bluuebunny/crossref_metadata_2025 huggingface dataset:
Psychological stress creates an immune-suppressive environment that increases the susceptibility of aged mice to Mycobacterium tuberculosis infection
Authors: Lafuse William P , Wu Qian , Saljoughian Noushin , Turner Joanne , Rajaram Murugesan
Published: January 2023

Age is a risk factor for chronic infections, including tuberculosis (TB). A second compounding factor of TB susceptibility is psychological stress, which is particularly acute in older individuals. While the influence of psychological stress in TB is well documented, the mechanisms by which stress influences M.tb susceptibility in the elderly are almost completely unknown. To study the effects of chronic psychological stress on M.tb infection in old mice, we employed a mouse social stressor model (called social disruption stress or SDR) which involves repeated social defeat in subordinate mice. Young (3 month) and old (18 month) C57BL/6 mice were subjected to SDR and bronchial fluid (BF) and alveolar macrophages (AMs) were isolated by lung lavage. Our studies showed that SDR created an immune-suppressive lung environment in old mice, based on lower levels of pro-inflammatory cytokines and chemokines in bronchial fluid (BF) or their mRNA expression by AMs. AMs from old SDR mice compared to non-stressed mice were also deficient in their ability to mount an inflammatory cytokine response or induce MHC class II mRNA expression. To determine the effect of SDR on M.tb growth, young and old mice were subjected to SDR and infected with M.tb. At 30 days, SDR in both young and old mice reduced M.tb burden. However, this was transient as at 60 days M.tb burden was higher in old mice compared to young mice and SDR further significantly increased M.tb burden in old mice. Also we found that SDR at 60 days in old mice induced significantly higher levels of IL-10 and IL-17 mRNA in the lung, which favors growth of M.tb. In conclusion, our studies suggest that psychological stress during the initial stage of Mtb infection has long-term impact on the control of the infection.
Neutrophil recruitment and dysregulation in an intradermal murine model of Orientia tsutsugamushi Infection
Authors: Liang Yuejin , Sun Jiaren , Soong Lynn
Published: January 2023

Scrub typhus is a life-threatening zoonosis caused by Orientia tsutsugamushi organisms that are transmitted by the larvae of trombiculid mites. However, the reason that innate immune cells fail to control local infection is unknown. By using a newly established intradermal (i.d.) infection mouse model, we analyzed myeloid cell subpopulations in the inoculation site at 4 hours post-infection (hpi) and found significant recruitment of neutrophils, langerhans cells and Ly6chi monocytes in the skin. To investigate what type of cells can be infected, we i.d. challenged mice with CFSE-labeled bacteria. We found that the majority of infected cells at 4 hpi were neutrophils and monocytes, which comprised around 90% of all the CFSE+ cells. To determine the fates of these immune cells carrying Orientia, we examined CFSE+ cells in various organs at 24 hpi. Our flow cytometry data demonstrated that langerhans cells were the only infected cell subset that migrated to draining lymph nodes (dLN) but were not found in the blood, spleen, bone marrow or lungs at this early stage. There were very few infected neutrophils in either the skin or dLN at 24 hpi, indicating that bacterial challenge may promote neutrophil death. To confirm this result, we infected bone marrow-derived neutrophils and monitored cell death for 36 h. Our data showed that Orientia infection increased neutrophil death from 12 to 36 hpi. Interestingly, infected neutrophils displayed an immunosuppressive phenotype, as evidenced by decreased TNF-a, CXCL10 and ICAM-1, but increased arginase-1, CCL2 and IL-4. Collectively, our data suggest that Orientia-triggered neutrophil dysregulation and death may contribute to bacterial dissemination and systemic infection in scrub typhus.
IFN-β and IL-10 induce mTOR phosphorylation and regulate IFN-γ-induced autophagy during mycobacterial infection
Authors: Teles Rosane M.B. , Tran Thanh , Ochoa Maria T , Sarno Euzenir N , Modlin Robert L
Published: January 2023

The ability of mycobacteria to block the antimicrobial activity through the IFN-β→IL-10 pathway is critical to suppress IFN-γ induced antimicrobial response and increase mycobacterial survival. Leprosy is an infectious disease, caused by Mycobacterium leprae and IFN-γ is prevalent in the self-limited tuberculoid (T-lep) and reversal reaction (RR) lesions, whereas, IFN-β is higher in the disseminated lepromatous (L-lep) lesions. Here we investigated the role of the IFN-β→IL-10 pathway in the regulation of the IFN-γ-induced autophagy in M. leprae-infected macrophages. IFN-γ induced 65% more colocalization of the autophagosome with M. leprae compared to media alone. IFN-β and IL-10 reduced by 60% of the colocalization of LC3 and M. leprae induced by IFN-γ. The blocking of IL-10 recovered in 70% of the IFN-γ-induced autophagosomal fusion. Autophagy is negatively regulated by the mTOR complex 1 activation and live M. leprae induced phosphorylation of mTOR in 40% compared to uninfected MDMs. IFN-γ decreased the mTOR phosphorylation in 50% compared to media alone in M. leprae-infected MDMs. IL-10 reversed in 50% the IFN-γ-inhibition of mTOR phosphorylation in M. leprae infected macrophages. The colocalization of LC3 and M. leprae PGL-1 is higher in T-lep and RR in comparison with L-lep patients. However, mTOR phosphorylation was strongly detected in L-lep lesions vs. T-lep/RR lesions. The ability of the IFN-β → IL-10 pathway to inhibit IFN-γ-induced autophagy through induction of mTOR phosphorylation can be key for mycobacterial surveillance and inhibition of antigen presentation leading to disease pathogenesis.
The inhibition of the particulate matter-induced intrinsic apoptosis through metal-chelating in MLE-12 cells
Authors: Kim H.J. , Herath KHINM , Jeon You-Jin , Jee Youngheun , Kim Hyun Jung
Published: January 2023

Particulate matter containing transition metals has known to cause adverse health impacts. Reactive oxygen species (ROS) are overproduced by Fenton reaction of transition metal ions and occur to oxidative DNA damage with the induction of intrinsic apoptosis. Sargassum horneri, a brown alga, is one of rich sources for health functional compounds which can chelate transition metals. In this study, the inhibition of the PM-induced intrinsic apoptosis pathway by S. horneri ethanol extract (SHE) through metal-chelating in MLE-12 cells was investigated. When MLE-12 cells were exposed to PM, the titanium and iron contents were 1.05 and 4.55 Wt.%, respectively. However, after the SHE treatment at 125 μg/mL, they were significantly reduced to 0.35 and 0.02 Wt.%. Additionally, the reduction of iron concentration was observed at 62.5 μg/mL of SHE treatment which revealed the metal-chelating effect of SHE. The expressions of P53, Bax, Cytochrome-c, and cleaved caspase-3 involved in the intrinsic apoptosis were activated in MLE-12 after PM exposure and they were decreased by the SHE treatment at 62.5 and 125 μg/mL. On the other hand, the expression of caspase-8 related to the extrinsic apoptosis was not cleaved by PM exposure in MLE-12, which meant PM induced only intrinsic cell death. These results suggested that SHE can inhibit the PM-induced intrinsic apoptosis through metal-chelating in MLE-12.

Funded by the Ministry of Oceans and Fisheries, Republic of Korea (Project NO. 20172085).
A trichlorobenzene-substituted azaaryl compound MTP0L145 blocks endosomal acidification to inhibit dengue virus infection
Authors: Lin Chiou-Feng
Published: January 2023

Blocking endosomal acidification retards dengue virus (DENV) infection. A trichlorobenzene-substituted azaaryl compound MTP0L145, originally designed as a novel fibroblast growth factor receptor (FGFR) inhibitor, confers an unexpected inhibition on endosomal acidification. Here, we examined for the blockade of DENV infection by MTP0L145. As similar to Bafilomycin A1, treatment of FGFR inhibitors MTP0L145 and BGJ39 all cause a blocking effect on endosomal acidification in cells with DENV infection. Inhibiting FGFR significantly causes inhibition on DENV infection, including dsRNA replication, protein expression, and virus release, but not viral entry as well as translation directly. FGFR antagonist NSC12, a blocker of FGF and FGFR interaction, neither inhibits viral entry nor causes blocking effects on endosomal acidification and viral replication, indicating a ligand-independent involvement. MTP0L145 and BGJ-398 inhibit receptor tyrosine kinase activity in FGFRs following DENV infection. Activation of vacuolar-type H+-ATPase for endosomal activation requires FGFR-mediated PI3-kinase/Akt and MAPK activation. Inhibiting the FGFR signaling pathway effectively retards DENV infection and mortality. These results demonstrate that MTP0L145 treatment reduces DENV infection principally by retarding FGFR-regulated endosomal acidification.

This study was supported by grants from the Ministry of Science and Technology (MOST108-2320-B-038-026), Taipei, Taiwan.
Differential innate immune response based on surgical treatment strategy after multiple trauma
Authors: Lackner Ina , Weber Birte , Gebhard Florian , Pfeifer Roman , Cinelli Paolo , Teuben Michel , Halvachizadeh Sascha , Lipiski Miriam , Cesarovic Nikola , Huber-Lang Markus , Pape Hans-Christoph , Kalbitz Miriam
Published: January 2023

Background

The complement system is part of the innate immunity, is activated immediately after trauma and is associated with ARDS, MOF and with death of multiply injured patients. The aim of the study was to investigate the complement activation in multiply injured pigs as well as its effects on the heart in vivo and in vitro. Moreover, the impact of reamed vs. non-reamed intramedullary nailing was examined with regard to the complement activation.

Methods

Male pigs received multiple trauma (blunt chest trauma, liver laceration, femur fracture, hemorrhagic shock), followed by femoral nailing with/without prior conventional reaming. Systemic complement hemolytic activity as well as the local cardiac expression of complement receptors was determined in vivo after trauma. Human cardiomyocytes were exposed to C3a or C5a and analyzed regarding calcium signaling and mitochondrial respiration.

Results

Systemic complement activation increased within 6h after trauma and was mediated via the classical and the alternative pathway. The expression of receptors for complement activation were altered locally in vivoin left ventricles. C3a and C5a acted detrimentally on human cardiomyocytes in vitro.

Conclusion

After multiple trauma, an early activation of the complement system is triggered, affecting the heart in vivo as well as in vitro, leading to complement-induced cardiac dysfunction. The intensity of complement activation after multiple trauma correlated with the invasiveness of fracture treatment. Consequently, the choice of fracture treatment might imply the clinical outcome of the critically injured patients and might be therefore crucial for their survival.
The Role of Dendritic Cells in Clostridium difficile Infection
Authors: Sun Xingmin , Wang Yuankai , kim Hyeun Bum , Zhuge Ronghua
Published: January 2023

C. difficile colonization and toxin production can lead to a wide range of symptoms from diarrhea to pseudomembranous colitis, systemic disease and death. The host immune status plays an important role in protection against C. difficile infection (CDI), especially the dendritic cells (DCs) as a bridge to connect innate and adaptive immunity. However, their role during CDI has not been characterized. In this study, we adopted a murine model of transgenic mice Tg-CD11c-DTR/EGFP (Tg), whose DCs can be conditionally depleted by injection of diphtheria toxin (DT), to investigate the role of DCs in CDI. We found that DT-injected Tg mice were more vulnerable to the CDI, compared with the PBS-injected Tg mice. DT-injection significantly depleted DCs, impaired the host capability to clear off C. difficile in the intestine, and increased the infiltration of other bacteria into organs. DC deletion did not seem to affect intestinal microbiota, nor neutrophil infiltration into lumen, but reduced macrophage infiltration into intestinal lumen. Interestingly, DC depletion impaired the intestinal barrier integrity, which could partially contribute to the increased infiltration of intestinal bacteria to organs, and severity of CDI. Transferring DCs to wild-type mice conferred host resistance to CDI. These data clearly demonstrate a protective role of DCs against CDI.
E3 Ubiquitin Ligase Nedd4 Is Essential for Anti-fungal Innate Immune Response
Authors: Nuro-Gyina Patrick Kwadwo , Guo Hui , Tang Na , Zhang Jian
Published: January 2023

C. albicans is the most common cause of fungal infections in humans, and disseminated candidiasis has become one of the leading causes of hospital-acquired blood stream infections with 45% to 75% mortality rate. The understanding of the host–pathogen interactions and the mechanisms of antifungal immunity is critical for developing immune-based strategies to combat candidemia. Nedd4 is a HECT-type E3 ubiquitin ligase which has been shown to positively regulate T cell activation and proliferation. However, the role of Nedd4 in innate immunity is unknown. To elucidate the role of Nedd4 in innate immune response against fungal infection, we generated Nedd4f/fRosa26-Cre-ERT2 and LysMCre.Nedd4f/f mice which lack Nedd4 systemically and in myeloid cells respectively. We found that although Nedd4 does not regulate the signalling via TLRs, it is required for the signalling through Dectin-1 and Dectin-2, two major fungal recognition receptors. Nedd4f/fRosa26-Cre-ERT2 and LysMCre.Nedd4f/f mice are highly susceptible to systemic C. albicans infection, which correlates with heightened organ fungal burden, defective inflammatory response, impaired leukocyte recruitment to the kidneys, and defective ROS expression by the granulocytes. At the molecular levels, Nedd4−/− macrophages from mice display impaired activation of NF-kB, p38, and ERK, but normal activation of Syk and PKC-d upon C. albicans yeast and hyphal infections. These data suggest that Nedd4 regulates the signaling event upstream of NF-kB/MAPK but downstream of PKC-d. Our data suggest Nedd4 expression in the myeloid cells is crucial for CLR-mediated innate immune response against systemic C. albicans infection.
A reparative neutrophil population released post-injury, increases host susceptibility to infection
Authors: Thanabalasuriar Ajitha , Keller Ashley , Cohen Taylor , DiGiandomenico Antonio
Published: January 2023

Patients suffering from severe thermal injury are susceptible to opportunistic infections, with pneumonia serving as the primary manifestation. The underlying mechanisms contributing to burn-induced infection susceptibility remains poorly understood. Using a combination of intravital microscopy, flow cytometry and a murine thermal injury infection model, we elucidate a novel population of neutrophils. Following injury, a population of neutrophils are released from the bone marrow and immediately travel to the lung where they appear trapped within the lung vasculature. These neutrophils express CD47, PDL-1, Cxcr4, and Cd11a suggesting that they are a unique ‘reparative’ subset. Consistent with this hypothesis, a significant number of these neutrophils migrated to injured tissue by 48h, correlating with wound healing. Interestingly, susceptibility to Pseudomonas aeruginosa lethal pneumonia was directly linked to neutrophil accumulation in the lung up to 24h post-injury. During this period, neutrophils were observed to phagocytose bacteria but were incapable of containing the infection. Intriguingly treatment with PDL-1 or TGFB neutralizing antibody, causes neutrophils to traffic directly to the skin without being stuck in the lungs. This alleviates susceptibility to infection but results in poor healing outcomes in the skin. In conclusion, we provide evidence that burn-injury promotes an imbalance in neutrophil subsets, which contributes to increased susceptibility to lung infection.
Rack1 is essential for activation of the Nlrp3 inflammasome
Authors: He Yuan , Duan Yanhui , Zhang Lingzhi , Nunez Gabriel , Kelley Nathan
Published: January 2023

The NLRP3 inflammasome is a critical component of the innate immune system that activates caspase-1 to induce secretion of pro-inflammatory cytokines interleukin (IL)-1β/IL-18 and cell death in response to microbial infection and cellular damage. However, aberrant activation of the NLRP3 inflammasome contributes to the pathogenesis of several inflammatory disorders, including cryopyrin-associated periodic syndromes, Alzheimer’s disease, type 2 diabetes, and atherosclerosis. Although extensively investigated, the mechanism of NLRP3 inflammasome activation remains not fully understood. Here we used a proteomic approach to identify Nlrp3-interacting proteins in mouse macrophages and found that Rack1 (Receptor for activated C-kinase 1) interacts with both Nlrp3 and Nek7. Inhibition of Rack1 expression abrogated caspase-1 activation and IL-1β release in response to Nlrp3 stimuli, but not Nlrc4 or Aim2 stimuli. Furthermore, this function of Rack1 was independent of its ribosomal association. Mechanistically, Rack1 was required for Nlrp3-mediated Asc speck formation and Nlrp3 stimuli-induced inflammasome complex assembly. Our work therefore identifies Rack1 as a potential therapeutic target for treating NLRP3 inflammasome-driven inflammatory diseases. Given the fact that Rack1 is a critical component of plant innate R protein complexes and contributes to the host defense against rice blast infection, our work further highlights a conserved link in innate immunity between mammals and plants.
A multi-component bacterial toxin incites host inflammation via the NLRP3 inflammasome
Authors: Mathur Anukriti , Feng Shouya , Hayward Jenni A. , Ngo Chinh , Fox Daniel , Man Si Ming
Published: January 2023

Innate immune recognition of microbial components serves as a cornerstone in mediating an effective immune response. The inflammasome is an intracellular signaling complex comprising of a sensor, an adaptor protein ASC and the cysteine protease caspase-1. Inflammasomes regulate secretion of the pro-inflammatory cytokines IL-1β and IL-18 and induction of a cell death pathway known as pyroptosis. Certain intracellular bacteria require cytosolic access to activate the inflammasome, however, how extracellular bacteria are sensed by the cytoplasmic inflammasome remains unclear. To understand innate immune recognition of extracellular bacteria, we analyzed a panel of clinically important intracellular and extracellular bacteria and identified an unknown secreted factor from the foodborne bacterium Bacillus cereus that activates the inflammasome without gaining cytosolic access. The tripartite enterotoxin called haemolysin BL (HBL) was identified as the novel activator of the NLRP3 inflammasome. The multi-component toxin assembled in a linear order on the mammalian cell membrane to form a pore, which induces potassium efflux and activates the NLRP3 inflammasome. Furthermore, HBL-producing B. cereus induced rapid lethality in the host via activation of the NLRP3 inflammasome. B. cereus-induced lethality was completely abrogated with administration of the NLRP3 inhibitor, MCC950. Our unpublished results further show that the transmembrane region of the apical subunit of HBL is dispensable for membrane binding but essential for inflammasome activation. Overall, our results indicate that intracellular sensing of a toxin secreted by an extracellular bacterium is critical for the innate immune recognition of infection.
Inflammasome activation by members of the Bacillus genus
Authors: Shen Cheng , Feng Shouya , Man Si Ming
Published: January 2023

Inflammasome activation induced by microbial components leads to an effective innate immune response. Bacillus anthracis is able to induce activation of the NLRP1b inflammasome, whereas B. cereus can induce activation of the NLRP3 inflammasome. However, whether different strains of B. cereus have differential ability to induce activation of the inflammasome has not been investigated. Further, whether other members of the Bacillus genus can induce activation of the inflammasome has remained unknown. Here, we screened 46 B. cereus strains from environmental and clinical sources to determine whether novel inflammasomes are activated. We infected primary mouse bone marrow-derived macrophages with bacteria or bacteria-free supernatant and observed for pyroptosis and other classical features of inflammasome activation. Strikingly, over 90% (42/46) of the B. cereus strains induced inflammasome activation as measured by caspase-1 cleavage and robust secretion of IL-1b, IL-18 and LDH. We identified that the inflammasome sensor NLRP3 was necessary to drive this response. Further, we confirmed that HBL and/or NHE toxins were expressed in all 42 B. cereus strains, suggesting a conserved inflammasome activation pattern amongst B. cereus strains. We also identified that four pathogenic Bacillus species induced inflammasome activation in an NLRP3-dependent manner, including B. idriensis, B. mycoides, B. pseudomycoides and B. thurigiensis. These findings highlight that the NLRP3 inflammasome sensor is important for immune recognition of several Bacillus species and suggest that therapeutic modulation of NLRP3 may enhance host protection against these Bacillus infections.
Regulation of the immune response by Siglec-1 through phosphorylation of Src at Ser17
Authors: Yang Darong , Wu Yin , Chen Guo-Yun
Published: January 2023

Introduction

Dysregulated host responses to infection usually cause severe immune diseases, including sepsis. TLR4 sensing of bacterial LPS is pivotal in the pathogenesis of sepsis. Identifying new regulatory mechanisms of TLR4 signaling will reveal the pathogenesis of inflammatory diseases and provide therapeutic targets.

Results

Silencing the immune regulatory receptor Siglec-1 in RAW 264.7 cells dramatically lowered LPS-induced Src phosphorylation at Ser17 (unknown function site) but not at Tyr416 and Tyr527, two critical sites that determine Src kinase activity. By contrast, P38, NF-kB, JNK, ERK, Syk and PKC phosphorylation triggered by LPS remained comparable between control and Siglec-1 knockdown cells. Strikingly, LPS induced Src phosphorylation at Ser17 in WT but not in Siglec-1−/− BMDMs; no defects were observed on Tyr416 and Tyr527 sites. Specifically blocking Ser17 phosphorylation of Src with H89 inhibitor remarkably decreased LPS-induced inflammatory cytokine secretion in WT BMDMs. Co-IP identified a novel interaction between Siglec-1 and Src. The S17A but not S17D mutation of Src attenuated its interaction with Siglec-1. Furthermore, to elucidate the role of Src-S17 in vivo, we successfully prepared three different knockin mice for Src-WT, Src-S17A or Src-S17D on the Src knockout background. Future investigations in Src knockin mice will determine how Src-Ser17 controls the immune response during infection with bacteria or LPS treatment.

Conclusion

Src phosphorylation at Ser17 is controlled by Siglec-1 and involved in the regulation of TLR4 inflammatory pathway. Pharmacological targeting of Src-Ser17 may provide promising strategies to combat inflammatory diseases.
Non-haemolytic enterotoxin induces pore-formation and activation of the inflammasome and pyroptosis
Authors: Fox Daniel R , Mathur Anukriti , Xue Yansong , Liu Yunqi , Tan Wei Hong , Feng Shouya , Pandey Abhimanu , Ngo Chinh , Hayward Jenni , Man Si Ming
Published: January 2023

Inflammasomes are central to innate immunity mediating host defence against pathogens and maintaining homeostasis with commensal microbes. The sensing of pathogens, danger, or homeostasis-altering signals by inflammasome sensor proteins is the molecular basis driving assembly of the inflammasome complex. Here, we identify non-haemolytic enterotoxin (NHE) as an activator of the NLRP3 inflammasome. We show that NHE, found in the neglected human foodborne pathogen Bacillus cereus, is a non-redundant toxin to haemolysin BL (HBL) despite exhibiting a similar mechanism of action. NHE-induced activation of NLRP3 has a delayed kinetic to that induced by HBL. Subunit C of NHE, via its putative transmembrane region, initiates binding to the plasma membrane, leading to the recruitment of subunit B and subunit A, forming a tripartite lytic pore permissive to efflux of potassium. NHE mediates killing of cells from multiple lineages and hosts, highlighting its versatile functional repertoire in different host species. Moreover, our data suggest that both NHE and HBL operate synergistically to induce inflammation in the host. Overall, our results highlight that multiple virulence factors from the same pathogen exhibiting conserved function and mechanism of action can be exploited for sensing by a single inflammasome, ultimately leading to increased capacity of the host to detect and defend against naturally-occurring genetic variants.
Functional macrophage chromatin landscapes depend on HMGN1/HMGN2
Authors: Sung Mia , Babagana Mahamat , Oh Kyu-Seon , Chakraborty Sayantan , Pacholewska Alicja , Furusawa Takashi , Bustin Michael
Published: January 2023

The High Mobility Group (HMG) proteins are ubiquitously present in the nuclei of all vertebrate cells. HMG proteins bind dynamically to chromatin without DNA sequence specificity, and they affect DNA-dependent functions such as replication, repair, and transcription, but only in the context of chromatin. HMGs are also suggested to play an important role in the immune system. Both HMGB1 and HMGN1 can act as alarmins. HMGN proteins also affect the response of B lymphocytes to bacterial LPS stimulation. HMGN proteins are typically enriched at regulatory sites such as enhancers and promoters, and have been shown to affect the chromatin landscape, suggesting a role in epigenetic regulatory processes. In LPS-stimulated bone marrow-derived macrophages (BMDMs), most HMG genes are downregulated in response to LPS (Oh KS et al, 2017 Immunity, PMID: 28801231. To understand the role of HMG proteins in the regulation of macrophage chromatin, we performed RNA-seq and ATAC-seq on BMDMs and two tissue-resident macrophage populations from mice lacking both HMGN1 and HMGN2. We identified several hundred differentially expressed genes in BMDMs with a large gene cluster showing female-specific downregulation in the double knockout. In peritoneal macrophages, the largest differentially expressed gene cluster was from the female-specific upregulated genes. The most dramatic transcriptome-wide changes were observed in microglia. Interestingly, MHC genes were commonly upregulated in macrophages from mutant mice. Together with alterations in chromatin, this raises the possibility that HMGNs maintain functional macrophage enhancers with specificity for the resident tissue.
Flightless-1 promotes lung CD103+ cDC phagocytosis and migration
Authors: O’Rourke Allison R , Hamerman Jessica A
Published: January 2023

Dendritic cells are specialized antigen-presenting cells integral for bridging the innate and adaptive immune responses. Critical to dendritic cell function is the need for a dynamic actin cytoskeleton. Flightless-1 is an actin capping protein linked to processes vital for dendritic cell immune functions including cell extension formation, phagocytosis, cell migration, and cell adhesion. Consistent with an important role in actin dynamics, whole body Flightless-1 knockouts are embryonic lethal. To enable further study of Flightless-1 in the immune response, we made mice with dendritic cell Flightless-1 deficiency using the CD11c-CRE driver. Homeostatic cDC1 and cDC2 populations in the spleen and lymph nodes were unchanged in DC-Flightless-1 knockouts relative to control animals. However, DC-Flightless-1 ablation led to a developmental disadvantage when in competition with WT DCs in mixed bone marrow chimeras. Upon LPS challenge in the airways, the Flightless-deficient cDC1 population showed reduced phagocytosis and migration to the lung draining lymph nodes. The DC migratory defect in the absence of Flightless-1 was supported by decreased CCR7 expression in both cDC1 and cDC2 populations. We hypothesize that the observed defects in phagocytosis and migration in Flightless-1-deficient dendritic cells are due to an altered actin cytoskeleton, which may also affect other actin-based immune structures. Current experiments are testing this hypothesis, and investigating the ability of Flightless-deficient DC to prime T cell responses.
Enhancing Anti-Tumor Activity of Natural Killer Cells by Upregulating Amino Acid Transporters
Authors: Lee Seung-Hwan , Jo Donghyeon , Almutairi Saeedah , Ali Alaa
Published: January 2023

In spite of the superior efficacy of NK cells against hematological cancers such as acute myeloid leukemia (AML), the poor ability of NK cells to infiltrate solid tumors, such as breast cancer or colorectal carcinoma, limits the potential NK cells for cancer immunotherapy. Diverse approaches are now being undertaken to overcome this limitation by modifying NK cells for better recognition and effector function of NK cells. One of the efforts is to genetically engineer NK cells to express chimeric antigen receptors (CARs) for new tumor recognition and migration to the tumor, resulting in enhanced tumor killing.

Here, we propose to enhance the anti-tumor activity of natural killer cells by upregulating amino acid transporters. Our laboratory recently showed that the upregulation of amino acid transporter is correlated with enhanced NK cell effector function. In particular, the upregulation of leucine amino acid transporter, CD98/LAT1, can induce leucine-driven mTORC1 activation and metabolic transformation, leading to enhanced proliferation and effector function of NK cells. Therefore, we will enhance the metabolism of NK cells by genetically modifying to upregulate the CD98/LAT1 nutrient transporter and make them more potent and sustained in vivo. In order to be the players in anti-tumor immunity, NK cells need to survive in the metabolically hostile conditions of the tumor microenvironment, where they have to compete for nutrients with metabolically active cancerous cells. We expect that combing our approach to augmenting the metabolism of NK cells with enhanced tumor recognition of CAR-NK cells could maximize the efficacy of cancer immunotherapy.
Resveratrol mediates regulation of genes through microRNAs in the gut epithelial cells that may play a role in the gut health and amelioration of EAE in mice
Authors: Singh Narendra Prasad , Chandler Zhane , Ezeanya Nneora , Nagarkatti Prakash S , Nagarkatti Mitzi
Published: January 2023

Resveratrol (RES), found abundantly in red grapes, blue berries and peanuts, is an anti-inflammatory, anticancer, and antioxidant compound. We have previously shown that RES ameliorated EAE in mice regulating immune cell functions and differentiation. In this study, we investigated the effect of RES on gene expression in the gut epithelial cells of mice with EAE. To this end, epithelial cells from small intestine (SI) and colons of mice with EAE treated with vehicle or RES were isolated. Transcriptome arrays using total RNAs from epithelial cells of SI and colons were performed and data obtained from the arrays were analyzed using Transcriptome Analysis Console software. Results showed that there were 1,591 (1165 in SI and 426 in colon) genes that were up- or down-regulated (>2-fold) in the epithelial cells of mice with EAE treated with RES, when compared to vehicle. We selected two genes, α-defensins and Mucin 3, for further analysis. Upon analysis of their expression in the gut epithelial cells, α-defensins was downregulated in SI while Mucin 3 was upregulated in colon of EAE mice treated with RES. To understand the regulation of these two genes in the gut, we investigated the role of microRNAs (miRs). There were two miRs, miR-378 and miR-338, that showed strong binding affinity for α-defensins and Mucin 3 respectively. Upon analysis of their expression in epithelial cells, miR-378 was upregulated but miR-338 was downregulated, demonstrating their possible role in the regulation of α-defensins and Mucin 3 genes. Data obtained from this study demonstrates a possible role of miRs in the regulation of genes in the gut epithelial cells may play a role in improving the health of the gut and amelioration of EAE in mice.
IL9 immune score predict better survival and IL9+ILC2 and Th9 are orchestrated by estrogen
Authors: Tong Huan , Feng Hao , Wan Xiaoping
Published: January 2023

PURPOSE

Phenotyping immune cell subtypes are essential to determine the role of the endometrial immune niche in endometrial carcinoma (EC) development, which could facilitate the development of therapeutics and to improve outcomes.

METHODS

The tumor-infiltrating lymphocytes isolated from EC and normal tissues were analyzed by mass cytometry (CyTOF) to evaluate the activity of antitumor immunity. Tissue microarrays (TMAs) of endometrial tumors including 128 subjects were used for immunofluorescence staining of parameters of interest. The combined impact of clinical and immune parameters (such as γδT cells, interleukin-9+cells, and ILC2 cells) on survival was analyzed using log-rank testing and Cox regression analyses.

RESULTS

CyTOF assessment revealed significant remodeling of both the lymphoid and myeloid intratumoral compartments. The density of IL9+immune cells was associated with improved overall survival(OS) in univariate analysis (p=0.0027). As in EC, >90% Vδ2 T cells expressed IL9, and Vδ2 T cells were reported as a major source of IL9, we supposed that the density of γδ T cells was also relevant to OS. However, in multivariate analysis, only IL9 and tumor stages but not the density of γδ T cells was linked to an altered prognosis.

Thus, we investigated the tissue resident IL9 expression and found that IL9 was mainly secreted by γδ T cells, ILC2, and Th9 cells in normal tissue. However, in tumor tissues, IL9 was hardly detected in ILC2 cells.

Next, the cytokine profile of infiltrating ILC2 and Th9 cells was investigated by CyTOF in endometrial hyperplasia, early and late stage of EC, and in PTEN genetic EC mice model. We found that the multifunctional roles of ILC2s and Th9 cells are tightly controlled by estrogen.
WNT/β-catenin directed metabolic choice uncouples lung-resident alveolar macrophage inflammatory activity from self-renewal
Authors: Zhu Bibo , Wu Yue , Huang Su , Zhang Ruixuan , Li Chaofan , Cheon In Su , Wang Min , Zhou Xian , Nguyen Quynh , Zeng Hu , Taketo M. Mark , Mack Matthias , Shapiro Virginia , Zhou Xiaobo , Goldrath Ananda , Kaplan Mark H , Sun Jie
Published: January 2023

Fetal-derived tissue-resident macrophages exhibit stem cell-like features of self-renewal in adulthood to maintain macrophage population during homeostasis and/or various insults. However, little is known about the cellular and molecular mechanisms modulating proliferative and inflammatory fate decisions of tissue-resident macrophages in vivo. Here, we show that WNT-β-catenin signaling inhibited lung-resident alveolar macrophage (AM) self-renewal, while simultaneously promoted AM inflammatory activities in vitro and in vivo during influenza virus infection. Mechanistically, WNT engagement facilitated the binding of β-catenin with HIF-1α over its conventional binding partner TCF-4. Such a binding choice led to the elevated macrophage inflammation in a glycolysis-dependent manner, while inhibited AM self-renewal by causing mitochondrial damage and impairing oxidative phosphorylation. Thus, AM self-renewal and inflammatory activity are uncoupled by WNT-β-catenin signaling through HIF-1α-mediated cellular metabolic choice. Importantly, we showed that AM with high HIF-1α activity had limited proliferative capacity and produced inflammatory cytokines, while AM with low HIF-1α activity were highly proliferative and expressed genes associated with tissue repair function in vivo during influenza virus infection. In accordance, we demonstrated that AM proliferation and repopulation were needed for optimal lung repair following the clearance of influenza virus in the respiratory tract. Our results have revealed key mechanisms modulating macrophage fate choice between progeny production versus inflammatory effector activity, and subsequent effects on tissue inflammation and repair.
Complement C3 deficiency exacerbates development of cardiovascular lesions in the LCWE-induced Kawasaki Disease Vasculitis
Authors: kocaturk begum , Lee Youngho , Moreira Debbie , Lane Malcolm , Shimada Kenichi , Rivas Magali Noval , Arditi Moshe
Published: January 2023

Background

Kawasaki disease (KD) is an acute febrile systemic vasculitis of childhood, mostly affecting the coronary arteries. A central feature of acute KD is the activation of the innate and adaptive immune systems. Serum complement component C3 is significantly increased in children during the acute stages of KD, and a polymorphism of a C type lectin, mannose binding lectin (MBL), has been associated with KD vasculitis and coronary artery lesions. However, the role of the complement pathway in KD vasculitis remains unclear.

Objective

Determine if the complement system contributes to KD pathogenesis in the Lactobacillus casei cell wall extract (LCWE)-mediated murine model of KD vasculitis.

Methods and Results

Serum levels of various complement proteins were elevated in LCWE-injected mice. We observed that the LCWE extract, rich in rhamnose and a TLR2 ligand, was able to bind both C3 and MBL in-vitro. Genetic deletion of C3 exacerbated LCWE-induced aortitis, coronary arteritis and abdominal aorta dilation and aneurysm. Furthermore, in-vivo blockade of C5 with a C5aR1 antagonist (PMX205) also increased these cardiovascular lesions in LCWE mice.

Conclusions

Blocking key components of the complement system, such as C3 and C5aR, significantly enhanced cardiovascular lesions in the LCWE-induced KD vasculitis mouse model. Thus, C3 or downstream activation fragments may play a protective role in the development of KD cardiovascular lesions. Whether this protection is due to complement mediated increased clearance of immune complexes or common carbohydrate pathogen molecules will need to be determined and may help our current understanding of the immunopathologic mechanisms of KD vasculitis. (Supported by NIH grant AI072726 to MA)
Regulation of RIG-I antiviral function by the RNA helicase DHX16 and unanchored polyubiquitin chains
Authors: Hage Adam , Bharaj Preeti , van Tol Sarah , Aguilera-Aguirre Leopoldo , Rajsbaum Ricardo
Published: January 2023

The type-I interferon (IFN-I) pathway mediates antiviral responses and is regulated to balance pathogen clearance while mitigating host damage. Our group recently showed that unanchored K48-linked poly-ubiquitin (K48 poly-ub) chains associate with the IKKɛ kinase to induce an optimal IFN-I mediated antiviral response. To identify other factors that may be regulated by unanchored poly-ub, we developed a novel method to isolate these chains from influenza A virus (IAV)-infected mouse lungs. Combining this approach with mass spectrometry analysis we identified DHX16, an RNA helicase previously known only as a pre-mRNA splicing factor. Next-Generation sequencing identified several IFN-stimulated genes whose expression was significantly downregulated in DHX16 knockdown cells. Consistent with this, in vitro and in vivo knockdown of DHX16 during IAV infection reduced IFN-I responses leading to increased IAV replication. DHX16-dependent IFN induction requires its intact RNA helicase motif and DHX16 ATPase activity is enhanced upon addition of unanchored K48 poly-ub in the presence of viral RNA, suggesting DHX16 first recognizes viral RNA and unanchored poly-ub regulate its activity. Furthermore, DHX16-induced IFN also requires RIG-I and this occurs in a RNA-dependent manner. Finally, we identified TRIM6 as the E3-Ub ligase that catalyzes the synthesis of unanchored K48 poly-Ub that positively regulate DHX16 activity. In conclusion, we uncovered a novel mechanism by which unanchored poly-Ub and DHX16 contribute to viral RNA recognition by RIG-I to induce an optimal IFN-I mediated antiviral response
Interleukin-22 controls RSV production in primary human airway epithelial cells by preserving cellular autophagy
Authors: Das Sudipta , Hu Sanmei , Chen Jie , St. Croix Claudette M , Kolls Jay K. , Ray Anuradha , Ray Prabir
Published: January 2023

OBJECTIVE

To study the ability of the host protective cytokine interleukin-22 (IL-22) to block productive infection of airway epithelial cells by Respiratory Syncytial Virus (RSV).

BACKGROUND

RSV infection can cause severe bronchiolitis and pneumonia in infants. However, no vaccine against RSV is currently available. RSV primarily infects both proximal and distal airways as well as alveolar epithelial cells in the lungs. IL-22 has mucosal-protective effects, known to preserve epithelial-barrier functions and promote host defense against pathogens. We, therefore, initiated studies to determine whether IL-22 can control RSV infection of airway epithelial cells (AECs).

METHODS

Primary human AECs differentiated under air-liquid interface (ALI) culture conditions were used for RSV infection (multiplicity of infection (MOI) 2) and then treated with IL-22 (50 ng/ml). 5-day old newborn C57BL/6 mice were intranasally infected with 1×106 PFU of RSV and then treated with an IL-22-Fc fusion protein (5 μg/mouse) intraperitoneally on day 3 p.i. Confocal imaging approach was utilized to determine co-localization of RSV and LC3B and to differentiate between autophagosomes and autolysosomes.

RESULTS

Treatment with IL-22 led to a significant decrease in viral load in primary human AECs as well as in vivo in mouse lungs. Confocal imaging and other approaches revealed subversion of the autophagic machinery by RSV for its own production, which was significantly inhibited by IL-22, thereby preserving autophagy in a STAT3 dependent manner.

CONCLUSIONS

IL-22 inhibits RSV production in both primary human airway epithelial cells and mouse lungs by preserving cellular autophagy thereby blocking hijacking of the autophagic apparatus by RSV.
Cytomegalovirus Blocks Macrophage TLR-mediated Tolerance through the Down-regulation of microRNA-146a to Enhance and Prolong Inducible Inflammatory Responses
Authors: Moran Monica Michelle , Dennis Evida A. , Smythies Lesley E. , Cong Yingzi , Sanchez Veronica , Britt William J. , Smith Phillip D.
Published: January 2023

Cytomegalovirus (CMV), a β-herpesvirus that persistently infects circulating monocytes, causes severe mucosal inflammation in the setting of immunosuppression. My laboratory has shown that CMV infection of monocytes upregulates toll-like receptor (TLR) expression and key components of the NF-κB signal transduction pathway, resulting in enhanced TLR-inducible inflammatory cytokine gene transcription and prompt, albeit transient, inflammatory cytokine production. However, the role of CMV in prolonged inflammation is not known. Here I investigated whether CMV prolongs macrophage-mediated inflammation by inhibiting tolerance to TLR stimulation, the process in which macrophages undergo profound down-regulation of inflammatory cytokine production after 24 hours. In addition to NF-κB induction of inflammatory cytokine production, NF-κB signaling induces microRNA146a (miR-146a), a regulatory miRNA that is induced 12 hours after the induction of inflammatory cytokine genes. Once transcribed, miR-146a feeds back to block IRAK1 and TRAF6, resulting in the downregulation of NF-κB-mediated gene transcription after 24 hours, thereby causing macrophages to become tolerant to subsequent TLR stimulation. Here we show that CMV potently suppresses miR-146a expression, preventing miR-146a blockade of the signal proteins IRAK1 and TRAF6 to promote NF-κB signal transduction. Thus, CMV suppression of macrophage miR-146a provides a mechanism by which CMV breaks macrophage tolerance to TLR stimulation, thereby promoting prolonged cytokine release. In conclusion, CMV enhances early TLR4- and TLR5-inducible cytokine production and blocks TLR tolerance, leading to prolonged cytokine-mediated inflammation.
The role of the bZIP transcription factor ZIP-1 in the Intracellular Pathogen Response of C. elegans
Authors: Lazetic Vladimir , Wu Fengting , Cohen Lianne B , Reddy Kirthi C , Troemel Emily R
Published: January 2023

How does the nematode Caenorhabditis elegans respond to natural pathogen infections with non-professional immune cells? To answer this question, we are studying the Intracellular Pathogen Response (IPR) – a host transcriptional response common to infection with molecularly diverse natural pathogens: microsporidia and the Orsay virus. In addition to intracellular pathogens, the IPR can be induced upon exposure to heat stress and proteasome inhibition. Mutant animals with constitutive IPR activation have increased intracellular pathogen resistance and thermotolerance, indicating that the IPR confers a protective response.

The IPR involves transcriptional activation of 80 genes, some of which are predicted to encode ubiquitin ligase components. One of the most highly induced IPR genes is pals-5, which serves as a robust readout for the IPR activation. Using pals-5::GFP reporters, we identified the predicted bZIP transcription factor ZIP-1 as a positive regulator of the IPR in two reverse genetic screens. Our qRT-PCR and smFISH studies demonstrated that ZIP-1 controls pals-5 mRNA expression early after proteasome blockade, but not at later time points. We further performed an RNA-Seq analysis, which revealed that ZIP-1 is required for mRNA expression of multiple IPR components. Based on the type of transcriptional regulation, we have identified three distinct types of IPR genes: a) completely ZIP-1-dependent, b) partially ZIP-1-dependent and c) ZIP-1-independent. Importantly, we found that ZIP-1 is required for the increased resistance to microsporidia infection in mutants with constitutive IPR expression, suggesting that ZIP-1-dependent genes play an important role in innate immunity.
Rhinovirus-infected COPD airway cells modulates innate immune function of alveolar macrophages via TGF-β1
Authors: Sajjan Uma
Published: January 2023

Airway epithelium, a primary target for rhinovirus (RV), modulates innate immune responses of other cell types including, pulmonary macrophages. The majority of RV-infected patients with chronic obstructive pulmonary disease (COPD) also acquire secondary bacterial infections. We hypothesized that COPD airway epithelial cells (AEC) affect pulmonary macrophage innate immune functions through secreted products, thus promoting secondary bacterial infections.

Pulmonary macrophages were pretreated with spent medium of RV- or sham-infected normal and COPD AEC and examined for innate immune responses to non-typeable H. influenzae (NTHI). Macrophages pretreated with spent medium from RV-infected COPD, but not normal AEC showed a defect in bacterial phagocytosis, and in the expression of cathelicidin, an antimicrobial peptide, and enhanced expression of CCL-20, TNF-α and CXCL-10 in response to NTHi. Proteomic analysis identified the presence of TGF-β1, tenascin C, fibronectin-1 in the secretome of RV-infected COPD, but not normal AEC. Neutralization of TGF-β1 abolished the inhibitory effects of spent medium from RV-infected COPD AEC on NTHi-induced cathelicidin and restored phagocytosis without significantly affecting NTHi-stimulated CCL-20, TNF-α and CXCL-10 in macrophages. These results indicate that RV-induced TGF-β1 secretion in COPD AEC attenuates the innate immune responses required for optimal clearing of bacteria by pulmonary macrophages and, this may increase the risk for acquiring secondary bacterial infections. At the same time, secretome from RV-infected COPD AEC may also increase inflammation via enhancing the bacteria-induced pro-inflammatory cytokines.