bluuebunny/crossref_metadata_2025 huggingface dataset:
Extracellular High mobility group box 1-induced epithelial mesenchymal transition through β-catenin pathway in human airway epithelial cells
Authors: Lee Chen-Chen , Chen Yu-Ching , Wu Reen , Wang Chien-Neng
Published: January 2023

High mobility group box 1 (HMGB1) protein belongs to late inflammatory phase cytokine which shows a crucial role in the pathology of chronic pulmonary inflammatory diseases. Epithelial–mesenchymal transition (EMT) is implicated in airway remodeling and lung dysfunction in chronic inflammatory airway diseases. At present, the effects and mechanisms of HMGB1 in EMT of human airway epithelial cells is still unclear. In this study, we found that HMGB1 treatment modulated EMT-related gene expression in human primary airway epithelial cells by RNA sequencing assay. HMGB1 induced downregulation of E-cadherin and ZO-1, and upregulation of vimentin mRNA transcription and protein translation in a dose-dependent manner. To dissect the mechanisms of HMGB1-induced EMT in human airway epithelial cells, we used western blotting, RNA interference and chemical inhibitors to investigate. Treatment with PI3K inhibitor (LY294006) and β-catenin shRNA reversed HMGB1-induced EMT. HMGB1 caused AKT phosphorylation, resulting in GSK3β inactivation, cytoplasmic accumulation, and nuclear translocation of β-catenin to induce EMT in human airway epithelial cells. Additionally, HMGB1 upregulated expression of receptor for advanced glycation products (RAGE), but not that of Toll-like receptor (TLR) 2 or TLR4, and RAGE shRNA attenuated HMGB1-induced EMT in human airway epithelial cells. In conclusion, we found that extracellular HMGB1 induced EMT through RAGE and β-catenin signaling pathway which might deteriorate bronchial epithelium which enhance disease severity and steroid resistance of airway.
HDAC inhibitors enhance CD1d-dependent NKT cell responses to lymphoma
Authors: Tiper Irina V , Webb Tonya J
Published: January 2023

Histone deacetylases (HDACs) influence expression of genes implicated in tumor initiation, progression and anti-tumor responses. We hypothesize that tumors use epigenetic mechanisms to dysregulate CD1d-mediated antigen processing and presentation, which leads to a functional impairment in the ability of Natural killer T (NKT) cells to recognize and destroy cancerous cells. In this study, we treated CD1d-expressing tumor cells with HDAC inhibitors (HDACi) to assess CD1d-dependent NKT cell responses to B cell lymphoma. Consistent with previous studies, we found that treatment with Trichostatin A, a pan-HDACi, enhanced both CD1d and MHC class II-mediated antigen presentation. Similarly, treatment of B cell lymphomas with other HDACi, Panobinostat and Vorinostat, resulted in enhanced CD1d-dependent NKT cell responses. Mechanistically, we found that HDAC2 binds to the CD1d promoter and knockdown of HDAC2 in tumor cells enhanced their immunogenicity. The observed changes are due at least in part to a dose-dependent increase in both CD1d mRNA and CD1d cell surface expression. In addition, treatment with HDCAi resulted in a decrease in inflammatory cytokine secretion by B cell lymphomas. To examine the therapeutic potential of HDACi, we treated human peripheral blood mononuclear cells with TSA and Panobinostat. Treatment with these HDACi did not suppress T cell activation; conversely, treatment with a novel HDAC4 inhibitor MC1568 suppressed T cell responses. Taken together, our studies demonstrate the efficacy of HDACi in restoring anti-tumor responses to B cell lymphomas through both cell-intrinsic and extrinsic factors and strongly implicate a role for select HDACi in cancer immunotherapeutic strategies.
NF-κB and MAPK signaling pathways regulate the IL6 mRNA stability under TLR4 by regulating the expression and degradation of Arid5a
Authors: Nyati Kishan K , Masuda Kazuya , Dubey Praveen , Zaman Mohammad Mahabub-Uz , Kishimoto Tadamitsu
Published: January 2023

AT-rich interactive domain (ARID) 5a controls the IL-6 mRNA stability by binding to its 3′UTR region and preventing it from degradation by Regnase-1. This mechanism was shown to contribute to the elevation of IL-6 levels, which causes the development of autoimmune diseases. However, almost nothing is known about the regulatory mechanisms of Arid5a mRNA and protein expression. In the present study, we have investigated the regulatory mechanisms of Arid5a at transcriptional, post-transcriptional and post-translational levels under TLR4 signaling. We found that in response to LPS stimulation NF-κB binds to the promoter region of Arid5a and induce its gene expression. After expression of Arid5a mRNA, MKP-1 signaling induces AUF-1 to translocate from the nucleus to the cytoplasm where it binds to the 3′UTR region of Arid5a and resulting in its destabilization. Inhibition of MKP-1 and AUF-1 results in the stabilization of Arid5a mRNA, which leads to higher expression of IL6, suggesting that this pathway has a significant role in regulating IL6 mRNA through Arid5a. We also show that during the late phase of LPS stimulation, p38 MAPK phosphorylates the Arid5a protein at Ser 253, 433 and 458 positions that lead to its ubiquitination at lysine 80 and 93 positions resulting in degradation. Inhibition of Arid5a phosphorylation and degradation causes the over-production IL6 mRNA further supports the importance of Arid5a in the regulation of IL6. Our data demonstrate that LPS signaling controls IL6 mRNA stability; therefore, its regulation through up-regulation of Arid5a by NF-κB at the early phase, and down-regulation of Arid5a at the mRNA and protein level by MKP-1-mediated AUF1 and P38 MAPK signaling, respectively at the later phase.
Controls of MDA5 Activation by The 14-3-3 Protein Family
Authors: Liu Helene Minyi , Lin Chih-Pu
Published: January 2023

During RNA virus infection, RIG-I like receptors (RLRs), such as Retinoic acid-inducible gene I (RIG-I) and Melanoma differentiation-associated protein 5 (MDA5), recognize cytoplasmic viral RNA and activate downstream adaptor proteins to produce type I interferon (IFN). RIG-I is known to bind short dsRNA (<200 bp) whereas MDA5 prefers to bind to dsRNA which are longer than 2000 bp. It has been reported that RIG-I can interact with TRIM25, which is the ubiquitin E3 ligase of RIG-I during viral infections, and with the chaperon protein 14-3-3ɛ, which facilitate RIG-I translocate to mitochondria. However, the molecular mechanisms of MDA5 activation remain unclear. Here we show that certain 14-3-3 isoforms are crucial for MDA5-dependent type I IFN induction. Transfection of dsRNAs in different lengths to Huh7 RIG-I knocked-down cells reveals that MDA5 may specifically binds to long dsRNA and activate IFNβ promoter activity 3 hours post-RNA transfection. Also, overexpression of full-length MDA5 or the N-terminal CARD domains may constitutively activate IFNβ promoter activity, while C-MDA5 lacking both CARD did not show any ability to signal itself. We then applied dsRNA transfection or MDA5 overexpression to assess which 14-3-3 isoform is critical for MDA5 activation. We found that certain 14-3-3 isoform may bind to full-length MDA5 and N-MDA5 shown by co-immunoprecipitation, and ectopic expression of 14-3-3 may enhance MDA5-dependent activation of IFNβ promoter. Further studies of the molecular mechanisms how 14-3-3 proteins participate and regulate in the MDA5-dependent signaling pathway will be investigated in order to define the critical steps of MDA5 activation during RNA virus infections.
Eosinophil migration is regulated by the transcription factor Aiolos
Authors: Bouffi Carine , Schollaert Kaila L , Morris David W , Fulkerson Patricia C
Published: January 2023

Accumulation of an abnormally high number of eosinophils (Eos) in tissues often results in damage and organ dysfunction in allergic disease. Preclinical and clinical studies have established that CCR3 ligands (CCL11, CCL24, CCL26) are critical for disease-associated tissue eosinophilia. Yet, blockade of the individual mediators (e.g. CCR3, CCL11) has been insufficient to completely resolve tissue eosinophilia, likely due to simultaneous local expression of multiple Eos-attracting mediators acting in parallel. Thus, delineating the molecular mechanisms that control Eos trafficking into tissues is clinically significant. We recently reported that the transcription factor Aiolos is expressed in murine Eos progenitors (EoPs) as well as mature Eos in mice and humans. We now show that the migratory response of Aiolos-deficient (AiolosKO) Eos is markedly impaired toward CCR3 ligands in vitro and in vivo, along with significantly reduced mRNA and surface expression of CCR3. Notably, migration of AiolosKO Eos toward leukotriene B4 is significantly reduced, suggesting that Aiolos-deficiency results in a global migratory defect. We also demonstrate a profound reduction in Eos accumulation in the small intestine of AiolosKO mice. These latter findings provide in vivo relevance to our findings as intestinal eosinophilia is CCL11 and CCR3 dependent. We further show that migration of adoptively transferred AiolosKO Eos into the airway of allergen-challenged mice was significantly reduced when compared to wild-type Eos in an experimental model of asthma, highlighting an intrinsic defect in tissue trafficking in the AiolosKO Eos. Collectively, our studies implicate Aiolos as a global regulator for eosinophil migration.
Global analyses of monocyte subsets revealed age-related alternations after stimulation of pathogen recognition receptors
Authors: Metcalf Talibah , Wilkinson Peter , Wertheimer Anne , Nikolich-Zugich Janko , Haddad Elias
Published: January 2023

In the aging population, intricate interactions between the innate and adaptive immune response is not as effective compared to younger adults. Monocytes play an important role in defense against microbes and express an array of pattern recognition receptors (PRRs). We applied a comprehensive approach to evaluate the effects of a broad range of innate immune agonists (LPS, CLO97, and 5′pppRNA) on monocyte subsets isolated from healthy non-frail adults and old subjects. We analyzed transcriptome data and measured cytokine and chemokine, ROS, and NO production. Under ex vivo conditions, we observed a larger proportion of genes were specifically upregulated in the classical (CD14+CD16−) and non-classical (CD14dimCD16+) monocytes while the intermediate (CD14+CD16+) monocytes showed a smaller proportion of upregulated genes. Distinct genes observed across both age groups for CD16+ monocytes included: CX3CR1, IL21R, IFITM1/2/3, SIGLEC10, and SOD1; whereas CD16− monocytes expressed CCR1/2, SELL, CD64, CD93, CD36, and CD14. Analysis of the transcriptional profiles elicited by innate agonists revealed age-related alternations in functional pathways. After stimulation of RIGI with 5′pppRNA, we observed that classical monocytes isolated from adults uniquely expressed the RIGI MDA5 mediated induction of IFNα and IFNβ pathway, as well as the IFNα transcript, which corresponded to higher levels of IFNα production. In addition, agonists elicited higher levels of IFNγ from non-classical monocytes isolated from adults. Our findings represent a comprehensive analysis of the influence of human aging on PRRs signaling and have implications for strategies to enhance the immune response in the context of infection or immunization.
Minocycline promotes the generation of dendritic cells with regulatory properties
Authors: Lee Chong-Kil , Kim Narae , Im Sun-A , Lee Jae-Hee , Park Chan-Su , Song Sukgil
Published: January 2023

Minocycline, which has long been used as a broad-spectrum antibiotic, also exhibits non-antibiotic properties such as inhibition of inflammation and angiogenesis. In this study, we show that minocycline significantly enhances the generation of dendritic cells (DCs) from mouse (BM) cells when used together with GM-CSF and IL-4. DCs generated from BM cells in the presence of minocycline (Mino-DCs) demonstrate the characteristics of regulatory DCs. Compared with control DCs, Mino-DCs are resistant to subsequent maturation stimuli, impaired in MHC class II-restricted exogenous Ag presentation, and show decreased cytokine secretion and allogeneic T cell stimulation in vitro. Mino-DCs also have a decreased ability to prime alloantigen-specific T cells when transferred to allogeneic mice. Splenic Mino-DCs generated in mice by injecting minocycline together with GM-CSF are also severely impaired in priming alloantigen-specific T cells in allogeneic mice. In allogeneic mice, Mino-DCs induce expansion of CD4+CD25+Foxp3+ T regulatory cells. Our study identifies minocycline as a new pharmacological agent that could be potentially used to increase the production of regulatory DCs for cell therapy to treat autoimmune disorders, allergy, and transplant rejection.
Inhalation injury causes recruitment of IL-12lo neutrophils late after injury
Authors: Kartchner Laurel B , Dunn Julia L , Stepp Wesley H , Vaughan Rachel , Glenn Lindsey I , Maile Robert , Cairns Bruce A
Published: January 2023

Inhalation injury causes increased morbidity and a 20% increase in mortality following burn injury. Although it is known that inhalation injury causes immune dysfunction among patients, few animal models of inhalation injury examine changes in the immune profile following inhalation injury. Our lab generated a clinically relevant murine model using female C57BL/6 mice (8–12 weeks, >18g) that were intubated and subjected to a woodsmoke inhalation injury. Additionally, a subset of mice were subjected to a combined injury consisting of inhalation and burn injury (20% total body surface area). Increased mortality was only observed following combined injury (50% mortality within 5 days of injury, p<0.05). Inhalation injury alone caused significant weight loss, peaking at 14 days post injury (p<0.05). Bronchioalveolar lavage (BAL) performed 16 days post injury demonstrated increased cell recruitment following inhalation injury (1.5 fold, p=0.15) and combined injury (2.4 fold, p<0.05) compared to sham controls. Cells recruited to the airway were identified as neutrophils and macrophages by flow cytometry. Intracellular staining demonstrated that neutrophils, but not macrophages, exhibited decreased expression of IL-10 and IL-12 following inhalation or combined injury. In our model, inhalation injury and combined injury cause cellular recruitment to the lungs and decreased production of cytokines known to be important for immune function. Thus we propose to use this model as a means to determine if changes to neutrophil activity contribute to increased susceptibility to bacterial infections and poor outcomes following inhalation or combined injury.
Cynomolgus macaque neutrophils express IL-4 and IL-10 in response to Mycobacterium tuberculosis bacilli
Authors: Mattila Joshua T , Flynn JoAnne L
Published: January 2023

Neutrophils are associated with increased disease severity in tuberculosis (TB) but mechanisms underlying their contribution to pathology are poorly understood. In granulomas, neutrophils localize to regions that are adjacent to macrophages, and we hypothesize that neutrophil-expressed cytokines can modify anti-mycobacterial immune responses. To investigate this, we examined pro- and anti-inflammatory cytokine production by neutrophils in Mycobacterium tuberculosis (Mtb) infected macaques, a nonhuman primate that replicates human TB. We found that intact bacteria were potent inducers of anti-inflammatory cytokine expression and viable Mtb elicited IL-10 while dead Mtb induced IL-4 expression. Bacterial stimulation and mycobacterial culture filtrate protein (CFP) induced TNF in peripheral blood neutrophils. Cytokine expression was not induced by stimulation with anti-CD3 antibody, poly I:C, or purified Mtb peptides and was independent of host infection status, suggesting this is an intrinsic property of neutrophils driven by mycobacterial TLR2/4 ligands. There was little polyfunctionality in neutrophils and most cells expressed either IL-4 or IL-10, or these cytokines in combination with TNF. Neutrophils were less efficient than T cells at cytokine production and expressed less cytokine on a per cell basis. Although there are more T cells than neutrophils in granulomas, we found similar frequencies of IL-4 or IL-10 expressing T cells and neutrophils, and identified IL-10+ neutrophils in caseum adjacent to epithelioid macrophages, a region associated with extracellular Mtb. These results suggest neutrophils are underappreciated sources of immunosuppressive cytokines that contribute to poor outcomes in TB.
Systemic infection ruined NK cell-mediated help on influenza virus-specific CTLs
Authors: Zheng Jian , Liu Yuan , Liu Yinping , Huang Lei , Xiang Zheng , Lam Kwok-Tai , Lv Aizhen , Mao Huawei , Lau Yu-Lung , Tu Wenwei
Published: January 2023

Host resistance against influenza A virus infection relies on the efficient collaboration of innate and adaptive immune response. Being a major component of innate immune system, NK cells have been confirmed to limit the replication and spread of influenza virus during early infection. However, their roles in the generation of virus-specific CTLs response still remain exclusive. Consistent with our in vitro study on human NK cells, here we firstly confirmed that murine NK cells could be directly infected with mouse-adaptive influenza A PR8 virus in vivo under both systemic and local infection. However, only systemic infection resulted in severe reduction and dysfunction of spleen NK cells, which was accompanied with the poor antigen-specific killing and IFN-γ secretion rather than the decrease in quantity of CTLs during the acute phase of infection. Importantly, the adoptive transfer of spleen NK cells from mice suffering from local infection rescued the impaired CTLs in systemic influenza virus-infected mice, whereas no similar effects could be observed by using NK cells from healthy mice. Taken together, our results demonstrated that NK cell-mediated help on the generation of functional influenza virus-specific CTLs was infection-dependent and determined by the dose of infection. This study provided some important information for developing novel NK-cell-targeted strategy against severe influenza infection.
CX3CR1 regulates type 2 immune response in respiratory syncytial virus infected newborn mice
Authors: Das Sudipta , Raundhal Mahesh Ramdas , Chemmannur Sijo Varghese , Huff Rachael , Morse Christina , Ray Anuradha , Ray Prabir
Published: January 2023

Respiratory syncytial virus (RSV) infection can cause severe bronchiolitis and pneumonia in infants requiring hospitalization. In mouse models of RSV infection, multiple studies have highlighted the ability of RSV to induce a type 2 immune response with associated mucus production and airway inflammation. Structurally RSV has two surface glycoproteins, F and G in the viral envelope. Previous studies have shown that the G-protein helps the virus to attach to the host cell by binding to the chemokine receptor CX3CR1 due to the presence of a CX3C motif and this interaction appears to be linked in the pathogenesis of RSV disease. However, the pathways that regulate RSV-induced immune response and pulmonary pathology are not well understood. Therefore, to study the functional relevance of CX3CR1 in the context of viral clearance and host pathology, we used CX3CR1gfp/gfp knock-in mice in which the homozygous mutant mice lack a functional CX3CR1 receptor. 5 day old newborn mice were infected with RSV intranasally and the lungs were harvested to isolate RNA and protein 4 day post infection. We observed that unlike WT mice, CX3CR1gfp/gfp mutant mice fails to induce the expression of type 2 cytokines IL-5 and IL-13 upon RSV infection. Additionally, two key genes Muc5ac and Gob5, involved in cell hyperplasia, mucus production and airway inflammation were found to be induced upon RSV infection in WT while those were suppressed in mutant mice. These observations suggest a regulatory function of CX3CR1 in the establishment of RSV-mediated type 2 immune response in the host. Our ongoing studies are focused on elucidating the mechanism by which CX3CR1 signaling regulates type 2 immune response and associated pathology in the RSV-infected host.
Early onset and persistence of metabolic alterations in exhausted T cells is regulated by PD-1
Authors: Bengsch Bertram R , Johnson Andy L , Kurachi Makoto , Odorizzi Pamela , Pauken Kristen E , Attanasio John , Wherry E. John
Published: January 2023

Dynamic reprogramming of metabolism is essential for T cell effector function and formation of memory. However, regulation of cellular metabolism in exhausted T cells in chronic infections and cancer is poorly understood. Here we found that as early as the first week of chronic LCMV infection, before severe T cell dysfunction becomes established, virus-specific CD8 T cells are already unable to match the bioenergetic demands of effector CD8 T cells generated during acutely resolving LCMV infection. Suppression of T cells bioenergetics involves restriction of glucose uptake and utilization, despite the up-regulation of multiple other metabolic pathways. The inhibitory receptor PD-1 controlled the development of this early glycolytic defect as well as mitochondrial mass and quality in the presence of persisting mTOR signaling. The suppression of glycolysis and mitochondrial metabolism in exhausted T cells persists into established chronic infection. Therapeutic reinvigoration of exhausted T cells by PD-L1 blockade reprogrammed the metabolism of PD-1Int but not the terminal PD-1Hi subset of exhausted T cells. These data highlight a key metabolic control event early in T cell exhaustion that precedes major transcriptional changes. Our findings also suggest that manipulating metabolism in combination with checkpoint blockade may enhance therapeutic outcomes.
Identification of a locus on chromosome 1 regulating IL-17-producing γδ T cells and mortality after influenza infection.
Authors: Dienz Oliver , DeVault Victoria L , Wallace Aaron M , Suratt Benjamin T. , Boyson Jonathan E
Published: January 2023

γδ T cells are an evolutionary conserved T cell subset with innate-like effector functions. We had previously identified a locus on chromosome 1 encompassing the Slam gene family that regulates the innate-like T cell compartment. To characterize this region further, we generated a B6.129 congenic mouse strain in which a 1.1 Mbp region (chr. 1: 171.05–172.12) from the 129X1/SvJ strain was introgressed onto the C57BL/6J background, and evaluated its effect on the host immune response in influenza-infected mice. Upon infection, we observed a significant and sustained increase in the number of lung γδ T cells in B6.129c2 congenic mice. A comparison of γδ T cell function revealed that a significantly greater fraction of B6.129c2 γδ T cells were producing IL-17A versus their B6 counterparts. These differences were subset-specific as they were observed in Vγ4 and at least one other Vγ subset, but not in Vγ1 T cells. Those IL-17-producing Vγ subsets accounted for most of the increase in B6.129c2 lung γδ T cell numbers and they displayed an attenuated cell surface expression profile of Slam family receptors. Finally, since enhanced IL-17 responses have been associated with immunopathology after influenza infection, we assessed the effect of the congenic interval on survival. We found that the survival of B6.129c2 congenic mice is significantly lower compared with B6 mice after influenza infection. Taken together, our data suggest that natural genetic variations at this locus significantly impact both IL-17A production by lung γδ T cells and mortality after influenza infection.
Modulation of cellular immune response by 2′-5′ Oligoadenylate Synthetase - like (OASL) proteins during DNA virus infection
Authors: Ghosh Arundhati , Sampath Padmavathi , Zhu Jianzhong , Hornung Veit , Thorne Stephen , Sarkar Saumendra N
Published: January 2023

Oligoadenylate Synthetase-like (OASL) is a type I interferon (IFN) stimulated gene, strongly induced following virus infection, but lacks enzymatic activity to synthesize 2′–5′ oligoadenylates. We show that human OASL, and its functional ortholog mouse Oasl2 are critical modulators of cellular antiviral signaling. During RNA virus infection, OASL binds the RNA-sensor, RIG-I, and enhances RIG-I signaling by mimicking one of the RIG-I-ligands, K63-linked polyubiquitin [p(Ub)], thus exerting an antiviral activity. In contrast, by binding the DNA-sensor, cyclic GMP-AMP synthase (cGAS), OASL negatively regulates IFN induction and restricts inflammation in the context of DNA virus infections, some of which can establish persistent infection.

Human OASL or mouse Oasl2 knockout cells showed enhanced IFN induction when stimulated either with intracellular DNA or DNA viruses such as Vaccinia and Herpes simplex virus. Consequently, Oasl2 knockout mice showed delayed and reduced Vaccinia virus spread and mortality. Additionally, OASL or Oasl2 affected IFN induction when stimulated with intracellular DNA, but not with cGAMP, indicating the upstream effect of OASL is on cGAS stimulation and not on STING stimulation. Mechanistically, OASL specifically interacted with cGAS, and inhibited its activity. Together these results establish the unique ability of OASL to differentially modulate RNA and DNA virus replication and IFN induction.
NLRP3 plays a major role in inflammasome activation during Mycoplasma pneumoniae infection
Authors: Segovia Jesus A , Chang Te-Hung , Pandranki Lavanya , Kannan Thirumalai R , Baseman Joel B
Published: January 2023

Mycoplasma pneumoniae (Mp) is a common bacterial pathogen that causes acute and chronic airway diseases in humans and is a leading cause of bacterial community-acquired pneumonia and is implicated in initiation and exacerbation of asthma. Mp infection triggers a strong inflammatory response driven by interleukin-1β (IL-1β). Mp produces the Community-Acquired Respiratory Distress Syndrome (CARDS) toxin, which is highly expressed during infection and specifically activates the nod-like receptor family protein 3 (NLRP3) inflammasome. However, the significance of the NLRP3 inflammasome during Mp infection is unclear. The goal of our study is to characterize the inflammasome-mediated response during Mp infection.

Wild-type (WT) and NLRP3 knockout (KO) bone-marrow derived macrophages (BMDMs) were infected with Mp and levels of proinflammatory cytokines and chemokines were measured by ELISA and immunoblot. Additionally, WT and NLRP3 KO C57 mice were inoculated intranasally with Mp and analyses were performed on cytokines and lung histopathology.

Following Mp infection, NLRP3 KO BMDMs secreted significantly less IL-1β compared to WT BMDMs, yet IL-6 levels were similar. Analyses of bronchoalveolar lavage and lung tissue from Mp-infected WT and NLRP3 KO mice revealed a unique proinflammatory cytokine response, considerably enhanced disease and impaired bacterial clearance in NLRP3 KO mice.

Our studies identified NLRP3 as a critical regulator of proinflammatory cytokine response and pathology during Mp infection. Future studies will focus on characterizing immune cell trafficking in the lungs during Mp infection and determining if Mp-related inflammasome activity is an ontogenic factor in initiating and exacerbating asthma.
Opposing roles of AIM2 inflammasome and type I interferon signaling in Francisella infection
Authors: Zhu Qifan , Man Si Ming , Karki Rajendra , Kanneganti Thirumala-Devi
Published: January 2023

Inflammasomes are crucial for mediating protection against microbial infection. Type I interferon (IFN) signaling is required for driving activation of the AIM2 inflammasome in macrophages in response to infection by Francisella tularensis subspecies novicida (F. novicida). Here, we showed that compared with infected wild-type mice, mice lacking the type I IFN receptor IFNAR2 exhibited increased resistance towards F. novicida infection, whereas mice lacking AIM2 were more susceptible. In addition, mice lacking IRF9, a core component of IFN-stimulated gene factor 3 that transduces type I IFN signaling, showed increased resistance following infection. These results suggested that while type I IFN signaling and AIM2 were essential for activation of the Francisella-induced inflammasome in macrophages, these components have strikingly contrasting effects in vivo. Importantly, we found that mice lacking both AIM2 and IFNAR2 were more resistant than infected wild-type mice and mice lacking AIM2. Taken together, we identified type I IFN signaling as a dominant regulator of anti-microbial activity during F. novicida infection in vivo even in the absence of AIM2 responses.
A protective role of hepcidin in bacterial pneumonia
Authors: Chen QiXing , Yang Yang , Song Shengwen , Fang XiangMing , Shu Qiang
Published: January 2023

Objective

Hepcidin is a b-defensin-like antimicrobial peptide and acts as a principal iron regulatory hormone. Hepcidin is mostly produced by hepatocytes, but is also expressed by other cells, such as airway epithelial cells. However, nothing is known about its function in lung infectious disease such as bacterial pneumonia.

Methods

Pneumonia was induced by intratracheally administration of Gram-negative bacterium Escherichia coli or Pseudomonas aeruginosa, or Gram-positive bacterium Staphylococcus aureus. Hepcidin level in lung tissue was measured by means of immunohistological staining. Hepcidin-overexpressing adenovirus and control adenovirus were intratracheally injected into mice. The survival rates of hepcidin genetically engineered mice suffered from pneumonia were monitored for 72 hours.

Results

Hepcidin was mainly expressed on airway epithelial cells in mouse lung tissue. The hepcidin levels were gradually decreased after pneumonia onset, and recovered at 48 hours after Escherichia coli and Staphylococcus aureus infection, and at 72 hours after Pseudomonas aeruginosa infection. Hepcidin knock-out mice were more susceptible to Escherichia coli pneumonia and only 37.5% of them were survived for 72 hours (80.0% for wild-type mice; P<0.05). In contrast, intratracheal overexpression of hepcidin protected mice from lethal Escherichia coli infection (survival rate: 100.0% for hepcidin overexpressing mice, and 41.7% for control mice; P<0.01).

Conclusions

Hepcidin plays an important role in protection against bacterial pneumonia, and the mechanism needs further investigation.
Mannose-binding lectin (MBL) is the main complement activator on Aspergillus fumigatus conidia under immunocompromised conditions
Authors: Rosbjerg Anne , Genster Ninette , Hansen Katrine Pilely , Skjoedt Mikkel-Ole , Gal Peter , Stahl Gregory L , Garred Peter
Published: January 2023

Aspergillus fumigatus is normally a non-pathogenic fungus, but in immunocompromised patients this fungus becomes highly pathogenic. Complement activation are thought to be of crucial importance for elimination of A. fumigatus. However, which of the three complement pathways that are important under immunocompromised conditions are only partly understood.

Conidia from a clinical isolate of A. fumigatus were co-incubated with serum before complement activation products C4b, C3b and TCC were measured in flow cytometry. Besides normal (NHS) and complement deficient sera plus complement inhibitors, we used umbilical cord serum (UCS) to mimic a compromised immune defense reflected in the low immunoglobulin (Ig) levels.

In NHS, the classical pathway was the dominant initiator with the alternative pathway providing a strong C3b amplification. Classical pathway is known to be initiated via C1q binding to Ig, whereas MBL and ficolin-2 from the lectin pathway bind the fungus directly. UCS conveyed low IgG and no IgM binding to the conidia, and we observed that complement activation on A. fumigatus in UCS was predominantly MBL dependent. UCS with low MBL had significantly lower C3b deposition than serum from NHS with low MBL. Moreover, a close positive correlation between MBL levels and C3b deposition in UCS (Spearman r = 0.79, p < 0.0001, n = 23) was observed. The significance of MBL was emphasized by decreased neutrophilic phagocytosis of conidia via MBL inhibition in C1q−/− serum.

We conclude that MBL is the main complement activator and phagocytosis initiator of A. fumigatus conidia in individuals with low Ig levels. This suggests a crucial role for the lectin pathway in protection against infection of A. fumigatus in immunocompromised patients.
Identification of an amphipathic helix within IFITM3 that promotes inhibition of influenza virus
Authors: Chesarino Nicholas M , McMichael Temet M , Zhang Lizhi , Davis Matthew J , Soewarna Victoria , Doering Rachel F , Yount Jacob S
Published: January 2023

Interferon-induced transmembrane protein 3 (IFITM3) inhibits influenza virus fusion with endosomes by altering cellular membrane properties through mechanisms that are largely uncharacterized. In order to identify regions of secondary structure within IFITM3 that may contribute to its mechanism of action, we performed structural predictions using the I-TASSER and PSSpred algorithms. Insterestingly, we identified a 10-amino acid region of likely helicity that possesses an amphipathic distribution of amino acids. Consistent with the known role of amphipathic helices in altering membrane properties, we observed that cells expressing a helix-deleted mutant of IFITM3 were infected with influenza virus at a 4-fold higher rate than cells expressing WT IFITM3. Confirming that the amphipathicity of this helix contributes to antiviral activity, single, double and triple alanine mutations of the three residues making up the hydrophilic face of the helix progressively decreased antiviral activity of IFITM3, as did single polar mutations in the hydrophobic face of the helix, or single amino acid insertions that disrupted amphipathicity. Amino acid sequence alignments suggest that amphipathicity in this region is highly conserved in IFITMs throughout evolution. Overall, we utilized bioinformatics methods coupled with functional infectivity assays to provide the first evidence that IFITM3 utilizes an amphipathicity-based mechanism for inhibition of viruses.
Reciprocal regulation of Th17 and Treg differentiation by PKC-θ-phosphorylated steroid receptor co-activator1
Authors: Sen Subha , Sun Zuoming
Published: January 2023

Interleukin 17 (IL-17) producing T helper cells (Th17) differentiation is regulated partially by TCR stimulated PKC-θ activation. In this report, we show that steroid receptor co-activator1 (SRC1), a key RORγT transcriptional co-activator is phosphorylated by PKC-θ and acts as a critical modulator to induce Th17 development while potentially inhibiting iTreg development. SRC1 phosphorylated by PKC-θ is required for optimal interaction between SRC1 and RORγT, the lineage defining transcription factor, to stably bind to the consensus DNA sites and induce productive transcriptional outcome. Moreover, SRC1 recruits CARM1, a protein arginine methyltransferase, to enhance the RORγT target site histone arginine methylation, thereby maintaining the target site availability for RORγT and hence contributing additionally to further stimulate the magnitude of IL-17 production. Herein we also reveal for the first time, that phosphor-SRC1 can reverse the inhibitory interaction between Foxp3 and RORγT leading to the displacement of inhibitory Foxp3 and thereby reversing the suppressive antagonizing effect of Foxp3 on RORγT, favoring Treg to Th17 balance shift. In mice, lack of SRC1 resulted in diminished Th17 development but enhanced Treg differentiation, resulting in better protection from autoimmune encephalomyelitis. Thus, PKC-θ modified SRC1 is identified as a major signaling checkpoint which determines the Th17-iTreg differentiation fate diverging from a common developmental pathway.
M. smegmatis vaccination reveals the mycobacterial ribosome as a potential CD4+ Tcell enhancing vaccine target
Authors: Kennedy Steven C , Johnson Alison J , Sette Alessandro , Chan John , Jacobs William R , Porcelli Steven A
Published: January 2023

Mycobacterium tuberculosis(Mtb) is an airborne pathogen that infects one third of the world’s population. To date no vaccine has had major success in reducing the enormous global burden of disease from this infection. Critical to the control of Mtb is the ability of a vaccine to induce a strong CD4+ Tcell response. The currently available vaccine, M. bovis BCG, is of limited efficacy, potentially due to its retention of immune evasion mechanisms that actively inhibit the host presentation of critical antigens to CD4+ Tcells. Examining antigens that are hidden during a BCG immunization may lead to identification of novel vaccine targets. Our lab has previously identified an M. smegmatis based vaccine, IKEPLUS, which elicits a strong CD4+ Tcell response leading to significantly enhanced protection to Mtb over BCG vaccination. M. smegmatis is a non-pathogenic mycobacterium that has not retained the ability to disrupt antigen presentation to the host immune system. Examination of the CD4+ Tcell response to IKEPLUS and BCG has identified the mycobacterial ribosome as a major immunogenic target in the M. smegmatis based IKEPLUS, but not in BCG. This dichotomy in the immune response led us to further characterize the ribosome and evaluate its protein constituents as potential vaccine targets. The ribosome is composed of 57 individual proteins; of these 57 proteins, we have identified 20 as immunogenic, and identified minimal CD4+ Tcell epitopes for 10 of the 20 identified proteins. These findings have led us to begin development of a mycobacterial ribosome based vaccine to enhance the currently existing BCG regimen. This approach will allow us to complement what we believe is a critical flaw of BCG while retaining the widely used BCG vaccination regimen.
IgG1 and Th1 responses to DosR, Rpf and active growth phase antigens of Mycobacterium tuberculosis in Brazilian tuberculosis patients.
Authors: Teixeira Henrique Couto , Mattos Ana Márcia Menezes , Almeida Caroline Souza , Abad Lily Paola Martinez , Franken Kees , Ottenhoff Tom H. M.
Published: January 2023

Tuberculosis (TB) is serious problem worldwide. New antigens from Mycobacterium tuberculosis are being investigated in order to improve the diagnosis of latent TB infection and TB disease. In this study, serum levels of IgG1 antibodies against active growth phase antigens (ESAT-6/CFP-10, Rv0717 and Rv3353), dormancy-related (DosR) antigens (Rv1733, Rv1737, Rv2029 and Rv2628), and resuscitation promoting factors (Rpfs Rv0867 and Rv2389) were evaluated in patients with TB before and after chemotherapy using ELISA. Antigen-induced IFN-γ, TNF-α, IL-17, IL-10 and IL-4 were evaluated in supernatants of PBMC cultures. Our results indicate that patients with pulmonary TB express high levels of IgG1 antibodies against ESAT-6/CFP-10, Rv0717, Rv3353, Rv1733, Rv2029 and Rv2628 Rv0867 in the active phase of the disease in comparison to healthy controls (p<0.001). These levels declined to control levels after the completion of six months treatment. ROC analysis confirmed the good performance of Rv0717, Rv1733, Rv3353, Rv0867, Rv2029 and Rv2628 antigens. Interestingly, Rv0717 and Rv1733 antigens induced an IgG1 peak response after 1–3 months of chemotherapy (P<0.01). Similarly, IFN-γ and TNF-α peaked early during treatment in response to ESAT-6/CFP-10, Rv1733 and Rv2029, and declined to control levels after 6 months of chemotherapy. The study groups did not differ in respect to IL-17, IL-10 and IL-4. Taken together, these data further reinforce a possible correlation of IgG1 production with Th1 responses in TB. Moreover, detecting IgG1 antibodies against M. tuberculosis antigens, including DosR and Rpf proteins, may represent an additional tool in the diagnosis of tuberculosis.

Financial support: FAPEMIG, CNPq and CAPES, EU and NWO-TOP.
Possible link between cystic fibrosis specific lipid A adaptations by Pseudomonas aeruginosa and disease associated hyperinflammation.
Authors: SenGupta Shuvasree , Uriarte Silvia M. , Ernst Robert K. , Mitchell Thomas C
Published: January 2023

Pseudomonas aeruginosa infection in cystic fibrosis (CF) lung disease causes airway neutrophilia and hyperinflammation without being cleared effectively. We evaluated the immunostimulatory activities of lipid A variants synthesized by P. aeruginosa (PA) exclusively in CF patients to determine if they correlate with CF disease severity and progression. One third of the PA isolates from CF patients with late severe stage express a unique hepta–acylated lipid A; hepta-1855 (m/z=1855) isoform. In primary cell cultures, we found that hepta-1855 functioned as a potent TLR4 agonist by priming neutrophil respiratory burst and stimulating strong chemokine response (interleukin-8 or IL-8) from monocytes and neutrophils. Hepta-1855 also had a potent survival effect on neutrophils. However, it was less efficient in inducing neutrophil granule exocytosis and was also less potent in triggering pro-inflammatory TNF-α response from monocytes. A precursor hexa-1616, found in hepta-1855 containing LPS mixtures, did not have direct inflammatory activity on neutrophils but induced moderate IL-8 response. Together, our data suggest a potential contribution of hepta-1855 to late CF stage associated excessive inflammatory burden by recruiting neutrophils via IL-8 and promoting their maintenance through its survival effect. Being a chemoattractant stimulus, hexa-1616, may serve as an accomplice to hepta-1855. Moreover, the relative inefficiency of hepta-1855 in triggering degranulation may partly explain the persistence of PA in CF disease in spite of neutrophil dominated inflammation.
HLA-DR3 transgenic mice expressing Nur77-GFP reveal early and robust activation of T cells by superantigens during Staphylococcus aureus pneumonia.
Authors: Rajagopalan Govindarajan , Karau Melissa , Krogman Ashton , Patel Robin , David Chella S
Published: January 2023

The role of superantigens (SAg) in the immunopathogenesis Staphylococcus aureus pneumonia has been shown by several indirect means. To directly demonstrate activation of T cells in vivo during staphylococcal pneumonia by SAg and to study the kinetics of T cell activation, HLA-DR3+.Nur77-eGFP+ mice were used. In these mice, rapid expression of green fluorescent protein (GFP) in T cells occurs only upon activation through the TCR, but not following other inflammatory stimuli. Experimental pneumonia was induced using isogenic strains of S. aureus either producing the SAg staphylococcal enterotoxin B (SEB), or not producing any SAg. Mice were sacrificed at different time points and expression of GFP was determined by flow cytometry. As SEB primarily activates CD4+ and CD8+ T cells bearing TCR Vβ8, the expression of GFP within the TCR Vβ8+ sub-populations was examined. Strong upregulation of GFP in the TCR Vβ8+ sub-population was appreciable in both CD4+ and CD8+ T cell subsets (20–25%) even as early as 4 hours only in mice infected with S. aureus strain producing SEB (SEB+SA), but not those infected with the isogenic strain that does not produce SEB (SEB-SA). Serum cytokine/chemokine levels mirrored this finding. By 24 hours, 80–90% of the CD4+ and CD8+ T cells bearing TCR Vβ8+ expressed GFP in mice infected with SEB+SA, compared to only 1–2% in mice infected with SEB-SA. The pattern of expression of CD69 expression, an early T cell activation marker, was distinct from that of GFP. Our novel study directly demonstrated T cell activation and established their activation kinetics in vivo during staphylococcal pneumonia. These results might be useful for timing of administration of novel drugs aimed at suppressing T cell activation during pneumonia.
A multi-prong global study provides insights to gut microbiota and mucosal immunity in dusp6-deficiency conferred obesity-resistance.
Authors: Ruan JW , Statt S , Huang CT , Tsai YT , Chan HL , Tan TH , Kao CY
Published: January 2023

Obesity is now a plague in developed countries and more than one third of U.S. adults are obese. Gut microbiota plays profound roles in host energy harvest, metabolism and inflammatory response associated with the development of obesity. Dusp6-deficient mice have been shown to be resistant to diet-induced-obesity (DIO) but the mechanism is still unclear. Using a gnotobiotic mouse model, we found that the transplantation of fecal/gut microbiota derived from dusp6-deficient mice can significantly increase energy expenditure and reduce weight-gain of the recipient mice. By analyzing fecal 16s rRNA genes, dusp6-deficient mice have different composition of gut microbiota when compared with wild-type mice. We also demonstrated that dusp6-deficient mice were resistant to DIO-mediated dysbiosis of gut microbiota. Upon further analysis of the intestinal transcriptome, we found that dusp6 deficiency is an important regulator of several metabolic pathways and genes related to intestinal barrier. Moreover, our analyses have shown that dusp6-deficient mice could alleviate DIO-induced inflammation.. This study demonstrates that dusp6 deficiency is a strong genetic factor that dominates diet effect on shaping gut microbiota and it remodels mucosal immunity to retain HFD-resistant homeostasis of gut microbiota. Our findings shed lights on obesity and obesity-derived metabolic disease treatment with novel therapeutic candidates/strategies such as DUSP6 pharmacological inhibitors and the development of microbiota-based therapeutics.
The presence of microbiota protects against Pseudomonas aeruginosa induced keratitis
Authors: Kugadas Abirami , Gadjeva Mihaela
Published: January 2023

The impact of microbiota on ocular immunity is unclear. We report that in health, the presence of microbiota strengthens the ocular innate immune barrier function and promotes resistance to infection. Consistent with this view, Germ-Free mice (GF) on a SW background that are typically resistant to P. aeruginosa-induced keratitis were rendered susceptible by the lack of microbiota. This was exemplified by increased corneal bacterial burden and elevated pathology of the GF when compared to conventionally maintained specific pathogen free SW mice (SPF). The elevated susceptibility correlated with significant decrease in the antimicrobial capacity of neutrophils derived from GF SW against P. aeruginosa (PA). Oral antibiotic treatment of SPF SW reduced the ability of neutrophils to kill PA in vitro and in vivo, demonstrating that microbiota-derived cues maintain neutrophil priming in the bone marrow against PA. In contrast, topical antibiotic treatment reduced ocular commensal presence at the conjunctival surface, but did not the resistance to infection, suggesting that microbiota resident at a site different from the ocular mucosal tissue are responsible for neutrophil priming. Consistent with this reconstitutions of GF with mouse or human-derived gut microbiota restored resistance to infectious ocular challenge. To determine the mechanims of commensal priming, RNAseq experiments comparing the phenotype of GF and SPF-derived neutrophils were carried out and showed alterations in type I interferon-dependent gene expression profiles, implicating IFN-dependent pathways in microbiota-dependent neutrophil maturation. Indeed, type I interferon priming of GF mice reconstituted neutrophil bactericidal activities against PA.