayley , Gessay Griffin , Hamerman Jessica A.
Published: January 2023

Systemic lupus erythematosus (SLE) severity is correlated with elevated serum type I interferons (IFN), mainly IFNα, which is produced in large amounts by pDC in response to nucleic acid sensing by TLR7 and TLR9. TLR7 and TLR9-induced IRF7 translocation to the nucleus and subsequent IFNα production by pDC is dependent on phosphatidylinositol-3 kinase (PI3K), but how PI3K regulates this process remains undefined. We showed that B cell adaptor for PI3K (BCAP) links TLRs to PI3K activation in macrophages, thus we asked if BCAP plays a role in pDC IFNα production and SLE pathogenesis. We show BCAP promoted many aspects of TLR7-driven lupus-like disease including interferon-stimulated gene expression in blood. BCAP promoted TLR7 and TLR9-induced IFNα production in pDC, and BCAP−/− mice produced significantly less serum IFNα after injection of TLR9 agonist than WT mice, consistent with a pDC IFNα defect. TLR-induced IFNα production in pDC involves dual signaling pathways that run in parallel and converge upon the phosphorylation and activation of IKKα. There is a TLR-independent pathway initiated by nucleic acid recognition at the plasma membrane that involves Dock2-mediated activation of Rac1, required for phosphorylation of IKKα. The TLR-dependent pathway occurs upon nucleic acid recognition by endosomal TLR7 or 9 and leads to MyD88 activation. BCAP regulated IFNα production independently of the TLR-MyD88 pathway and both BCAP and PI3K were required for CpG DNA-induced early actin remodeling, a readout of Rac1 activation, and IKKα phosphorylation in pDC. Our data suggest BCAP and PI3K specifically regulate the TLR-parallel pathway in pDC. Overall, we show a novel role for BCAP in regulating IFNα production in pDC and SLE pathogenesis.
N-Glycanase 1 Deficiency Triggers Innate Immune Activation Through Dysregulated Mitophagy
Authors: YANG KUN , YAN NAN
Published: January 2023

The cytosolic N-glycanase 1 (Ngly1) is an evolutionarily conserved de-glycosylating enzyme, which plays a critical role in the quality control of newly synthesized glycoproteins. Misfolded N-glycoproteins retro-translocated from endoplasmic reticulum lumen to cytosol are deglycosylated by Ngly1 prior to proteasome degradation. Mutations in the human NGLY1 gene are associated with a severe rare congenital disorder characterized by global development delay, neurological abnormalities and liver diseases. The pathogenesis of the NGLY1-associated disease remains largely unknown. In this study, we observed an aberrant innate immune activation in both human and mouse Ngly1−/− cells, as evidenced by elevated expression of interferon-stimulated genes that are collectively known as the ‘type I interferon gene signature’. Using pharmacological inhibitors and RNAi knockdown, we identified the cytosolic DNA sensing pathway as the key innate immune pathway activated in Ngly1−/− cells. We also found that Ngly1−/− cells exhibited fragmented mitochondria and mtDNA leakage into the cytosol, which likely serve as the source for self DNA sensed by the innate immunity. Mitochondria are dynamic organelles, and aged or damaged mitochondria are cleared through mitophagy. We found that mitophagy was impaired in the absence of NGLY1. Together, our data reveal the first evidence of immune dysregulation associated with NGLY1 deficiency, as well as defects in mitophagy that likely lead to accumulation of damaged mitochondria, mtDNA leakage into the cytosol and activation of cytosolic DNA sensing pathway.
Lipocalin 2 deficiency exacerbates collagen antibody induced arthritis in mice
Authors: Lopez Maria , Meyers Keya R. , samuels janaiya , Shashidharamurthy Rangaiah
Published: January 2023

Rheumatoid arthritis (RA) is associated with elevated levels of a numerous acute phase proteins (APPs), including lipocalin 2 (Lcn2), that drive/dampen the inflammatory response. Lcn2, a 25KDa innate immune protein is known to be significantly up regulated during various inflammatory disorders including antibody-mediated arthritis such as rheumatoid arthritis (RA). However, its biological role remains unclear. Herein we have demonstrated that Lcn2 levels were significantly upregulated during collagen antibody induced arthritis (CAIA). Lcn-2-knockout mice (Lcn2KO) developed more severe serum-induced arthritis compared to wild-type (WT) mice. Histological analysis revealed extensive tissue and bone destruction in Lcn2KO mice compared to WT mice. Furthermore, we have observed reduced immune cell infiltration in Lcn2KO mice compared to WT mice. These studies suggest a crucial role of Lcn2 in resolution of arthritic inflammation, making it a promising target in designing better therapeutic strategies for the treatment of rheumatoid arthritis.
An upregulation of TLR7 in renal dendritic cells is associated with glomerulonephritis across multiple B6. Sle1-associated strains.
Authors: Fairhurst Ann-Marie
Published: January 2023

Toll-like receptors (TLRs) 7 and 9 are important innate signalling molecules with opposing roles in the development and progression of Systemic Lupus Erythematosus (SLE). While multiple studies support a dependency on TLR7 for disease development, genetic ablation of TLR9 results in severe disease with glomerulonephritis (GN) by a largely unknown mechanism. In the present study we show that the autoimmune phenotypes exhibited by TLR9-deficient Sle1 mice (Sle1TLR9−/−) mice are akin to those shown by the Sle1Tg7 strain, a model with a 2-fold increase in TLR7 expression. These include increased autoantibody titers, systemic immune activation and glomerulonephritis (GN). Kidney disease in Sle1TLR9−/− mice is associated with an increase in renal CD11b+CD11c+MHC+F480− dendritic cells (DC). These cDCs have higher levels of TLR7 protein with a corresponding increased ability to stimulate T cell proliferation following R848 exposure. Our analyses of TLR7 using flow cytometry in the Sle1Tg7 and Sle123 murine lupus models indicated similar increases in protein expression within cDCs, suggesting their importance across multiple model systems.

Genetic ablation of TLR9 in Sle1 mice also resulted in increased TLR7-reactive RNA/anti-RNA-associated autoantibodies, indicating that TLR9 is required for B cell tolerance to RNA-associated antigens.

In summary, the increase in TLR7-reactive immune complexes (IC) and the concomitant enhanced expression of their receptor, promotes Inflammation and Disease in Sle1TLR9−/− mice.
CD40 expression on specific antigen-presenting cells mediates experimental autoimmune encephalomyelitis
Authors: Lu Ying , Hodes Richard J.
Published: January 2023

CD40 is a cell surface receptor that belongs tothe tumor necrosis factor receptor (TNFR) family and serves as a costimulatory molecule on the antigen presenting cells (APCs). The interaction between CD40 on B cells and its ligand CD154 on activated T helper cells is known to be essential for the development of thymus-dependent humoral immunity. Administration of blocking anti-CD40L antibody or CD40 gene knockout make mice resistant to experimental autoimmune encephalomyelitis(EAE), an animal model of human multiple sclerosis (MS), but the complex cellular interactions mediating the role of CD40 in EAE are not fully understood. We are using conditional CD40 knockout mice to selectively delete CD40 on specific APC populations and evaluate the effects by recombinant human MOG (rhMOG) induced EAE, a model is both T cell and B cell dependent. We found that conditional knockout of CD40 on B cells or dendritic cells led to significantly reduced severity of EAE. Studies of the mechanisms mediating CD40 function in immune response and CNS immunopathology are underway.
Myeloid NF-κB signaling contributes to pathogenesis of immune-mediated nephritis
Authors: Chalmers Samantha A. , Garcia Sayra , Shum Justine , Herlitz Leal , Putterman Chaim
Published: January 2023

Immune-mediated glomerulonephritis is a serious end organ pathology that affects patients with systemic lupus erythematosus (SLE). A common murine model used to study lupus nephritis (LN) is nephrotoxic serum nephritis (NTN) in which mice are passively transferred nephrotoxic antibodies. We have previously shown that macrophages are important for the pathogenesis of LN. To further investigate the mechanism and determine if the contribution of macrophages is mediated via the NF-κB pathway we created B6 mice which had RelA knocked out in myeloid cells, thus inhibiting classical NF-κB signaling in this cell lineage. We induced NTN in this strain to assess the importance of macrophage derived NF-κB signaling in contributing to disease progression.

Myeloid cell RelA knock out (KO) mice injected with nephrotoxic serum had significantly ameliorated proteinuria (p<0.01) and serum BUN levels (p<0.05) compared to control injected wildtype B6 mice (WT). Inhibiting myeloid NF-κB signaling also decreased inflammatory modulators within the kidneys. We measured significant decreases of IL-1a, IFNg, and IL-6 in kidneys from KO mice, but higher IL-10 expression. Flow cytometry revealed decreased numbers of kidney infiltrating classically activated macrophages in KO mice as well. Analysis of renal histopathology is pending.

Taken together, our studies indicate that macrophage NF-κB signaling is instrumental in the contribution of this cell type to the pathogenesis of NTN. Our results suggest that while approaches which decrease macrophage numbers can be effective in immune mediated nephritis, more targeted treatments directed at modulating macrophage signaling and/or function could be beneficial, at least in the early stages of disease.
Macrophage dependent pathways modulate the pathogenesis of cytokine storm syndrome
Authors: Mahajan Sahil , Decker Corinne , Mellins Elizabeth , Faccio Roberta
Published: January 2023

Cytokine storm syndromes (CSS) are life-threatening conditions characterized by excessive activation of T cells, accumulation of well-differentiated macrophages in various organs and exuberant release of inflammatory cytokines. CSS are associated with a wide variety of infectious and noninfectious diseases, often occurring in patients with familial hemophagocytic lymphohistiocytosis and systemic juvenile idiopathic arthritis (sJIA). Although increased number of macrophages is observed in CSS, their role in disease initiation has never been investigated. Here we report that depletion of macrophages at disease onset decreases lethality and limits CSS in two clinically relevant animal models. Mechanistic studies reveal that phospholipase C gamma 2 (Plcʏ2) is critical for macrophage responses during CSS. To dissect how Plcʏ2 induces macrophage activation during CSS, we evaluated two Plcʏ2 downstream pathways, namely calcium and diacylglycerol (DAG). Calcium and DAG levels are tightly controlled by expression of Tmem178, a Plcʏ2-dependent protein and negative regulator of calcium fluxes, and Dgkζ, an enzyme converting DAG into phosphatidic acid, respectively. Intriguingly, by using Tmem178−/− or Dgkζ−/− mouse models, we discovered that calcium and DAG exert opposite effects, with increased calcium augmenting macrophage activation and CSS severity while accumulation of DAG limiting these excessive inflammatory responses. Importantly, macrophages exposed to plasma from sJIA patients that are at risk of developing CSS show skewing toward increased calcium and reduced DAG, via modulation of Tmem178 and Dgkζ expression. In conclusion our data indicate a potential role for macrophage-based therapies for CSS.
The role of innate lymphoid cells in the heart and cardiac inflammation
Authors: Choi Hee Sun , Hou Xuezhou , Bracamonte-Baran William , Diny Nicola , Talor Monica , Cihakova Daniela
Published: January 2023

Studies have shown the diverse role of innate lymphoid cells (ILCs) in mucosal organs, however, the role of ILCs in the heart has not been studied. The aim of the work was to examine the role of group 2 innate lymphoid cells (ILC2s) in the heart and inflammatory heart diseases. To study the role of ILCs in the heart, we used a pericarditis model where IL-33 treatment induces pericarditis in mice. In this model, diastolic dysfunction was observed in the heart of IL-33-treated mice and eosinophils were heavily infiltrated into the heart. We found that the number of ILC2s was increased in the heart after IL-33 administration. We hypothesized that IL-33 induces cardiac ILC2s to produce IL-5 and IL-13, which drives pericarditis development. To examine whether ILCs are required for pericarditis development, we treated WT, Rag2−/− and Rag2−/−γc−/− mice with IL-33. Rag2−/−γc−/− mice which are deficient in all lymphoid cells including ILCs were completely protected from developing pericarditis, while WT and Rag2−/− mice developed pericarditis, indicating the adaptive immunity is not required for the development of IL-33-induced pericarditis. This data suggests that ILCs might play a pathogenic role in the heart. To determine the function of ILC2s, we analyzed cytokine production by ILC2s in the heart. Cardiac ILC2s were a major producer of IL-5 and IL-13. With anti-IL-5 treatment, the number of eosinophils in the heart was reduced, which prevented the heart from developing pericarditis. IL-13−/− mice showed less severe pericarditis upon IL-33 treatment than WT mice. Collectively, our data suggest that ILCs play a critical role in pericarditis, and could provide insight into novel strategies for treating this disease.
IL-17A induced hematopoietic reprogramming produces both PMN and MDSC at the post-acute stage of inflammation.
Authors: Culpepper Courtney D. , Tremblay Alexandra V. , Bian Zhen , Niu Shuo , Liu Yuan
Published: January 2023

Our previous study suggests that polymorphonuclear leukocyte (PMN) response is enhanced at the post-acute stage of murine colitis due to changes in bone marrow (BM) myelopoiesis. Herein, we report that hematopoietic reprogramming during the post-acute phase of inflammation produces myeloid derived suppressor cells (MDSC) that co-infiltrate the intestine with PMN. Employing Percoll density gradients, we analyzed dynamic changes to the myeloid compartment by separating BM leukocytes from DSS colitis-to-recovery mice into four density-increasing bands with myeloid leukocytes enriched in bands III (50–60%) and IV (60–70%). Band III was comprised of monocytes and low-density granulocytes; both confirmed to be M-MDSCs and G-MDSCs, respectively, by displaying potent inhibition of T-cell proliferation. Despite significant decreases in the PMN population, presumably caused by mobilization into inflamed tissues, MDSC numbers significantly increase beginning at the post-acute stage of colitis. Furthermore, MDSC numbers do not return to baseline until late in the recovery stage. The expanded G-MDSCs also showed increased CXCR2 expression, which guides egress out of BM, and produced ROS upon PMA activation. Adoptive transfer assays demonstrated that functionally enhanced PMN infiltrated the intestine at the post-acute stage to clear the existing inflammatory reaction, while MDSC accumulated in the intestine during recovery suggesting a role in tissue repair. IL-17A signaling at the post-acute stage of inflammation likely induced hematopoietic reprogramming of the myeloid compartment, as its neutralization blocked expansion and infiltration of both PMN and MDSC.
Loss of MSP-dependent Ron receptor signaling exacerbates liver fibrosis in a high fat high cholesterol diet-induced ApoE KO mouse model
Authors: Allen Joselyn Natasha , Kennett Mary , Patterson Andrew , Hankey-Giblin Pamela A.
Published: January 2023

Liver fibrosis marks the turning point of non-alcoholic steatohepatitis and with no specific and effective anti-fibrotic therapies available, this disease is a major global health burden. Here, we established the novel role of the Ron receptor in mitigating liver fibrosis. For 18 weeks, apolipoprotein E (ApoE KO) and ApoE x Ron double knockout (DKO) mice were fed a fat and cholesterol-rich diet. Livers from DKO animals exhibited increased accumulation of sirius red-stained collagen. Immunohistochemistry of αSMA revealed that DKO livers had increased activation of collagen-producing hepatic stellate cells. In agreement with this, DKO livers exhibited higher expression of pro-fibrogenic molecules, PDGF, CTGF, and TGFβ. Additionally, DKO mice exhibited increased hepatic expression of collagens (Types 1, 3, and 4) and ECM remodeling proteins (MMP-2 and TIMP-1). The expression of pro-fibrogenic inflammatory cytokines (TNFα and IL-12b), receptors (TLR-4), and chemokines (MIP-2, CCL2, and CCL5) were significantly higher in DKO livers. DKO livers also exhibited increased infiltration of F4/80+ Kupffer cells, which are well-known to mediate the majority of cytokine and chemokine expression in fibrotic livers. Ron also attenuated several risk factors of liver fibrosis including insulin resistance, steatosis, hepatocellular damage, and inflammation. Despite lifestyle changes and pharmacotherapies for treating etiologies of liver fibrosis, the disrupted liver homeostasis often remains and requires effective treatments. Investigating the protective functions of Ron offers a pharmacological target for resolving the steatohepatitis spectrum.
Regulators of mycobacterial granuloma formation – CCL2 and VEGF-A
Authors: Herbath Melinda , Harding Jeffrey S. , Marcus Sarah , Hasko George , Nagy Andras , Fabry Zsuzsanna , Sandor Matyas
Published: January 2023

Granulomas are macrophage dominated lesions and are a central feature of mycobacterial infections. These cell aggregates are dynamically changing structures as the life span of the granuloma recruited effector cells is relatively short and the cells have to be replaced. CCL2 and VEGF-A are required for granuloma maintenance as blocking the action of these chemokines results in reduction of granuloma size and number after BCG or Mtb infections. We seek to elucidate the relevance of these two cytokines in the timeline of mycobacterial infections and their relative contribution to granuloma formation. CD11c+ cell immigration is impaired, costimulatory molecule expression is lower and granulomas are smaller while the bacterial burden is higher in the liver when CCR2KO mice are infected i.p. with M. bovis BCG. Similarly, in animals that are selectively deficient in macrophage VEGF-A production the granulomas are smaller.

Granuloma cells produce VEGF-A. Caseating granulomas, like the ones induced in C3HeB/FeJ (Kramnik) mice are hypoxic and hypoxia is a strong inducer of VEGF-A production. Sarcoid lesions induced by BCG or Mtb in B6 mice are not hypoxic but we show that ATP released from dead cells induces VEGF-A in a subpopulation of macrophages in the granulomas.

Additionally, VEGF production proved to be dependent on granuloma size: bigger granulomas produce larger quantities of VEGF-A while smaller lesions produce less or none. This finding suggests that unlike other chemokines such as CCL2, which is described to be involved in both granuloma initiation and maintenance, VEGF-A may support an increased lesion size in the late acute phase of infection.

Regulating cellular recruitment may provide new therapies for granulomatous diseases.
Using Multiplex and Single-Molecule Bead-Based Technologies to Assess Inflammatory Changes Following Skeletal Muscle Injury
Authors: McFarlin Brian Keith , Bowman Erin M. , Gary Melody A. , Stelly Steven , Tanner Elizabeth A. , Davis Asheal
Published: January 2023

Inflammation is a key component in the healing and recovery of injured skeletal muscle. Despite the known importance of inflammation, its response has not been consistently demonstrated in human experimental model systems. One plausible explanation for the inconsistent outcomes in human skeletal muscle injury models may be caused by the analytical methods selected. Traditional methods (i.e. ELISA, Western Blotting, etc.) used to measure serum inflammatory/muscle damage biomarkers lack the bandwidth and/or sensitivity to measure multiple biomarkers or femtomolar changes. The purpose of this investigation was to describe a comprehensive method for the measurement of serum inflammatory biomarkers in a human skeletal muscle injury model. To accomplish this objective, we combined 384-well, bead-based multiplex assays (MILLIPLEX® and Luminex® FM3D) with a novel, bead-based single molecule detection technology (MilliporeSigma SMCxPro). The samples used in the present investigation were collected from an IRB-approved study and consented subjects (in accordance with the latest Declaration of Helsinki). Subjects completed a muscle damage exercise session and blood samples were collected at 0, 24, 48, and 72-h after muscle damage. We observed a significant change in inflammation post-damage using both detection technologies. Combining multiplexing with the precise, single molecule counting detection method allowed for a better clarification and description of the inflammatory response in otherwise undetectable sample concentrations. More research is needed to demonstrate repeatability for this assay approach as well as validation for other inflammatory states (i.e. chronic inflammatory disease).
Neutrophil extracellular traps are a heterogeneous feature in sickle cell disease
Authors: Barbu Emilia Alina , Dominical Venina M. , Mendelsohn Laurel , Thein Swee Lay
Published: January 2023

Sickle cell disease (SCD) is an inherited hemoglobin disorder caused by a single base change (A-T) in the beta-globin gene of hemoglobin with ensuing recurring inflammation that results in progressive end-organ damage and early mortality. Production of neutrophil extracellular traps (NETs) have been shown to be a key component of this inflammatory process in SCD pathophysiology.

We sought to determine the relevance of NETosis versus a different type of programmed cell death (apoptosis) in SCD. We used an indirect assay using healthy neutrophils exposed to paired plasma samples (n=16) from SCD patients in steady-state and crisis for 30 min to 7 hrs, and a direct approach of observing neutrophils (3 healthy and 3 SCD) themselves up to 4 hrs. Features of NETosis (histone citrullination, nuclear decondensation, string nets) and apoptosis (piknotic nuclei, c-Parp, membrane damage) were assessed by imaging flow cytometry and fluorescence microscopy. Additionally, plasma titers for 20 inflammatory analytes were determined for the 16 paired SCD samples and 4 healthy controls. High circulating IL-6 levels at crisis found in 9/16 of the paired samples correlated with an increased number of NETs at crisis compared to steady-state. NETs were also observed directly in SCD neutrophils at steady state but not consistently. When NETs or histone citrullination were absent or low, c-Parp was typically detected. Additionally, membrane damage was observed in neutrophils from all SCD donors. The data suggest that the SCD environment disrupts neutrophil homeostasis and activate multiple cell death programs. SCD NETosis appears to be heterogeneous, its contribution to the disease pathophysiology remains to be determined.
Accelerated onset of arthritis by systemic infection of Mycoplasma fermentans in the knock-in mice gp130F759
Authors: YAHAGI Ayano , SAIKA Taro , HIRANO Hiroyasu , ISEKI Masanori , ISHIHARA Katsuhiko
Published: January 2023

Development of autoimmune diseases is influenced by both genetic and environmental factors. The knock-in mouse gp130F759 having a point mutation Y759F in gp130, a signaling receptor subunit for IL-6 family cytokines, spontaneously develops arthritis like rheumatoid arthritis (RA) at 8 months old (M.O.) by aberrant IL-6 signals. Mycoplasma fermentans (Mf) was reported to exist in the synovial fluid of RA, but its pathogenic role has not been clarified. In this study, we examined the effects of systemic infection with Mf on the arthritis of gp130F759, and the cellular changes in the synovium. Gp130F759 at 3 M.O. with no symptoms of arthritis were infected with Mf via vein. The clinical scores based on the restriction of joint flexibility were determined from day 3 to 28 after infection. At 1 month after infection, the group with Mf infection had significantly higher total scores of 4 limbs than the group at the same age without infection. Additionally, the cellular populations and numbers in the synovium were analyzed with flowcytometry. The kinetics study revealed that the numbers of the synovial cells predominated by neutrophils increased at day 10 and 28 after infection. To verify the roles for neutrophils increased, 0.25 mg of anti-Gr-1 antibody was injected intraperitoneally at 9 days after infection. The group of treated mice showed no increases of the clinical scores and the synovial cells at 1 month after infection. These data indicated that systemic infection of Mf induces neutrophil-dependent facilitation of arthritis in gp130F759. We propose that this model is useful to clarify the molecular events caused by microorganisms at the preclinical phase of RA.
Signal crosstalk between cytokine RANKL and AhR signaling in osteoclasts controls bone homeostasis
Authors: Izawa Takashi , Arakaki Rieko , Tanaka Eiji , Ishimaru Naozumi
Published: January 2023

The nuclear receptor aryl hydrocarbon receptor (AhR) plays a crucial role in receptor activator of nuclear factor (NF)-kB ligand (RANKL)-mediated osteoclastogenesis. A considerable amount of evidence reveals that AhR is also a key modulator of skeletal remodeling. However, the mechanism underlying the regulation of AhR expression in osteoclasts and the signaling pathway through which AhR controls osteoclastogenesis remain unclear. We found that the expression of AhR in bone marrow-derived osteoclasts was upregulated by RANKL at an earlier stage than the expression of signature osteoclast genes such as NFATc1, c-Src, integrin avb3, and cathepsin K. In response to RANKL, bone marrow macrophages isolated from AhR−/− mice exhibited impaired phosphorylation of Akt and mitogen-activated protein kinase as well as NF-kB, while their response to the macrophage colony-stimulating factor remained unchanged. Osteoclast differentiation mediated by the AhR signaling pathway was also regulated in a RANKL/c-Fos-dependent manner. Further, ligand activation of AhR by the smoke toxin benzo [a] pyrene (BaP) accelerated osteoclast differentiation in a receptor-dependent manner, and AhR-dependent regulation of mitochondrial biogenesis in osteoclasts was observed. Moreover, AhR−/− mice exhibited impaired bone healing with delayed endochondral ossification. Taken together, the present results suggest the new role that the RANKL-AhR-c-Fos signaling axis plays a critical role in osteoclastogenesis, thereby identifying the potential of AhR in treating pathological, inflammatory, or metabolic disorders of the bone.
C-C motif chemokine receptor 2 drives protective immunity by mediating alveolar macrophage localization in tuberculosis granulomas
Authors: Dunlap Micah , Howard Nicole , Das Shibali , Ahmed Mushtaq , Prince Oliver , Rangel-Moreno Javier , Rosa Bruce , Mitreva Makedonka , Randolph Gwendalyn J. , Khader Shabaana
Published: January 2023

C-C motif chemokine receptor 2 (CCR2) axis is a major chemokine axis that recruits myeloid cells including monocytes and macrophages. Thus far, CCR2−/− mice have not been found to be more susceptible to infection with Euro-American lineage 4 strains of Mycobacterium tuberculosis (Mtb). Here, using a prototype W-Beijing family lineage 2 clinical Mtb strain, HN878, we show that CCR2−/− mice exhibit increased susceptibility to tuberculosis (TB). Following exposure to Mtb HN878, alveolar macrophages (AMs) are amongst the earliest cells infected. Using a novel labeling technique, we show that AMs accumulate early in the airways following Mtb HN878 infection and express CCR2. During disease progression, CCR2-expressing AMs exit the airways and localize within the TB granulomas to mediate protective immunity. RNA-sequencing of sorted airway and non-airway AMs show distinct gene expression profiles, suggesting that upon exit from airways, AMs become classically activated. Furthermore, absence of CCR2+ cells specifically at the time of AM egress from the airways resulted in enhanced susceptibility to Mtb infection, increased accumulation of neutrophils, and loss of Mtb control. Interestingly, we provide new evidence that infection with an Mtb strain HN878 mutant lacking phenolic glycolipid (PGL) expression still resulted in increased susceptibility in CCR2−/− mice. Together, our data provide novel evidence for a critical protective role for CCR2 in AM localization within the TB granulomas to mediate protective immunity against clinically relevant and emerging Mtb infections.