bluuebunny/crossref_metadata_2025 huggingface dataset:
The maternal IL-17a pathway in mice promotes autism-like phenotypes in offspring
Authors: Huh Jun R
Published: January 2023

Human studies suggest that maternal viral infection during pregnancy correlates with an increased frequency of Autism Spectrum Disorder (ASD) in the offspring. This observation has been modeled in rodents subjected to maternal immune activation (MIA). The immune cell populations involved in induction of ASD-like behavior in the MIA model have not been identified. Using both genetic mutants and blocking antibodies, here we show that RORgt-expressing T lymphocytes (e.g. Th17 cells) and the effector cytokine interleukin-17a (IL-17a) in mice are required in mothers for MIA-induced behavioral abnormalities in the offspring. We also find that MIA induces an abnormal cortical phenotype, which is dependent on maternal IL-17a, in the fetal brain. Our data suggest that therapeutic targeting of Th17 cells in susceptible pregnant mothers may reduce the likelihood of bearing children with inflammation-induced ASD-like phenotypes.
Vitamin A deficiency disrupts conversion of inflammatory monocyte derived M2 macrophages into a tissue resident macrophage phenotype.
Authors: Gonzalez Michael A , Girgis Natasha , Gundra Uma Mahesh , Tang Mei San , Vozhilla Nikollaq , Loke Png
Published: January 2023

Alternatively activated M2 macrophages accumulate during inflammation from the recruitment of inflammatory monocytes, or through the proliferation of tissue resident macrophages. Monocyte derived and tissue resident M2 macrophages are phenotypically and functionally distinct, with UCP1 expression being a unique feature of tissue resident cells. We now show that monocyte derived M2 macrophages can acquire some of the phenotypic features of tissue resident cells after long-term residence in tissues. In liver granulomas of S. mansoni infected mice, UCP1 expression and proliferating cells accumulate in the periphery of mature granulomas. Genetic fate mapping macrophages derived from CX3CR1(+) monocytes indicate that UCP1 expressing cells are derived from monocyte precursors. Conversion to a tissue resident phenotype is independent of Stat6, although Stat6 regulates macrophage proliferation both intrinsically and extrinsically. Vitamin A deficiency (VAD) leads to dysregulation of the balance between monocyte and tissue macrophage derived M2 cells. Liver granulomas of S. mansoni infected VAD no longer express UCP1 and GATA6, indicating a failure to adopt tissue resident phenotype, and are associated with increased mortality. Inflammatory macrophages transferred into VAD mice fail to convert to a tissue resident phenotype. Genetic fate mapping experiments in VAD mice confirmed the failure of inflammatory macrophages to adopt tissue resident macrophage features despite long term tissue residence. Retinoic acid treatment of VAD mice reverses some of the defects. Hence, retinoic acid may regulate the conversion of monocyte derived inflammatory M2 macrophages into a tissue resident phenotype.
Independent production of distinct monocyte subsets by granulocyte-monocyte progenitors (GMPs) and monocyte-dendritic cell progenitors (MDPs)
Authors: Boyer Alberto Yanez , Goodridge Helen
Published: January 2023

Myeloid cells – neutrophils, monocytes and dendritic cells (DCs) – play key roles in inflammation, immune defense against pathogens, and tissue repair and remodeling. Myelopoiesis, the production of myeloid cells by hematopoietic stem and progenitor cells, must be carefully regulated to support immune surveillance in the steady-state and meet the additional demands of emergency conditions such as infections. The current widely-accepted model of myelopoiesis describes the production of neutrophils by granulocyte-monocyte progenitors (GMPs), which also yield monocytes and DCs via monocyte-DC progenitors (MDPs). However, our data show that murine GMPs do not give rise to MDPs and that conventional and plasmacytoid DCs (cDCs and pDCs) are produced by MDPs but not GMPs. Thus GMPs and MDPs arise separately but both yield monocytes. Our in vitro and in vivo studies of the pathways and mechanisms of monocyte production have revealed that both GMPs and MDPs yield Ly6C+ inflammatory monocytes and Ly6C− CD43+ monocytes, but that GMP-derived and MDP-derived Ly6C+ inflammatory monocytes exhibit distinct patterns of gene expression. In ongoing studies we are comparing the phenotype and function of GMP- and MDP-derived monocytes, and determining the relative contributions of GMPs and MDPs to the monocyte and tissue macrophage pools under steady-state and emergency conditions.
In vitro immunosuppressive effects of mesenchymal stem cells on allergic asthma
Authors: Liou Yih-Mei , Chuang Ya-Hui
Published: January 2023

Allergic asthma is an airway inflammation in the present of high serum IgE and large numbers of eosinophils which mediated by an imbalanced type II helper T cell (Th2) immune response. Mesenchymal stem cell (MSC) is a multipotent progenitor which shows immunomodulatory ability and thought to be a potential immunotherapy. In this study, we evaluated whether allogenic MSC suppress immune cells from allergic asthma patients and whether Th2 microenvironment cytokine affect the immunomodulatory properties of MSCs. To this end, peripheral blood mononuclear cells (PBMCs) from allergic asthma patients were cocultured with MSC and proliferation and cytokine production of PBMCs were assayed. In addition, MSCs were treated with Th2 cytokine IL-4 and immunosuppressive properties of MSCs were examined. Our results showed that MSC suppressed proliferation and the expression of activation marker CD25 of both CD4+ and CD8+ T cells form allergic asthma patients. Otherwise, Th2 cytokine production was also reduced. However, IL-4 did not affect the immunosuppressive properties of MSCs. It seems that MSCs did not response to IL-4 since the immunosuppression related genes COX2, IDO, IL6 and IL8 did not increase. In conclusion, MSC is able to reduce Th2 response in allergic disease and Th2 environment might not affect their immunosuppressive function.
Direct aeroallergen exposure disrupts the oxidant and antioxidant balance in human bronchial epithelial cells
Authors: Wong W.S. Fred , Chan Tze Khee , Tan W.S. Daniel , Peh Hong Yong , Engelward Bevin P.
Published: January 2023

Exposure to aeroallergens is a major risk factor for asthma development. Allergen possessing proteolytic activity is capable of disrupting airway epithelium. Increasing evidence pointing to asthma as an epithelial disease, however, the underlying mechanisms that drive asthma are not fully elucidated. Here, we investigated the DNA damage potential of aeroallergens on human bronchial epithelial cells BEAS-2B, and elucidated the damaging mechanisms. We exposed BEAS-2B cells to house dust mite (HDM), and measured DNA damage by CometChip and DNA double strand breaks (DSBs) by γH2AX staining. Intracellular oxidative stress was evaluated by staining cells with CellROS and MitoTracker. Antioxidant genes expression were determined using RT-PCR. Cell viability was determined by XTT assay. Cell death was studied using flow cytometry. To elucidate the underlying mechanism, cells were treated with Toll-like receptor 4 (TLR4) agonist or antagonist, or antioxidants glutathione or catalase. BEAS-2B cells exposed to HDM showed enhanced DNA damage, elevated ROS production, and increased mitochondrial oxidative stress and nitrite level. Expression of Nrf2-dependent antioxidant genes was reduced by HDM exposure. HDM also reduced cell viability and induced cell death. Importantly, HDM-induced DNA damage could be prevented by antioxidants. Our results suggest that direct HDM exposure to bronchial epithelial cells leads to production of RONS that damage DNA and induce cytotoxicity. Antioxidants can prevent HDM-induced DNA damage, revealing a novel role for antioxidants in preventing genotoxicity. [This study was partially supported by NMRC/CBRG/0027/2012, NUHS Seed Grant R-184-000-238-112, NRF, NIEHS R44-ES0211116 and Trevigen Inc.]
Effects of immunomodulatory supplementation with pre and probiotic on Th2-mediated airway inflammation in A/J and C57BL/6 mice
Authors: Ferreira Caroline Marcantonio , Casaro Mateus , Fukumori Claudio , Câmara Niels Olsen Saraiva , Curi Rui , Vieira Angelica Thomaz , martins Flaviano S
Published: January 2023

Studies have demonstrated that some probiotics and prebiotics could improve Th2 airway inflammation, such as asthma disease. However, it is unclear if a specific probiotic and prebiotic could regulate asthma disease independent of genetic host and gut microbiota composition. Our objective is to elucidate if the habitual probiotic and prebiotic consumption can affect Th2-mediated airway inflammation in two inbred mice, A/J and C57BL/6 (B6). It is known that A/J have a greater Th2 airway inflammation predisposition than B6 and this fact was attributed to genetic difference. The purpose of this study is to examine the role of gut microbiota composition on Th2 inflammation predisposition and the effects of the probiotic (Bifidobacterium longum) and the prebiotic (pectin fiber) on experimental asthma on both mouse strains. Before treatment, an Ion-Torrent 16S rRNA gene-based amplification and sequencing protocol was used to determine the fecal microbiota profile. Ten days before sensitization, they were treated with probiotic or prebiotic. Experimental asthma was performed by sensitization and challenges with ovalbumin. Twenty-four hours after the last challenge, pulmonary inflammation and lung function were analyzed. Our results show that A/J mice have lower microbiota diversity than B6. Interestingly, probiotic and prebiotic treatment reduced lung inflammation and airway hyperresponsiveness, only on A/J mice. The results suggest that low microbiota diversity and genetic background can predispose Th2 airway inflammation. Moreover, high gut microbiota diversity can to strengthen the barrier function of the mucous membrane and to interfere with the effects of pre and probiotics, such as we observed in B6 mice.
Dipeptidyl peptidase-4 promotes T cell migration
Authors: Zhong Jixin , Rao Xiaoquan , Braunstein Zachary , Bromberg Jonathan S , Rajagopalan Sanjay
Published: January 2023

Dipeptidyl peptidase-4 (DPP4) is well known as a post-prandial blood glucose regulating protein by inactivation of incretin hormones. DPP4 also regulates T cell activation by interacting with adenosine deaminase. We investigated the role of hematopoietic DPP4 in T cell-mediated vascular inflammation and atherosclerosis. DPP4 highly expressed on the plasma membrane of T cells especially CD4+ T cells. DPP4 expression increased on circulating T cells in atherosclerotic patients (% DPP4+ CD4+ T cells: 27.46 ± 2.67 vs. 48.06 ± 1.96 for healthy control vs. atherosclerotic patients, p<0.0001). Deficiency of DPP4 on hematopoietic cells protected Ldlr−/− mice from high fat diet-induced atherosclerosis progression and reduced T cell infiltration in aortic plaque. Aorta from Ldlr−/− mice with Dpp4−/− bone marrows showed a much less DPP4 expression, suggesting bone marrow derived cells are the major sources of DPP4 within the aorta. Transwell® migration assay showed T cells with high migratory activity had higher levels of DPP4 expression. Both in vitro and in vivo migration assays suggest T cells deficient for DPP4 displayed a reduced migratory ability. Futher investigation suggests both enzymatic and non-enzymatic actions of DPP4 are involved in the regulation of T cell migration. Taken together, DPP4 increased on T cells in atherosclerosis and is important for T cell migratory activity, which promotes T cell-mediated vascular inflammation and atherosclerotic progression. Our results suggest an essential role for DPP4 in regulating T cell migration.
Anti-inflammaging effect of human alpha 1 antitrypsin
Authors: Yuan Ye , Li Ying , Titov Denis , Brenner Brian , Seifer Lee , El Shikha Ahmed , Akbar Mohammad , Zhou Lei , Song Sihong
Published: January 2023

An important feature of aging is a low grade, persistent, systemic inflammation without overt infection which is defined as “inflammaging”. Inflammaging plays an important role in both morbidity and mortality of most age-related diseases. However, the mechanism of inflammaging is largely unknown, and no treatment has been discovered. Human alpha-1 antitrypsin (hAAT), with anti-inflammatory properties, holds potential to decelerate the aging process. To test the anti-aging effect of hAAT, we generate hAAT transgenic Drosophila lines and showed that over-expression of hAAT in Drosophila fat body (liver) significantly extended the life span. Importantly, real-time PCR and RNA sequencing analyses showed that age-related inflammatory genes (Relish and Diptericin), and immune responsive genes were significantly inhibited in hAAT transgenic lines. To further investigate this anti-inflammaging effect in human cells, we employed a widely used irradiation-induced senescence cell model and tested the effect of hAAT treatment on Senescence-associated Secretory Phenotype (SASP). Our results from this study showed that hAAT treatments significantly decreased gene transcription and protein secretion of IL-6 and IL-8 (the 2 major SASP factors). In contrast, no effect from hAAT on senescence formation was detected. Intriguingly, it is strongly suggesting the epigenetic effect of hAAT since the inhibitory effect can last at least 7 days after withdraw of the treatment. In conclusion, our results have shown that hAAT has the anti-inflammaging effects and may extend life span by inhibiting transcription of inflammatory factors of SASP. Hence, as a FDA-approved drug, hAAT is a promising candidate to combat aging and aging-related diseases.
Systems analysis of microRNAs reveals a novel link between CD8 T cell dysfunction and TGF-β signaling in immune aging
Authors: Gustafson Claire Ellen , Cavanagh Mary , Hu Bin , Zhang David , Weyand Cornelia M , Goronzy Jorg J
Published: January 2023

MicroRNAs (miRNA) have been identified as important regulators of effector CD8 T cell responses and memory differentiation. Here, we examined whether miRNA expression is altered in CD8 T cells of older individuals and whether functional consequences of miRNA expression account for defective CD8 T cell responses with age. We characterized the expression of 44 immune-related miRNAs in CD8 T cells subsets in young and elderly adults using microfluidic qPCR. 32 out of the 44 miRNA investigated were expressed in CD8 T cells. Out of those expressed, 16 miRNAs had significantly altered expression between young and elderly adult CD8 T cells, with 11 of these being altered in naive CD8 T cells. These 11 miRNAs were not directly related to miRNA profiles seen with TCR-activation or T cell differentiation in CD8 T cells. Integrated pathway analysis of these miRNAs using the DIANA MirPath software identified the TGF-β receptor signaling pathway as a highly enriched (p=1.70E-08) target of the decreased miRNAs. Decreased expression of these five miRNAs suggested increased TGF-β receptors and/or signaling in naïve CD8 T cells during aging. Consistently, expression of TGFBR3 and PU.1, a TGF-β-induced transcription factor, was up-regulated in elderly naïve CD8 T cells compared with young. Moreover, overexpression of these miRNAs in elderly naïve CD8 T cells decreased TGFBR3 expression, demonstrating a direct effect of miRNAs on the TGF-β pathway. Thus, using a systems approach to interrogate the role of miRNAs, we identified TGF-β receptor signaling as a potential mediator of altered CD8 T cell responses during aging. Further studies into CD8 T cells and TGF-β signaling in the context of vaccination and aging are currently underway.
Retinoic acid-induced upregulation of αE integrin (CD103) is TGFβ-dependent in human dendritic cells
Authors: Roe Mandi M , Swain Steven D , Bimczok Diane
Published: January 2023

CD103 (αE integrin) is thought to mediate the cross-talk between dendritic and epithelial cells. Based on a recent study showing retinoic acid (RA) may enhance CD103 expression at high concentrations (Hartog, Allergy and Immunology. 2013), we examined monocyte-derived dendritic cell (MoDC) CD103 expression levels with physiological concentrations of RA (100nM) under serum-free conditions. FACS analysis revealed that RA induced a significant increase of MoDCs positive for both extracellular and intracellular CD103 expression. Gene expression analysis corroborated these results with a 57-fold increase in ITGAE upon RA culture. Moreover, expression of β7, which forms the αEβ7 integrin heterodimer with CD103, was similarly upregulated by RA. We next evaluated the TGFβ-dependence of RA-induced CD103 regulation based on a study showing that itgαE gene expression in mice is regulated by TGFβ in T-cells (Robinson, Immunology. 2001). MoDCs were cultured with RA and SB431542, a TGFβR inhibitor that blocks SMAD phosphorylation. When MoDCs were cultured with RA and SB431542, CD103 expression was significantly reduced when compared to MoDCs cultured with RA alone (p<0.01). Similarly, qPCR analysis revealed RA-induced ITGAE gene expression was significantly decreased by 2.4-fold with SB431542 treatment. We also showed that TGFβ directly enhanced CD103 expression in human MoDCs. However, RA-treatment of the MoDCs did not result in enhanced secretion of TGFβ1/2/3 or enhanced expression of TGFβRI/II. Therefore, RA modulation of cellular TGFβ signaling pathways may be involved in the upregulation of CD103 expression. In summary, our studies reveal a TGFβ-dependent mechanism for RA-induced regulation of CD103 in human dendritic cells.
Distinct roles of dendritic cell-derived iNOS in the control of effective and regulative dendritic cell differentiation.
Authors: Si Chuanping , Zhang Ruihua , Hu Yuan , Zhang Hui , Xiong Huabao
Published: January 2023

The immune system exists in a delicate equilibrium between response and tolerance. Dendritic cells (DC) are a plastic lineage able to process and integrate signals from the microenvironment. But the regulation how DCs differentiate to effective or regulatory DC cells are incompletely understood. Inducible nitric oxide synthase (iNOS) derived NO plays critical roles in immune suppression of immune cells. But, it is still not clear what the function of DC cells-derived iNOS is in the regulation in inflammatory diseases. In this study, we demonstrated that DC-derived iNOS regulates balance of effective and regulatory DC cell differentiation. iNOS deficient mice displayed an increased effective DC phenotypes, whereas the percentage of regulatory DCs were comparable in wild-type and iNOS deficient mice in vivo and in vitro. The results were further supported by increased effective DCs from iNOS−/− BMDC cells. Activation of DCs by LPS/IFNg resulted in the expression of iNOS in WT mice. The iNOS inhibitor L-NIL enhanced effective DCs differentiation, mimicking the effect observed in iNOS deficient mice. NO donor SNAP suppressed effective DCs. iNOS−/− DCs result in more enhanced T cell activation. iNOS−/− mice infected with Citrobacter Rodentium led to more severe intestinal inflammation compared to WT mice and the results were correlated with more inflammatory cells infiltration in iNOS−/− in colon tissues. And iNOS−/− mice showed increased effective DCs in colon tissues than that in WT mice. Our results suggest that DC-derived iNOS negatively controls effective DC development and targeting DC-derived iNOS would lead to the new therapies for autoimmune/inflammatory diseases.
Activator of G-protein Signaling 3 (AGS3) modulates CXCR2/GRK6 activation and functions: Role of the GPR domain.
Authors: Richardson Micheler , Adekoya Timothy O , Mowatt Tonelia A , Smith Nikia
Published: January 2023

Activator of G-protein Signaling 3 (AGS3) is a receptor-independent activator of G-protein signaling which comprises seven tetratricopeptide (TPR) motifs and four GPR motifs joined by a linker sequence. Previous studies have demonstrated that the TPR domain of AGS3 is important for determining the positioning of AGS3 within the cell, whereas the GPR domain serves as a docking site for Gαi-GDP leading to AGS3-Gαi signaling. We have recently shown that GRK6 complexes with AGS3-Gαi to regulate CXCR2-mediated cellular functions including Ca2+ mobilization, mitogen-activated protein kinase (MAPK) activation, receptor desensitization and down-regulation. In the current study, mutants from the GPR and TPR domains were used to determine which motif(s) of AGS3 interact with GRK6 to modulate CXCR2-mediated inflammatory responses. Co-immunoprecipitation studies in transiently transfected HEK 293 cells, and RBL-2H3 cells stably expressing wild-type and mutants AGS3 demonstrated that interleukin 8 (IL-8/CXCL8) activation increases GRK6 interaction with the GPR domain not the TPR. Interestingly, the TPR motif displayed a robust agonist-independent association with GRK6 which was prevented by CXCL8 activation of the receptor. Overexpression of AGS3 and the GPR motif, not the TPR, inhibited CXCR2 mediated intracellular Ca2+ mobilization and MAPK activation. Taken together, these results indicate that GRK6 interacts with the GPR domain of AGS3 to modulate CXCR2/Gai interaction and signal transduction.
Dengue virus infection causes Nrf2 activation to induce CLEC5A-facilitated TNF-α production in mononuclear phagocytes
Authors: Lin Chiou-Feng
Published: January 2023

Infection of dengue virus (DENV), an arthropod-borne flavivirus, threatens global public health due to its high prevalence and the lack of effective treatment. Multiple risk factors are believed to be involved, including virus virulence, antibody-dependent enhancement, T cell response, complement activation, autoimmune response, host factors, cytokine storm, and vascular leakage. Regarding host factors may contribute to DENV infection, replication, and pathogenesis, here we investigate the role for nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which is activated by DENV in mononuclear phagocytes. DENV infection selectively activates Nrf2 through a mechanism involving endoplasmic reticulum stress and protein kinase R-like ER kinase activation. After DENV infection, activation of Nrf2 facilitates an increase in C-type lectin domain family 5, member A (CLEC5A) expression through the binding of activated Nrf2 to the CLEC5A promoter. The downstream signaling of the Nrf2-CLEC5A interaction enhances TLR3-independent TNF-α production following DENV infection. Forced expressing the viral protein NS2B3 induces Nrf2 activation and CLEC5A expression to increase DENV-induced TNF-α production. Further animal studies confirm Nrf2-induced CLEC5A and TNF-α in brains from DENV-infected mice. These results demonstrate that, in addition to TLR3, DENV infection also causes Nrf2-facilitated TNF-α production by increasing levels of CLEC5A.
A fluorescent reporter mouse for inflammasome assembly demonstrates an important role for both cell bound and free ASC specks during in vivo infection
Authors: Tzeng Te-Chen
Published: January 2023

Inflammasome activation is associated with numerous diseases. However, in vivo detection of the activated inflammasome complex has been limited by a dearth of tools. We developed transgenic mice that ectopically express the fluorescent adaptor protein, ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), and characterized the formation of assembled inflammasome complexes (“specks”) in primary cells and tissues. In addition to hematopoietic cells, we found that a stromal population in the lung tissues forms specks during the early phase of influenza infection whereas myeloid cells showed speck formation after two days. In a peritonitis and Group B streptococcus infection models, a higher percentage of neutrophils formed specks at early phases of infection, while dendritic cells formed specks at later time points. Furthermore, speck-forming cells underwent pyroptosis, and extensive release of specks to the extracellular milieu in vivo. These data underscore the importance of free specks during inflammatory processes in vivo.
‘Farm dust extract increases the epithelial barrier function of human bronchial epithelial cells partly via MyD88 signalling’
Authors: van der Vlugt Luciën E.P.M. , Eger K. , Amatngalim G.D. , Müller C. , Bracher F. , von Mutius E. , Smits H. H. , Hiemstra P. S.
Published: January 2023

Epidemiological studies showed that farmer’s children have a lower incidence of childhood asthma, which was partly attributed to a higher microbial exposure. Recently, distorted airway epithelium is considered an active player in pathogenesis of asthma. Interestingly, Toll-like receptor (TLR) ligation in the gut was found to enhance the gut epithelial barrier function. We hypothesize that exposure of the airways to microbial-rich farm dust may have barrier-enhancing effects on the airway epithelium and thereby may contribute to the protection against asthma in farmer’s children.

In this study, the effect of farm dust extracts (FDE) collected from traditional German farms was investigated on barrier function and -repair in primary bronchial epithelial cells (PBECs). The expression of tight (TJs) and adherens junctions (AJs)-important to maintain the barrier function- and the involvement of TLR-induced MyD88 signalling were investigated.

FDE exposure induced a dose-dependent two-fold increase in resistance measured by ECIS and up to 70% reduction in permeability in undifferentiated PBECs. Furthermore, restoration of barrier function of fully differentiated PBECs after cigarette smoke exposure was accelerated in the presence of FDE. The increase in barrier function was accompanied by an 1.5 to 2-fold enhanced mRNA expression of TJs occludin-1, claudin-1 and −4 and AJ E-cadherin. In search for the underlying mechanisms, we found that the increased barrier resistance could be significantly attenuated by blocking MyD88 signalling.

These data altogether suggest that FDE exposure strengthen the epithelial barrier function partly via MyD88 signalling and, thereby, possibly reduce the risk of development of asthma and allergies.
Mechanisms governing human memory-like natural killer cell function.
Authors: Wagner Julia A , Berrien-Elliott Melissa M , Rosario Maximillian , Jewell Brea A , Leong Jeffrey W , Schappe Timothy K , Abdel-Latif Sara , Fehniger Todd A
Published: January 2023

Natural killer (NK) cells are innate lymphoid cells critical for host defense against viral infection and malignant transformation. NK cells exhibit innate immunologic memory in response to specific haptens, viruses, or combined cytokine pre-activation. Human cytokine-induced memory-like NK cells, generated by overnight pre-activation with IL-12, IL-15, and IL-18, respond more robustly to numerous stimuli (including leukemia target cells) for weeks to months following the initial activation. The mechanisms responsible for the enhanced effector function of human memory-like NK cells are poorly understood. We hypothesized that memory-like NK cell differentiation alters the balance of signal integration downstream of activating and inhibitory receptors. NK cells achieve functional competence through self-MHC class I interactions (licensing) during maturation; if this does not occur, NK cells remain anergic. We tested this hypothesis using normal donors with licensed and unlicensed NK cell populations. We observed that cytokine pre-activation increases IFN-γ production by both licensed and unlicensed NK cells in response to tumor targets, activating receptor (FcγRIIIa) ligation, or cytokine stimulation. In fact, in response to cytokine stimulation, memory-like NK cells produce significantly more IFN-γ than controls with no impact of licensing. We observed no difference in the expression level of several signaling adaptor molecules or in phospho-signaling downstream of FcγRIIIa ligation in memory-like vs. control NK cells. We are currently investigating epigenetic modification of the IFN-γ gene locus in memory-like NK cells as a potential mechanism for their enhanced function; updated results will be presented.
Influence of M2b macrophage elimination on the wound healing responses in severely burned mice
Authors: Ito Ichiaki , Herndon David N , Suzuki Fujio , Kobayashi Makiko
Published: January 2023

To restore the impaired antibacterial resistance of burn patients, we performed M2bMϕ elimination using CCL1 antisense ODN. However, our treatment with the M2bMϕ polarizer includes a concern about the delay or disturbance of wound healing, because M2a and M2cMϕ distributed in tissues surrounding the burn area are known to be indispensable for host wound healing responses. In this study, the influence of M2bMϕ elimination on wound healing responses was investigated in severely burned mice. Mice 10 days after burn injury (day-10 burn mice) were treated with the ODN (s.c., 10 μg/mouse). Dermal F4/80+ cells (Mϕ) from these mice were analyzed for the expression of IL-10 (a biomarker of M2Mϕ), Arg1 and Ym1 (biomarkers of M2a and M2cMϕ), and LIGHT (a biomarker of M2bMϕ). Also, these dermal Mϕ were tested for the production of fibronectin and collagen (wound healing-related soluble factors) by ELISA. In the results, the increased expression of IL-10, Arg1, Ym1, and LIGHT mRNA was shown in dermal F4/80+ cells of day-10 burn mice, as compared to that of normal mice. Similarly, Arg1 and Ym1 mRNAs, but not LIGHT mRNA, were expressed by the same cells of day-10 burned mice treated with the ODN. Similar amounts of wound healing-related soluble factors were produced by dermal Mϕ from day-10 burned mice and the same mice treated with the ODN. These results indicate that the delay or disturbance of wound healing is minimal in severely burned hosts with eliminated M2bMϕ.
IL-15 complex-stimulated NK cells protect mice from cerebral malaria
Authors: Burrack Kristina Stoermer , Hamilton Sara E. , Jameson Stephen C.
Published: January 2023

Cerebral malaria (CM) is one of the most lethal complications of Plasmodium falciparum infection, responsible for a large fraction of the nearly one million malaria-related deaths annually. Infection of susceptible mouse strains such as C57BL/6 with Plasmodium berghei ANKA (PbA) induces a fatal neurological syndrome from 6–10 days post-infection (dpi) resulting from CD8 T cell-mediated damage to the CNS. We found that treatment of C57BL/6 mice with interleukin (IL)-15 complexes (IL-15C; IL-15 bound to an IL-15Rα-Fc fusion protein) prevented the development of PbA-induced CM. Rescue from CM was not associated with reduced parasitemia at 5 dpi. Instead, IL-15C treatment resulted in reduced CD8 T cell activation in the brain at 6 dpi and reduced blood brain barrier breakdown. In addition, adoptive transfer of IL-15C-stimulated natural killer (NK) cells (but not CD8+ T cells) was sufficient to prevent CM. IL-15C and similar complexes formed with IL-2 (IL-2C; IL-2 bound to the anti-IL-2 S4B6 antibody) both cause robust expansion and activation of NK cells but, unexpectedly, NK cells from mice treated with IL-2C failed to protect against CM. Comparative RNAseq analysis of IL-15C and IL-2C-treated NK cells identified novel gene expression patterns, demonstrating previously unappreciated differences between these cytokine complex signaling cascades in NK cells; the functional significance of these differences is being assessed. These data indicate that NK cells – which are typically involved in promoting inflammatory responses – can restrain damaging immune responses, and that differences between the activation of NK cells by IL-2C and IL-15C regulates their capacity to control the inflammatory response to blood stage malaria infection.
NLRX1 modulates the balance between NLRP3 inflammasome activation and NF-kB signaling during Fusobacterium nucleatum infection.
Authors: Hung Shu-Chen , Huang Pei-Rong , Bui Fiona Q , Ojcius David
Published: January 2023

NOD-like receptors (NLRs) play an important role in regulation of host innate immunity, yet their role in periodontitis remains to be defined. NLRX1, a member of the NLR family that localizes to mitochondria, modulates mitochondrial ROS (mROS) generation. mROS has been shown to activate the NLRP3 inflammasome, yet the role of NLRX1 in NLRP3 inflammasome activation has not been examined. In this study, we revealed the mechanism by which NLRX1 positively regulates ATP-elicited NLRP3 inflammasome activation through mROS in gingival epithelial cells (GECs). Fluorescence microscopy showed that depletion of NLRX1 by shRNA attenuates ATP-induced mROS generation and redistribution of the NLRP3 inflammasome adaptor protein, ASC. Furthermore, depletion of NLRX1 inhibits Fusobacterium nucleatum infection-activated caspase-1, suggesting that it also inhibits the NLRP3 inflammasome. Therefore, we propose that NLRX1 may promote F. nucleatum-caused dysregulated pro-inflammatory responses in periodontitis. On the other hand, the mechanism by which F. nucleatum infection-induces IL-8 expression is still unclear. We showed that NLRX1 also acts as a negative regulator in NF-kB signaling to modulate IL-8 expression. Thus, NLRX1 stimulates detection of the pathogen F. nucleatum via the inflammasome, while dampening cytokine production. We expect that commensals should not activate the inflammasome, but NLRX1 should still decrease their ability to stimulate inflammation. We conclude that NLRX1 may act as a potential switch in regards to the virulence of F. nucleatum in healthy or diseased oral cavity.
Cytosolic entry of LPS and activation of noncanonical inflammasome via bacterial outer membrane vesicles
Authors: Rathinam Vijay , Vanaja Sivapriya Kailasan , Russo Ashley , Behl Bharat , Banerjee Ishita
Published: January 2023

Sensing of lipopolysaccharide (LPS) in the cytosol triggers caspase-11 activation and is central to host defense against Gram-negative bacterial infections and to the pathogenesis of sepsis. Most Gram-negative bacteria that activate caspase-11 however are not cytosolic and the underlying mechanism by which LPS from these bacteria gains access to caspase-11 in the cytosol remains elusive. Here we identify outer membrane vesicles (OMV) produced by Gram-negative bacteria as a vehicle that delivers LPS into the cytosol triggering caspase-11-dependent effector responses in vitro and in vivo. OMV are internalized by macrophages and LPS is released into the cytosol from early and late endosomes. The use of hypovesiculating bacterial mutants, compromised in their ability to generate OMV, reveal the importance of OMV in mediating the cytosolic localization of LPS. Collectively, these findings demonstrate a critical role for OMV in enabling the cytosolic entry of LPS and consequently caspase-11 activation during Gram-negative bacterial infections. In broader terms, our data reveal a fundamental mechanism that serves as a conduit to deliver pathogen-associated molecular patterns (PAMP) to the cytosol to alert the immune system. Our data suggest that OMV with the cargo of immunostimulatory PAMP represent a cardinal sign of infection with not only viable but also multiplying bacteria consistent with the “patterns of pathogenesis” hypothesis.
Unanchored Polyubiquitin Chains and Innate Immune Sensing of Influenza Virus Infection
Authors: Rajsbaum Ricardo , Bharaj Preeti
Published: January 2023

Innate immune responses are initiated when pattern recognition receptors detect incoming microbes and trigger signaling pathways leading to production of inflammatory cytokines and type-I interferons (IFNs). These signaling pathways are regulated by different cellular processes including the ubiquitin (Ub) system. Proteins covalently attached to K48-linked polyUb are targeted for degradation by the proteasome, whereas protein modification with K63-linked polyUb may have non-proteolytic-activating functions. Unanchored Ub chains that are not covalently attached to any protein are also important for kinase activation. We recently reported that the E3 ligase TRIM6 synthesizes unanchored K48-linked polyUb chains, which activate the IKKɛ kinase for induction of an antiviral response. To better understand the physiological role of unanchored polyUb during virus infection, we developed a system to isolate ubiquitin-interacting proteins from the lungs of influenza-infected mice. By using a proteomics approach we identified three RNA helicases that interact with unanchored polyUb chains in vivo. Knockdown analyses of these RNA helicases resulted in decreased IFN induction upon influenza virus infection, suggesting that they may be involved in viral RNA recognition. Therefore, unanchored polyUb chains are emerging as important players in viral sensing and innate immune signaling. Our approach provides a new strategy for the identification of Ub-binding proteins from in vivo samples.
The pro-apoptotic factor Bax regulates macrophage necrosis during Mycobacterial infection
Authors: Soler Francisco Jose Roca , Redmond Sarah , Ramakrishnan Lalita
Published: January 2023

In the zebrafish-Mycobacterium marinum model for tuberculosis infection, excess TNF triggers necrosis of infected macrophages which leads to exuberant extracellular bacterial growth and host susceptibility (Tobin et al., 2012; Roca and Ramakrishnan, 2013). It has been shown that in infected macrophages, excess TNF induces production of mitochondrial reactive oxygen species (ROS) which activate two death pathways that converge in macrophage necrosis: cyclophilin D-dependent mitochondrial transition pore and acid sphingomyelinase-dependent ceramide production that leads to lysosomal permeabilization.

In exploring the events downstream of lysosomal permeabilization, we found that ceramide acts in a Cathepsin D-dependent manner to induce cell death. In the cytosol Cathepsin D initiates the activation of pro-apoptotic factors including Bid and Bax. We engineered Bax mutants that lack its different functional domains and studied the ability of each mutant protein to regulate apoptosis and necrosis. Then we infected Bax-deficient zebrafish larvae expressing the Bax mutants that were unable to induce apoptosis. We found the BH3 domain, which is required for Bax oligomerization during apoptosis, not to be required for necrosis under excess TNF conditions. Rather, an N-terminal transmembrane helix suggested to be the first portion of Bax in contact with the mitochondrial membrane was found to be required for necrosis. Current studies are focused on understanding post-translational modifications of Bax that determine whether activation of the same protein promotes apoptosis or necrosis.
Influence of Ectromelia virus (ECTV-Mos) infection on mRNA transcript levels of selected genes encoding antiviral proteins in a macrophage cell line RAW 264.7 (in vitro studies).
Authors: Toka Felix N , Dolega Patryk , Gregorczyk Karolina , Bossowska Magdalena , Mielcarska Matylda , Szulc-Dabrowska Lidia
Published: January 2023

Poxviruses have evolved a number of mechanisms to avoid immune response by the infected host. We investigated the impact of poxvirus infection on the level of mRNA transcripts of selected genes encoding antiviral proteins in the macrophage cell line (RAW 264.7). We observed reduction of mRNA transcript level in four groups of signalling pathways (Toll-like receptor signalling, NOD-like receptor signalling, RIG-I-like receptor signalling, and Type I interferon signalling) involved in innate immune response. Seventy-three genes had a statistically significantly decreased mRNA expression, 4 genes had a statistically significantly sustained mRNA expression. Only Cxcl11 and Ifna2 were statistically significantly increased. The results confirmed that the mouse poxvirus ECTV may interfere with or inhibit many signalling pathways, which are involved in inducing an antiviral immune response in infected macrophages.
Neutrophilic inflammation promotes bacterial growth during Tuberculosis
Authors: Mishra Bibhuti B , Sassetti Christopher
Published: January 2023

Nitric oxide is generally assumed to protect from tuberculosis by inhibiting the growth of Mycobacterium tuberculosis (Mtb), thereby preventing tissue damage. In contrast, we report that nitric oxide protects the host by repressing a neutrophil recruitment cascade that requires IL-1, CXCR2 and 12-lipoxygenase (12-LOX)-derived eicosanoids. Inhibition of the neutrophil recruitment alone enables the susceptible NOS2 KO mice to control Mtb growth, suggesting that neutrophilic inflammation, not an intrinsic anti-bacterial defect, drives susceptibility. By using a library of bacterial transposon mutants as reporters of host derived stress conditions, we show that granulocytic inflammation generates a nutrient-replete growth-permissive environment for Mtb which obviates the need for essential virulence factors, including the siderophore, mycobactin, and the virulence-related lipid, phthiocerol dimycocerosate, that are otherwise indispensable for establishing infection. A similar inflammatory pathway promotes TB in humans, as human genetic polymorphism that increases 12-LOX expression is associated with TB risk. The concentration of 12-LOX products correlates with neutrophil numbers and bacterial burden in the bronchoalveolar lavage of patients with pulmonary tuberculosis. We propose that Mtb exploits the inflammatory response to preferentially replicate at sites of tissue damage that promote contagion.
A novel antimicrobial agent differentially impacts the immunological response of stimulated bovine monocytes.
Authors: Dawes Maisie Ernestene , Aguilar Jose S , Chitko-McKown Carol G.
Published: January 2023

Despite advances in anti-inflammatory therapy, the US cattle industry now loses over $1 billion annually due to Gram-negative infections. In light of reduced lymphocyte proliferation concurrent with decreased cyclooxygenase-2 and matrix metalloproteinase-9 expression in bovine lactoferrin (bLF)-supplemented cells, we intended to show that bLF and the lactoferricin B (LFcin B) peptide, proven antibiotic agents, can also serve as low-risk anti-inflammatory agents, and hypothesized their ability to influence p38 MAPK signaling which is critical in macrophage-mediated inflammation. While LF is present in neutrophils, it is anticipated that enzymatic activity within infective sites will heighten local LFcin B concentrations.

Following pre-treatment with either bLF, the selective p38 MAPK inhibitor SB203580 (SB), or LFcin B, monocytes isolated from healthy 1 wk. to 5 mo old cattle stimulated with either lipopolysaccharide (LPS) or anisomycin (Aniso), were evaluated for iNOS, IL-1β and TNF-α expression. Preliminary data show that like SB, both bLF and LFcin B significantly abrogate LPS- and Aniso-induced TNF- α expression. In contrast, iNOS expression was increased in cells co-cultured with either LPS or Aniso and each of the three inhibitors. While these results support our hypothesis, additional work is needed to decipher the mechanistic rationale for the immunomodulatory effects of bLF and LFcin B, and the basis for differential cytokine release.
CX3CR1 distinguishes three antigen-experienced CD8 T cell subsets with distinct migratory, functional and homeostatic properties
Authors: Gerlach Carmen , Moseman Ashley , Loughhead Scott , Alvarez David , von Andrian Ulrich H
Published: January 2023

Following infection, pathogen-specific T cells differentiate into diverse short-lived effectors (TEff) that give rise to long-lived memory (TMem) subsets. These T cell fate decisions and lineage relationships are poorly understood, in part, because available subset-defining markers do not adequately reflect existing heterogeneities. Here, we report that CX3CR1 identifies three distinct CD8+ TEff and TMem subsets.

CX3CR1 expression levels on TEff were indicative of TEff differentiation, and predictive of the magnitude and type of memory progeny. Classical central memory (TCM) and effector memory cells (TEM) were CX3CR1– and CX3CR1high, respectively. In addition, we identified a hitherto unrecognized CX3CR1int population. The fraction of CX3CR1int cells among antigen-experienced TMem was remarkably stable over the course of a year, while TEM gradually declined and TCM increased to be the largest subset by 10 months. Interestingly, CX3CR1int TMem had the highest proliferation rate of the three TMem subsets. While TCM and TEM were phenotypically stable over >10 weeks at steady state, CX3CR1int TMem not only self-renewed, but also gave rise to bonafide TCM. This suggests that CX3CR1int TMem may contribute to the gradual increase in TCM. In contrast, re-infection resulted in unidirectional differentiation from TCM to CX3CR1int TMem to TEM.

The newly identified CX3CR1int TMem displayed unique migratory properties. Unlike CX3CR1high TEM, CX3CR1int gradually acquired lymph node homing capability. Moreover, parabiosis experiments and analysis of recirculating memory cells in thoracic duct lymph revealed that CX3CR1int TMem, not TEM, are the predominant T cell subset surveying non-lymphoid peripheral tissues.
High resolution phenotyping identifies NK cell subsets that distinguish healthy children and adults
Authors: Mahapatra Sanjana , Banerjee Pinaki , Minard Charles , Mace Emily , Vargas-Hernandez Alexander , Canter Debra , Forbes Lisa , Makedonas George , Lee Dean , Duryea Teresa , Shearer William T , Orange Jordan S
Published: January 2023

Natural killer (NK) cells possess an array of germline-encoded activating, inhibitory and adhesion receptors, the balance of which in concert with their developmental stage, determine their cytotoxic and cytokine-producing potential. Although NK cells develop in the bone marrow and secondary lymphoid tissues, substantive differentiation is apparent in the peripheral blood including known age-related variation. In order to gain greater insight into phenotypic and functional variation within peripheral blood NK cells across age groups, we used multi-parameter, polyfunctional flow cytometry to interrogate the NK cell variability in 20 healthy adults and 15 5–10, 11–15 and 16–20 year-old children. We found that the normative ranges in both adults and children displayed great inter-individual variation for most subsets. Children possessed >76% unstimulated NK subsets coexpressing perforin and granzyme B as opposed to >65% in adults. Children also had >28% NK subsets expressing all adhesion receptors in our panel compared to half of that in adults. As examples, terminally differentiated CD16+CD57+CD11b+ NK cells were reduced after 16 years of age as well as NKp46, 2B4, CD107a and newly-synthesized perforin in adults. Although CD107a intensity and new perforin synthesis was lower in adults, induced levels of IFNγ (by intensity) was higher. Despite most receptors not differing in their overall expression levels, major phenotypic differences were revealed in the combinatorial expression of NK receptors between adults and children. Our findings identify previously unappreciated NK cell subsets potentially distinguishing children from adults and suggest functional correlates that may have relevance in age-specific host defense.