bluuebunny/crossref_metadata_2025 huggingface dataset:
Influenza infection exacerbates disease symptoms in relapsing models of multiple sclerosis
Authors: Diaz Katiria Soto , Kim Justin , Wetzel Lisa , McKim Daniel B. , Oelze Michael L. , Steelman Andrew J.
Published: January 2023

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Most patients present with a relapsing-remitting form (RRMS), wherein they experience new or exacerbated neurologic symptoms known as relapses followed by periods of partial or complete recovery (remissions). Previous evidence suggest that upper-respiratory infection increases the risk of relapse, but the mechanisms are ill-defined. The aim of this study was to elucidate the effects of upper-respiratory viral infection on disease progression in three models of relapsing experimental autoimmune encephalomyelitis (REAE). Relapsing EAE was induced in SJL/J, C57BL/6J and NOD/SHILtJ mice. At the peak of disease, mice were randomized into treatment groups, then inoculated with saline or mouse-adapted human influenza A virus (strain A/Puerto Rico/8/1934 H1N1; 0.7 HAU) two days later. Weights and scores were recorded daily. At day 42 post induction mice were euthanized and antigen recall responses measured from lymphocytes isolated from the spleen and cervical lymph nodes. Infection exacerbated symptoms of disease in both SJL/J and NOD/SHILtJ and tended to exacerbate disease in C57BL/6 mice. Following antigen stimulation, splenocytes and cervical lymph node lymphocytes from infected mice had increased production of IFNγ compared to cells isolated from saline inoculated controls. There was no effect of infection on IL-17A or GM-CSF production. These data indicate that infection may function to expand myelin-specific Th1 cell clones by an undefined mechanism. Together, these findings indicate influenza infection can exacerbate symptoms of REAE in SJL/J and NOD/SHILtJ mice.
STAT3 modulates metabolic programming of inflammation-induced macrophages
Authors: Patel Bhakti , Zhang Huiyuan , Zal Malgorzata A. , Wan Xianxiu , Zal Tomasz , Watowich Stephanie S.
Published: January 2023

Redox homeostasis in an inflammatory environment is crucial to ensure a balanced immune response. Several inflammatory disorders are associated with impairment of redox homeostasis, leading to DNA damage and subsequent immune dysregulation. The transcription factor STAT3 has anti-inflammatory activity in myeloid cells, mediated by inhibition of Ubc13, a key Toll-like receptor (TLR) signaling component. STAT3 anti-inflammatory activity is crucial in protection from systemic inflammation, maintenance of lineage-balanced hematopoiesis, and inhibition of DNA damage accumulation in hematopoietic progenitors. The molecular cues by which STAT3 mitigates DNA damage in an inflammatory environment, however, are poorly understood. Recently, we found that STAT3-deficient bone marrow derived-macrophages (BMDMs) exhibited increased oxidative stress upon TLR4 stimulation, including reduced antioxidant amounts and elevated production of reactive oxygen species (ROS). These responses were accompanied by increased gH2AX foci formation, suggesting accumulation of DNA damage. By contrast, BMDMs lacking Ubc13 exhibited increased expression of antioxidants and reduced DNA damage. RNA-sequencing analysis identified transcriptional targets of STAT3 and Ubc13 in steady state and TLR-stimulated BMDMs. Collectively, our findings suggest STAT3-mediated control of Ubc13 is important in regulating inflammation-induced oxidative stress and DNA damage by modulating antioxidant signaling and DNA damage repair pathways. Thus, STAT3 plays a crucial role in metabolic reprogramming of macrophages during inflammation to preserve redox homeostasis.
Validation of a chronic imiquimod-induced psoriasiform dermatitis model in Balb/c mice
Authors: Kemeny Agnes , Horvath Szabina , Perkecz Aniko , Pinter Erika , Gyulai Rolland
Published: January 2023

We developed a modified imiquimod (IMQ)-induced dermatitis protocol (Horvath et al, Scientific Reports 2019) using Finn chambers to minimize the unwanted systemic side effects of this widely used psoriasis animal model. It enables to perform prolonged imiquimod treatment in a self-control way as well. In the present study we investigate the long-term application of IMQ in this localized model.

20 mg Aldara cream (5% IMQ) or 20 mg vaseline were applied in two Finn chambers on the dorsal skin of Balb/c mice during the first 4 days of the experiment and every second or third day from day 5 to day 15, in separate groups. Vaseline or corticosteroid cream were applied on the intermediate days. Skin thickness, blood perfusion, body weight and skin scaling were measured daily. Histopathological alterations were evaluated on H&E stained sections, inflammatory cytokine concentrations (IL-1β, TNF-α, IL-17, IL-22, IL-23) were measured with Luminex technology.

Skin edema increased to 80–120% in the every 2nd or 3rd days treated group. Blood perfusion reached maximal response on day 4 and then gradually decreased until the end of the experiment. Moderate body weight loss was observed on the first 5 days of the experiment. Inflammatory cytokine concentrations in the skin were elevated during the onset of the symptoms (1–5 days) but dropped in the chronic phase (day 10 or 15). Topical corticosteroid treatment significantly reduced skin edema from day 8, but it had no effect on blood perfusion.

Our results proved that long-term Aldara application in Finn chambers enables the study of chronic psoriatic skin inflammation. Furthermore, our chronic model may be used to investigate topically applied anti-inflammatory drug candidates in psoriasiform dermatitis.
Inhibition of thrombin signalling on the surface of monocytes reduces inflammation in oxazolone-induced delayed type hypersensitivity
Authors: Wilkinson Hannah , Chen Daxin , Dorling Anthony
Published: January 2023

Thrombin can directly signal to innate immune cells via protease activating receptor-1 (PAR-1) but the functional consequence of receptor activation has yet to be fully defined. This study aims to assess the outcome of signalling through PAR-1 on monocytes in vivo. In a model of oxazolone-induced delayed type hypersensitivity (DTH)-contact dermatitis; inhibiting thrombin signalling via transgenic expression of hirudin on murine CD31 cells (Hir-Tg) significantly reduced ear swelling (ES) vs. wild type (WT) at 24 and 48 hours. The Hir-Tg mice had reduced CD68 cell recruitment and the recruited cells had a phenotype more polarised towards anti-inflammatory M2, with reduced iNOS, IFNγ & CCR2 expression, and increased expression of IL-10 and ABCA1. This phenotype was shown to be due to inhibition of thrombin signalling on the monocytes, as WT recipients of Hir-Tg bone marrow had a reduced ES, CD68 infiltration, granuloma and iNOS expression. Whereas Hir-Tg recipients (who would still express hirudin on the surface of endothelial cells) of WT bone marrow did not show a reduction in ES and behaved phenotypically akin to WT mice. Mice receiving IP-PAR1 antagonist prior to re-challenge had a similar reduction in ES and similar CD68 infiltration. This data shows that inhibiting thrombin signalling on the surface of innate immune cells both reduces recruitment in models of DTH and alters the behaviour of the cells to a more anti-inflammatory phenotype.
Single dose of anti-high mobility group box 1 protein (HMGB-1) neutralizing antibody ameliorates dysregulated inflammation and restores fracture healing in a polytrauma rat (Rattus norvegicus) model
Authors: Muire Preeti Judith , Mangum Lauren H , Avila Joshua J , Lofgren Alicia L , Wenke Joseph C
Published: January 2023

Polytrauma (PT) is associated with dysregulated inflammation (DI) and complicated/delayed fracture healing. We recently reported delayed fracture healing and increased circulating levels of HMGB-1 in a rat model of PT compared to osteotomy (OST). Since HMGB-1 binds to and activates TLR2, TLR4 and RAGE surface receptors to induce pro-inflammation, we investigated this protein’s role in delayed healing. We hypothesized that neutralization of cell-free HMGB-1 immediately after PT would resolve DI and restore fracture healing in PT. Anti-HMGB-1 polyclonal antibody (2mg/kg) was administered via IP injection following PT (PT-Ab) and bone/tissue volume (BV/TV) of fractures was evaluated by micro-CT imaging at 5 weeks post PT. As hypothesized, by neutralizing HMGB-1, fracture healing was improved with significantly higher BV/TV ratio in PT-Ab compared to PT rats. Changes to circulating CD68+ CD86+ (M1) and CD68+CD163+ (M2) monocyte/macrophage populations, as well as alterations to CD4+ and CD8+ T cells and their RAGE and TLR4 surface expression, were assessed on 1 and 3 days post-injury (d). While circulating M1 and M2 populations are scarce in naïve rats, these populations elevated post trauma. M1 was significantly higher in PT than in OST and PT-Ab at 1d but not at 3d. M2 was significantly high in only OST at 1d. In contrast to OST, we observed decreased RAGE and TLR4 expression on circulating T cells at 1d in PT and PT-Ab and at 3d in PT but not in OST and PT-Ab. Our observations suggest that elevated levels of HMGB-1 following severe trauma initiate immune dysregulation causing delayed fracture repair and the data presented herein indicates that HMGB-1 may be a therapeutic target to restore normal immune response and normal fracture healing in PT.
Epigenetic Regulation of Notch1/2 Signaling Modulates CD4+ T cell Phenotype and Impairs Diabetic Wound Healing
Authors: Davis Frank M , Wolfe Sonya , Dendekker Aaron , Joshi Amrita , Audu Christopher , Moore Bethany B , Lukacs Nicholas W , Kunkel Steven L , Gallagher Katherine
Published: January 2023

T cell plasticity, allowing for transition of CD4+ T cells from an inflammatory to a reparative phenotype, is critical for normal wound healing. In pathologic conditions, such as type 2 diabetes (T2D), wounds fail to heal due to persistent inflammation. Notch activation has been associated with CD4+ TH17/T regulatory (Treg) polarization, in several inflammatory diseases; however, it has not been examined in diabetic wounds. Recently, there is increasing evidence that epigenetic mechanisms control T cell plasticity in tissue. Using FoxP3GFP+ reporter mice we find that FoxP3+CD4+ Tregs were markedly upregulated in normal wounds at late time points. In contrast, wounds from a murine T2D model (DIO) displayed decreased FoxP3+CD4+ Tregs and an overabundance of RORγt+CD4+ TH17 cells with elevated IL17A production. We find that Notch 1 and 2 expression were markedly upregulated in DIO CD4+ T cells. Our prior work has demonstrated that MLL1, a histone methyltransferase, increases gene expression via H3K4 trimethylation (H3K4me3). DIO CD4+ T cells displayed increased Mll1 resulting in elevated H3K4me3 on the Notch 1 and 2 gene promoters increasing receptor expression and activation. Mice with a CD4+ T cell specific depletion of MLL1 (Mll1f/fCD4Cre+) had decreased H3K4me3 on the Notch 1 and 2 promoters with decreased Notch expression. Further, genetic depletion of Notch signaling in CD4+ T cells (DNMAMLf/fCD4Cre+) reduced IL17A production and TH17 cells in diabetic wound tissue. In conclusion, MLL1-mediated activation of Notch 1 and 2 in diabetic wound CD4+ T cells contributes to a persistent TH17 inflammatory response and results in impaired diabetic tissue repair.
A potential association of an interferon gene signature with RBC alloimmunization in sickle cell disease
Authors: Madany Emaan , Kadi Najwa El , Pandya Sumaarg , Hendrickson Jeanne , Gibb David R
Published: January 2023

RBC transfusion can lead to the production of alloantibodies against RBC antigens. RBC alloimmunization can result in hemolytic events and severely limit the availability of compatible RBCs. Patients with sickle cell disease (SCD) have the highest rate of alloimmunization, compared to any other disease population. However, mechanisms underlying this increased frequency are poorly understood. Inflammation in the recipient has been shown to promote alloimmunization in mice and humans. In mouse transfusion models, type 1 interferons (IFNα/β) and interferon-stimulated genes (ISGs) have been shown to promote alloimmunization. Given the chronic inflammatory state in patients with SCD, we hypothesized that SCD patients may also have an IFNα/β gene signature that may contribute to the increased frequency of alloimmunization. To test this hypothesis, we measured the expression of multiple ISGs, including myxovirus resistance protein 1 (MxA) and Siglec-1 by whole blood immunoassay and flow cytometry, respectively. MxA and Siglec-1 levels were significantly elevated in SCD patients, compared to transfused controls. We then determined the degree to which ISG expression correlated with alloimmunization frequency by linear regression. There was a trend toward elevated MxA expression in patients with 1 or more alloantibodies. Patients with 2 or more alloantibodies had significantly elevated MxA compared to non-alloimmunized transfused patients. These findings suggest the presence of an IFNα/β gene signature in patients with SCD and warrant further investigation of the contribution of IFNα/β pathways toward the profoundly elevated frequency of RBC alloimmunization in patients with SCD.
Inflammatory Response to Chronic Opioid Exposure in Humans
Authors: Mathi Kavita , Dobrota Sylvie D , Lee Timothy J , Sayyid Zahra N , Rouholiman Dara , Gamble Jamison G , Encisco Ellen M , Rico Tom , Maecker Holden T , Chu Larry F
Published: January 2023

Objective

In this study, we investigated if chronic opioid exposure leads to changes in immune signaling pathways.

Method

We used phospho-flow cytometry to study intracellular signaling in immune cells of patients with chronic opioid exposure. PBMCs were stimulated with cytokines (IFN-α, IFN-γ, IL-2, IL-6, IL-7, IL-10, IL-21), then stained to analyze 6 lymphocyte subsets and monocytes for phosphorylated STATs (signal transducers and activators of transcription), in particular p-STATs 1, 3 and 5.

Results

After one month of opioid exposure as compared to baseline, IL-2 stimulation of B cells resulted in significantly elevated pSTAT3 levels whereas IFN-γ stimulation resulted in significantly reduced pSTAT5 levels. CD4+ T cell subsets showed reduced pSTAT3 induction in response to multiple cytokine stimuli, and reduced pSTAT1 and pSTAT5 induction with IFN-γ. Similar reductions in pSTAT3 and pSTAT5 induction were seen in CD8+CD45RA+ T cells with several cytokine stimuli. In monocytes, IL-7 and IL-21 stimulation resulted in significantly decreased pSTAT1 levels. No significant changes in pSTAT signaling was found in non-B non-T cells after stimulation with various cytokines. In addition, acute opioid withdrawal via intravenous naloxone resulted in significantly increased pSTAT1 and pSTAT5 levels after IFN-γ stimulation in CD8+ T cells.

Conclusion

One month of opioid exposure significantly reduced cytokine-induced levels of phospho-STAT signaling. These findings suggest that chronic opioid exposure suppresses immune signaling pathways.
Anti-CD3 Mono-Fab co-potentiates TCR antigen recognition in human T cells
Authors: Becher Laura R , Nevala Wendy , Sutor Shari , Abergel Megan , Hoffmann Michele , Parks Christopher , Pease Larry R , Schrum Adam G , Markovic Svetomir N , Pages Diana Gil
Published: January 2023

TCR mediated antigen recognition results in a signaling cascade leading to T cell activation. Antigen binding to its cognate TCR drives receptor aggregation and conformational changes that trigger signaling from the receptor. Conformational change in the CD3 complex (CD3Δc) is required for robust signaling and productive T cell responses. In contrast, weakly recognized antigens do not induce CD3Δc and the resulting T cell response is poor. Thus, T cell responses to weak antigens may be improved by induction of CD3Δc. We previously described an anti-mouse CD3ɛ monovalent Fab (Mono-7D6-Fab) that induced CD3Δc and synergized with weak antigens to enhance the T cell response, a process we termed co-potentiation. However, Mono-7D6-Fab did not induce classical T cell signaling in the absence of antigenic ligation. Mono-7D6-Fab effectively co-potentiated T cells against murine B16-F10 melanoma cells and reduced tumor burden in vivo in a T cell dependent manner. We exploited a model of human cytomegalovirus (CMV) to generate proof of concept that co-potentiation may enhance T cell antigen recognition in humans. Anti-human CD3ɛ monovalent Fab (Mono-OKT3-Fab) improved the CD8 T cell response to the CMV pp65495–503 peptide as measured by increased T cell proliferation, cytokine production, and the ability to kill target cells. Co-potentiation was dependent on antigen specificity, as 1) the effects were dampened when antigen recognition was impaired and 2) sequencing of the TCRs revealed a restricted number of expanded TCR clones. Our results suggest that Mono-OKT3-Fab may have therapeutic clinical applications to achieve a more robust antigen-specific T cell response in the context of diverse human disease, from cancer to chronic infection.
The Role of Circadian Clock Period 2 (per 2) gene and the expression of per2 related gene in atopic dermatitis animal model
Authors: Kang Tae Jin , Prakash Annamneedi Venkata Venkata , Seong Juwon
Published: January 2023

Atopic dermatitis is a chronic, inflammatory skin disorder associated with defective skin barrier and epidermal hyper reactivity. AD is a Th2 mediated allergic disease. GATA3 is a transcription factor that plays an important role in Th2 differentiation. In this study we investigated the per2 gene role and the expression of per2 related genes in atopic dermatitis. We established the atopic dermatitis animal model in per2 knock out and C57BL/6 by 2,4-dinitrochlorobenzene (DNCB). A subset of mice in both per2 knock out and C57BL/6 were treated with saline as control group. The skin lesions severity score was alleviated in per2 knock out mice DNCB group than wild type group. We examined the TNF-α mRNA expression in skin samples that showed that per2 knock out mice protected from DNCB induced inflammation than wild type mice. We next examined BMAL1 mRNA expression in skin samples that showed BMAL1 functioned as anti-inflammatory molecule in per2 knock out mice and levels are high than wild type mice and GATA3 expression levels were not initiated in per2 knock out mice than wild type mice. According to these results we hypothesized per2 knock out mice protected from allergic atopic dermatitis by increasing anti-inflammatory molecule BMAL1 through supressing GATA3.
Biomarker prediction of pediatric atopic dermatitis severity
Authors: Kaplan Mark H , Engle Sarah , Chang Ching-Yun , Satterwhite Allyson , Ulrich Benjamin , Hayes Tristan , Tepper Robert , Sims Jonathan
Published: January 2023

Atopic dermatitis (AD) is a chronic inflammatory skin disease that affects 25% of children and 1–3% of adults worldwide, and has increased 2- to 3-fold over the past several decades. The pathogenesis of AD may result from complex interactions between environmental and genetic factors, barrier defects and immune dysregulation resulting in epidermal hyperplasia and increased penetration of allergens and microbial pathogens. Although most cases of AD are transient, and AD is often thought of as the first step of the atopic march, some AD patients have active disease throughout life. In this study, we analyzed a population of infants that were at high risk for atopic disease and sampled serum, analyzed peripheral blood mononuclear cells, and clinical parameters upon entry (mean age 10.3 months) and five years later. We tested 161 serum analytes using a combination of single-plex, multiplex, Olink, and Quanterix assays. Broadly, the concentration of many analytes fell over time, while SDF and sCD40L concentration increased at the later time point. Several analytes correlated with AD severity as assessed by SCORAD, and most significantly, IL-13 and MCP-4 correlated with SCORAD at both time points. Subsets of cytokines were significantly correlated with each other, consistent with early disease skewing toward type 2 immune response, and two subsets were correlated with percentages of NKT cells or Th2 cells in the peripheral blood. Importantly, forward selection modeling identified 33 infant serum analytes that could be used to predict AD severity five years later (r2=0.85, p=0.042). This dataset will likely have predictive value for AD persistence past infancy and will be useful in further defining the pathogenesis of atopic disease.
A critical period of innate-immune development: Fetal origins of allergic asthma susceptibility
Authors: Lopez Diego A , Beaudin Anna E.
Published: January 2023

Our lab previously identified a developmentally-restricted hematopoietic stem cell (drHSC) that only exists during fetal development and specifically gives rise to innate-like lymphocytes that persist across the lifespan. The identification of a developmentally-limited cell of origin for innate-like immune cells defines a “critical window” for immune development, in which the phenotype of the developing immune system can be shaped via extrinsic inputs. To test how perturbation during this critical window drives immune dysfunction, we examined underlying changes to innate lymphoid cells (ILCs) in the lung. ILCs are a recently identified family of fetal-derived innate-like lymphocytes that mimic the adaptive T-helper arm of our immune system. In the lung, type-2 innate lymphoid cells (ILC2s) produce IL5 and IL13, cytokines important for eosinophil recruitment, activation and goblet cell hyperplasia during allergic airway inflammation. Surprisingly, a single low-dose injection of poly (i:c) at mid-gestation robustly increased proliferative capacity and cellularity of lung ILC2s in offspring at postnatal day (P)9 and P14, respectively, concomitant with drHSC and common-helper innate lymphoid progenitor (ChILP) cell expansion during fetal development. Additionally, in poly (i:c) perturbed offspring, lung ILC2s exhibited heightened IL5 and IL13 production upon in-vitro stimulation with PMA and ionomycin. Ongoing experiments will examine how perturbation of transient progenitors during fetal development may impact the ChILP cell immune trajectory, altering the establishment and function of neonatal lung ILC2s, and ultimately contributing to allergic asthma susceptibility into adulthood.
Mast cell activation enhances the TLR4-mediated antigen-specific CD4 T cell response
Authors: D’Souza Shaina J , Phong Binh , Immethun Vicki E , Bauer David L , McLachlan James B
Published: January 2023

Mast cells are important immune mediators located at host environment interfaces and are most well-known as the major mediators of allergy and asthma. When activated, they have the ability to release prestored granules that contain immune modulating compounds, such as histamine and inflammatory cytokines. In skin, mast cell activation happens via crosslinking of the high-affinity FcɛRI receptor which is activated by the antibody IgE on the mast cell surface binding to a pathogen or allergen inducing intracellular signaling. While there is some evidence that mast cell activation alone does not induce expansion of endogenous antigen-specific CD4 helper T cells, it is not clear whether mast cells have the capacity to fine tune the expansion of T cells that are responding to other stimuli. To assess this, we focused on determining the effects of mast cell activation through FcɛRI alone or in combination with bacterial products on the generation of antigen specific CD4 T cells. Using an in vivo skin model of mast cell activation, combined with MHC class II tetramers, we show that crosslinking FcɛRI alone does not induce antigen-specific CD4 T cell immunity; however, cross-linking FcɛRI in the presence of the Toll-like receptor 4 (TLR4) agonist LPS provides a synergistic co-stimulatory signal that significantly increases T cell expansion. This effect is lost when stimulating with CpG, a TLR9 agonist. We also show that this synergy is lost when dendritic cells are ablated prior to activation. Further, we demonstrate that TLR4 expression by the mast cells themselves is essential to maintain this synergy. This study provides insights into the ability of mast cells to alter antigen-specific CD4 T cell responses in vivo in response to inflammatory stimuli.
Establishment of airway tolerance in early life to inhaled house dust mite involves mitochondrial H2O2
Authors: Yuan Huijuan , Chen Jie , Hu Sanmei , Sinha Ayan , Ray Prabir , Ray Anuradha
Published: January 2023

BACKGROUND

Exposure to innocuous inhaled foreign antigens such as dust mite and pollen triggers aberrant immune responses in the respiratory tract of individuals genetically predisposed to asthma. Immune tolerance restricts such overreaction and plays a key role in preventing unwanted inflammatory responses. Yet the mechanisms underlying this protection are still largely unclear. Our studies of immune tolerance in regulation of experimental asthma and those of others in the context of cancer demonstrated PPARγ-facilitated fatty acid oxidation (FAO) resulting in mitochondrial H2O2 production in dendritic cells (DCs) in promotion of immune tolerance. However, neither study addressed whether mitochondrial H2O2 is essential for suppression of inflammation in response to antigens.

OBJECTIVE

In this study, we sought to establish a mouse model of airway tolerance to inhaled house dust mite (HDM) allergen in newborn mice and study the role of mitochondrial H2O2 in tolerance establishment.

METHODS AND RESULTS

Repeated exposure of wild type (WT) mice to a low dose of the common allergen HDM in early life suppressed airway inflammation and airway hyperreactivity when the mice were challenged by a high dose of the allergen in adulthood. This protective effect was abolished in MCAT transgenic mice, which overexpress the catalase gene in their mitochondria. The inflammatory phenotype in the MCAT mice was associated with a high TH2 response facilitated by lung dendritic cells (DCs).

CONCLUSIONS

Our study illustrates a crucial role of mitochondrial H2O2 in the establishment of airway tolerance in early life, which suppresses DC-mediated TH2 response.
Plasmactoid dendritic cells express EphA2 to negatively regulate type I interferon production in response to viral infection
Authors: FANG MINGLI , XING JUNJI , ZHANG ZHIQIANG
Published: January 2023

Plasmacytoid dendritic cells (pDCs) are primary producers of type I interferon (IFN-I) in response to viral infection. But how such responses are negatively regulated remains poorly defined. Here, we report that the EPH receptor 2 (EphA2), a member of the protein-tyrosine kinase subfamily of ephrin receptors, is selectively expressed in pDCs. Knockout of EphA2 enhanced IFN-I production in pDCs from infection with influenza virus or DNA virus of HSV-1. The expression of Epha2 was dramatically down-regulated after pDCs activation. Surprisingly, challenge of EphA2−/− mice with HSV-I resulted in lethal inflammation and tissues including stomach, intestine, lung and liver damage due to massive production of IFN-I, while depletion of pDCs, partially blocking of IFN-I receptor (IFNAR) signaling, or transplantation of wild type bone marrow cells into irradiated EphA2−/− mice protected mice from this lethal infection. Mechanistically, EphA2 phosphorylates tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6), a key adaptor protein in pDCs function, and inhibits IRF7 activation to reduce IFN-I production in pDCs. These data demonstrate that pDCs expressed EphA2 limits excessive IFN-I production in response to viral infection to avoid uncontrolled inflammation and multiple organ failure. And this finding may provide attractive opportunities for therapeutic targeting pDCs mediated acute inflammation.
Differences in NK and Memory CD8 T cell responses to antigen-nonspecific stimulation by interleukin-15
Authors: Judge Sean J , Dunai Cordelia , Le Catherine T. , Khuat Lam T. , Vick Logan V. , Stoffel Kevin M. , Monjazeb Arta M. , Canter Robert J. , Murphy William J.
Published: January 2023

Natural killer (NK) cells are innate lymphoid cells that exert immediate functions which can be further augmented and sustained with immunostimulatory cytokines. Memory CD8 T cells, due to expression of CD132 and CD122, can be activated by similar cytokines in the absence of TCR engagement (termed “bystander” activation). This results in activation and proliferation but necessitates high amounts of cytokine as high-affinity IL2R complexes (CD25) are not induced. Interestingly, both cell types can then elicit similar effector functions via NKG2D-mediated target cell recognition. As these cell types can fill a similar immunologic niche, we set out to compare NK and memory CD8 T cell responses following IL-15 exposure in vitro from healthy human donors. Cell analysis was performed by flow cytometry and qRT-PCR. At baseline, CD25 expression is negligible at <5% on both human NK and memory CD8 T cells. Surprisingly, culture with rhIL-15 (10 ng/mL) for 4–6 days resulted in marked CD25 upregulation on CD56+CD3− NK cells but not bystander-activated CD45RA-CD95+ CD8 T cells (72±9.2% vs 11±3.8%, P=0.003) despite comparable expansion. Additionally, cytokine-activated NK cells expressed higher levels of inhibitory receptor TIGIT (85±4% vs 57±2%, P=0.01) and activation marker CD69 (99±1% vs 27±9%, P=0.008). Functionally, NK cells had increased expression of granzyme B compared to bystander-activated CD8 T cells. Thus, although NK and bystander CD8 T cells can fill a similar immunologic niche regarding target cell killing, there are significant differences in expression of critical markers following activation. These differences may have consequences in the regulation of these cell types and impact anti-viral and anti-tumor responses.
NATURAL KILLER CELL KILLING OF EXTRACELLULAR PSEUDOMONAS AERUGINOSA
Authors: Feehan David D , Jamil Khusraw , Mody Christopher H
Published: January 2023

Pseudomonas aeruginosa is an opportunistic pathogen that commonly infects individuals with the genetic illness, Cystic Fibrosis and contributes to airway blockage and loss of lung function. NK cells are cytotoxic, granular lymphocytes that are part of the innate immune system. NK cell secretory granules contain the cytolytic proteins granulysin, perforin and granzymes. NK cells, in addition to their cytotoxic effects on cancer and virally infected cells have been shown to play a role in an innate defense against microbes.

Hypothesis

NK cells will kill P. aeruginosa using cytolytic effector protein(perforin, granulysin or granzymes) alone or working synergistically.

Results

Live-cell imaging of a co-culture of YT cells, a human NK cell line, incubated with GFP P. aeruginosa in the presence of the viability dye, propidium iodide, demonstrated that YT cell killing of P. aeruginosa is contact-mediated. CRISPR knockout of granulysin or perforin in YT cells had no significant affect on NK cell killing of P. aeruginosa, as determined by CFU counts. Pre-treatment of YT and NK cells with the serine protease inhibitor 3,4-Dichloroisocoumarin (DCI) to inhibit granzymes, resulted in an inhibition of killing. CRISPR knockout of granzyme B in YT cells, in addition to treatment with the granzyme A inhibitor Futhan, significantly inhibited killing of P. aeruginosa, as determined by CFU counts. These results suggest that NK cells induce membrane damage in P. aeruginosa through a contact dependent process, and that killing requires granzymes.
The PB1-F2 virulence protein regulates host ILC2 responses during influenza
Authors: Barman Tarani Kanta , Califano Danielle , Huber Victor C. , Metzger Dennis W.
Published: January 2023

Influenza remains a major public health concern. The H1N1 PR8 and CA04 influenza viruses are both pandemic strains, yet they have distinct origins and pathogenicity. In PR8, the PB1-F2 viral protein promotes apoptosis of immune cells and negatively influences viral clearance. In the less virulent CA04 strain, however, the PB1-F2 protein is truncated and nonfunctional. Our aim was to examine the role of PB1-F2 in regulating group II innate lymphoid cell (ILC2) function and host susceptibility to viral infection. We found that in the absence of IFN-γ, ILC2s stimulated by CA04 challenge expressed a robust IL-5 response whereas following PR8 challenge, ILC2s predominantly produced IL-13. We observed increased resistance to CA04 challenge in IFN-g−/− mice, which was dependent on ILC2 production of IL-5 and improved tissue integrity. Conversely, during PR8 infection, ILC2s produced only limited amounts of IL-5, with no effect on tissue integrity. We next investigated infection with PB1-F2 recombinant CA04 and PR8 viruses that were created by gene reassortment. CA04 expressing recombinant PR8 PB1-F2 and PR8 expressing recombinant CA04 PB1-F2 induced an ILC2 phenotype and tissue pathogenicity that were similar to the PB1-F2 donor strains, indicating a detrimental role of full-length PB1-F2 protein on ILC2 function and host resistance. Our results suggest that activation of ILC2s can improve host survival following CA04 infection and that the PR8 PB1-F2 inhibits this ILC2-mediated protection. Our findings suggest the concept of a strain-specific contribution of PB1-F2 and ILC2s in protection against influenza virus infection. (supported by NIH Grant R01 HL140496).
The expression of NKG2D ligand is induced in the cutaneous ischemia-reperfusion injury and mitigates pressure ulcer via NKG2D-NKG2D ligand interaction
Authors: Otsuka Noriyuki , Makita Keishi , Tomaru Utano , Kasahara Masanori
Published: January 2023

NKG2D is an activating receptor on NK cells, NKT cells, and several types of T-cells. The ligands for NKG2D are expressed on distressed cells, and NKG2D-NKG2D ligand interaction plays a major role in the surveillance against tumors and viral infection. Several studies have reported the physiological role of NKG2D-positive cells, such as NK cells, iNKT cells, and dendritic epidermal T-cells (DETCs) in skin tissue repair and wound healing. It has been proposed that a major etiology of pressure ulcers is reperfusion injury following ischemia, though the role of NKG2D and its ligands in the process remains to be clarified. Here we report the involvement of NKG2D-NKG2D ligand interaction in the cutaneous ischemia-reperfusion (I-R) injury. The wound size of cutaneous I-R injury was expanded in NKG2D-deficient mice, compared with wild-type and TCRδ knock-out mice. Histopathological evaluation revealed that the accumulation of macrophages and neutrophils was prominent at the periphery of the I-R injury site in NKG2D-deficient mice. The transcripts and the protein expression of murine NKG2D ligands, retinoic acid early inducible gene 1 (RAE-1), were detected in fibrocytes at the I-R injury site. The expression of RAE-1 transcripts was accelerated in primary fibroblasts cultures with sodium arsenite treatment. These results suggest that NKG2D ligand expression is induced by the oxidative stress at the I-R injury site. The pressure ulcer formation by the I-R injury is mitigated via NKG2D-NKG2D interaction. This process may be mediated by the influx of NKG2D-positive cells, which regulate the degree of acute inflammatory cells accumulation at the I-R injury site.
Protective role of lung mesenchymal stem cells in pneumonia caused by the Carbapenem-resistant Klebsiella Pneumoniae.
Authors: Rangasamy Tirumalai , Le John , Ghimire Laxman , Jin Liliang , Jeyaseelan Samithamby
Published: January 2023

Rationale

Although the therapeutic benefits of bone marrow-derived mesenchymal stem cells have been extensively studied in host protection against bacterial pneumonia, the role of lung derived mesenchymal stem cells in bacterial pneumonia has not been explored.

Methods

We purified the stem cell antigen 1+ mesenchymal stem cells from the lung (Sca1+ LMSCs) of C57BL/6J mice using fluorescence activated cell sorter. We subsequently determined the colony forming units, differentiation capacity, proliferation potential, ultrastructural features, and phagocytotic capacity of Sca1+ LMSCs. We also investigated the therapeutic potential of LMSCs in pneumonia caused by the carbapenem resistant K. pneumoniae (CRKP) using a pre-clinical (mouse) model.

Results

Flow cytometric analysis revealed the expression of different mesenchymal stem cell markers in Sca1+ LMSCs. The Sca1+ LMSCs formed colonies, differentiated into osteogenic and adipogenic lineages, and exhibited high proliferation potential in vitro. SEM and TEM images show that the Sca1+ LMSCs are morphologically similar to mouse embryonic fibroblasts but ultra structurally they differ. LMSCs are potent in intracellular killing of CRKPs than BMDMs. Pre-treatment with 3-Methyladenine enhanced the replication of CRKP in both Sca1+ LMSCs and BMDMs. Intriguingly, administration of Sca1+ LMSCs attenuated the infiltration of inflammatory cells (predominantly neutrophils) in the airways and decreased bacterial burden in the lung and in extrapulmonary organs of CRKP-infected mice.

Conclusions

These results show for the first time that the phagocytic capacity and intracellular bacterial killing ability, as well as protective role of LMSCs in pneumonia caused by CRKP.
Differences in the M1 and M2 macrophage subtypes between the sexes determine susceptibility to Francisella tularensis infection
Authors: Sanchez Michelle A , Spencer Charles T
Published: January 2023

Francisella tularensis is the causative agent of the human disease tularemia. F. tularensis infects a variety of cells, including macrophages, in order to cause pathogenicity. As few as 10 inhaled microorganisms cause a lethal infection by over-activating the host’s own inflammatory response. Differences in the intensity of the inflammatory response exist between the sexes which leads to differences in sensitivity to autoimmune and infectious disease. Males tend to be more susceptible to infectious diseases whereas females tend to be more susceptible to autoimmune diseases. However, our preliminary data unexpectedly demonstrated that female mice were more susceptible to F. tularensis--mediated disease than male mice. We hypothesized that female macrophages respond to F. tularensis infection by generating a more intense inflammatory response which makes females more susceptible to F. tularensis. Indeed, female mice infected with F. tularensis generate significantly more inflammatory M1 macrophages compared to male mice. Furthermore, naïve M1 and M2 macrophages were infected in vitro to directly compare cellular responses between male and female mice. On a per cell basis, M1 macrophages from female mice generated a more robust inflammatory response compared with M1 macrophages from male mice. This suggests that M1 macrophages from females respond more strongly to infection resulting in the heightened inflammatory response.
Early innate interactions drive alveolar macrophage localization and effector function during Mycobacterium tuberculosis infection
Authors: Dunlap Micah , Howard Nicole , Das Shibali , Bobba Suhas , Rangel-Moreno Javier , Khader Shabaana A
Published: January 2023

Mycobacterium tuberculosis (Mtb) is the leading cause of death by infectious agent in the world today, infecting roughly ¼ of humans. Despite this, the mechanisms of early pathogenesis and host protective innate immune responses remain poorly understood and uncharacterized.

Lung resident Alveolar Macrophages (AMs) are the first host contact with Mtb bacilli after inhalation and thus key mediators of the early pulmonary immune response. AMs are generally believed to reside entirely in the airway, but it was recently demonstrated that they have the capacity to egress and enter into granulomas during pulmonary infection with hypervirulent Mtb. Furthermore, we found that airway and non-airway AMs display distinct transcriptional profiles that suggest differential effector functions based on compartmental localization. The variety of effector function pathways expressed by non-airway AMs are primarily mediated by NFkB signaling and are indicative of an M1 activation phenotype, which shifts the classic paradigm of AMs as permissive reservoirs for Mtb replication.

In the current work, we examine the host and Mtb factors/signals that modulate these compartmentally distinct AM effector functions and how these specific interactions determine protective or detrimental outcomes for the host. We examine the various functions that AMs contribute to the early immune response, focusing on migration from the airway, cellular interactions with epithelial or recruited immune cells, Mtb phagocytosis and killing, and inflammatory cytokine production. We also examine how specific Mtb cell wall lipid factors that mediate drug resistance and virulence can modulate these AM effector functions, thus skewing the host immune response.
Elucidation of the respective roles of itaconate and nitric oxide in the nuclear accumulation and activation of Nrf2 in activated macrophages
Authors: Krepel Stacey A , Gonzalez-Cotto Marieli , Palmieri Erika M , McVicar Daniel W
Published: January 2023

Nrf2 is a transcription factor that, under basal conditions, is produced in the cytosol and is marked for degradation by its repressor Keap1. Oxidative stress causes Keap1 to release Nrf2, and it accumulates in the nucleus to upregulate certain antioxidant gene targets. Itaconate, the metabolic product of IRG1, has been suggested to play a role in alkylating Keap1, leading to the release of Nrf2. However, there is much ambiguity and even controversy surrounding this role of itaconate. Our study therefore seeks to definitively elucidate the role of itaconate in the regulation and activation of Nrf2. Here we utilize expression analysis and targeted metabolomics in bone marrow-derived macrophages isolated from varying murine backgrounds (Acod1−/−, Nos2−/−, and double knockouts). Despite previously published data suggesting a role for itaconate in Nrf2 activity, our data demonstrate a significant role of Nos2, but not itaconate, in Nrf2 activation, as Acod1−/− mice show little change in the activity of Nrf2 transcription targets, while Nos2−/− mice show severe impairment of Nrf2 gene target activity. For Nrf2 to properly upregulate its gene targets, it must be released from Keap1, phosphorylated, and then allowed to translocate to the nucleus; all three must occur. This may help explain why some studies have found itaconate to be important in Keap1’s regulation of Nrf2, while others demonstrate that this same itaconate mechanism produces comparatively little Nrf2 activity.
Basal TCR signaling drives the functional heterogeneity of naïve CD4+ T cells
Authors: Au-Yeung Byron B , Weiss Arthur , Zinzow-Kramer Wendy
Published: January 2023

Prior to their activation with high affinity foreign peptides presented by MHC Class II (pMHC), naïve CD4+ T cells experience weak “tonic” or “basal” T cell receptor (TCR) signals in response to self-pMHC. We used a combination of the Nur77-GFP reporter transgene and the surface protein Ly6C to visualize the strength of basal TCR signaling in peripheral T cells, and aimed to determine how the strength of basal TCR signaling affects the function of naïve CD4+ cells. The combination of Nur77-GFP and Ly6C enables visualization of a broad range of basal TCR signaling; cells experiencing the weakest basal TCR signals are GFP low Ly6C+, whereas cells experiencing the strongest basal TCR signals are GFP high Ly6C–. We find that there is functional heterogeneity in the naïve CD4+ T cell population, even if they express identical transgenic TCRs, such as the OT2 or AND TCR. Cells experiencing weak basal TCR signaling generate more sustained IL-2 responses and proliferate to a greater extent than cells experiencing moderate basal TCR signal strength. The GFP high Ly6C–cells consistently mounted attenuated IL-2 and proliferative responses, and expressed proteins associated with anergy, such as Grail and Cbl-b. This model is supported by RNA-seq analysis, which identified approximately 100 genes that were more highly expressed in cells experiencing strong basal TCR signaling. Furthermore, ATAC-seq analyses revealed over 3000 loci that were differentially accessible in cells experiencing strong basal TCR signals. We propose that naïve CD4+ cells exhibit phenotypic and functional heterogeneity, and that the source of their heterogeneity is due to the varying strengths of basal TCR signaling experienced by individual T cells.
CNS lymphangiogenesis regulates fluid homeostasis and immunity during neuroinflammation
Authors: Hsu Martin , Choi Yun Hwa , Laaker Collin , Karpus William J. , Sandor Matyas , Fabry Zsuzsanna
Published: January 2023

Recent reports have described meningeal lymphatic vessels residing in the dural layer surrounding the dorsal and basal regions of the brain as well as near the cribriform plate. While all three regions are able to uptake CSF macromolecules, it is unknown how cells and fluid from the CSF-filled subarachnoid space gain access through the blood-CSF arachnoid barrier and into dural lymphatics. In this study, we expand on our previous findings demonstrating the capability of neuroinflammation-induced cribriform plate lymphangiogenic vessels in draining CSF and leukocytes. These lymphatics reside in an optimal location for CSF drainage due to gaps in the arachnoid epithelial layer separating the dura from the subarachnoid space and correlate with increased CSF accumulation near the cribriform plate during neuroinflammation. This is in contrast to other lymphatics residing in the dural layer dorsal and basal to the brain, which are separated from CSF by a complete and uninterrupted arachnoid layer. Additionally, we show lymphangiogenic cribriform plate lymphatic vessels dynamically up-regulate proteins to increase dendritic cell binding and T-cell tolerance during autoimmunity. These data identify cribriform plate lymphatic vessels as dynamic structures that are able to undergo lymphangiogenesis to facilitate fluid drainage and regulate adaptive immunity during neuroinflammation.
In Silico Modeling of Metabolic State in Single Th17 Cells Reveals Novel Regulators of Inflammation and Autoimmunity
Authors: Wagner Allon , Wang Chao , DeTomaso David , Pacheco Julian Avila , Zaghouani Sarah , Fessler Johannes , Akama-Garren Elliot , Pierce Kerry , Ron-Harel Noga , Haigis Marcia , Sobel Raymond A , Clish Clary , Regev Aviv , Kuchroo Vijay K , Yosef Nir
Published: January 2023

Cellular metabolism is a major regulator of immune response, but it is difficult to study the metabolic status of an individual immune cell using current technologies. Here, we present Compass, an algorithm to characterize the metabolic landscape of single cells in silico based on single-cell RNA-Seq and flux balance analysis. We used Compass to study the landscape of metabolic heterogeneity in Th17 cells and search for novel metabolic regulators of their inflammatory function. In central carbon metabolism, Compass predicted a metabolic switch between glycolysis and fatty acid oxidation that mirrors the Th17 vs. Treg phenotype, which we validated through transcriptomic, metabolic and functional assays. The TCA cycle was predicted to break at two points, both of which have been independently identified by other groups in M1 macrophage polarization. Surprisingly, and contrary to common immunometabolic understanding, Compass predicted that glycolysis too was divided into functional modules, and that one of them supported an anti-inflammatory phenotype. We validate the paradoxical prediction and demonstrate that inhibition of this module promotes a pro-inflammatory transcriptional program in Th17 cells, resulting in neuroinflammation in an adoptive transfer model of autoimmune disease. In conclusion, Compass is a widely applicable algorithm to characterize metabolic states at single cell resolution. It allows associating cellular metabolic states with effector functions and detection of metabolic targets that regulate effector phenotypes. We expect it to become a widely used tool with the increasing availability of single-cell RNA-Seq data, spurred by efforts such as the human cell atlas.