bluuebunny/crossref_metadata_2025 huggingface dataset:
Unexpected separation of functions at the level of miR-146a targets regulates immune cell activation and malignant transformation
Authors: Magilnick Nathaniel , Reyes Estefany , Wang Wei-Le , Kramer Nicholas , Boldin Mark
Published: January 2023

microRNA-146a (miR-146a) has been previously implicated as an essential molecular brake that prevents immune overreaction and malignant transformation by attenuating NF-κB signaling. miR-146a−/− mice display an autoimmune disorder, and also develop frank tumors in secondary lymphoid organs and a progressive myelodysplastic syndrome (MDS) as they age. miR-146a was proposed to exert its regulatory activity by targeting Traf6 and Irak1, genes which encode two key adapter proteins that function upstream of NF-κB activation. The exact contribution of miR-146a-mediated silencing of Traf6 and Irak1 to the control of immune activation is currently unknown. Therefore, we defined the role of the miR-146a-Traf6 axis in the regulation of immune homeostasis using mouse genetic epistasis analysis. Our studies have uncovered a unique separation of functions at the level of miR-146a targets. Lowering the Traf6 gene dose and consequent attenuation of NF-κB activation rescued many significant miR-146a−/− phenotypes such as splenomegaly, aberrant myeloproliferation, and excessive inflammatory responses. In contrast, decreasing Traf6 expression had no effect on the development of the MDS-like phenotype in aged miR-146a−/− mice, indicating that miR-146a controls hematopoietic stem cell (HSC) homeostasis through a different molecular mechanism. Our genetic data implicate excessive IFNγ production as the main driver of HSC exhaustion and bone marrow failure in miR-146a−/− mice. Furthermore, our findings suggest that control of the Traf6-NF-κB regulatory axis by miR-146a is not essential for its tumor suppressor activity, because lowering the Traf6 gene dose did not affect the rate of development of miR-146a-deficient lymphomas.
Role of IL-1β and the gut-lung axis in sterile inflammation following lung ischemia reperfusion injury
Authors: Prakash Arun , Tian Xiaoli , Hellman Judith
Published: January 2023

Background

Ischemia reperfusion (IR) injury is a source of sterile inflammation that can complicate the clinical course of severely injured trauma patients in shock, organ transplantation, and thrombotic/embolic events. The lungs are a portal to the external environment and a barrier organ. As such, they are vulnerable to infectious and sterile insults that can be life threatening if their ability to deliver oxygen and eliminate CO2 are compromised. We previously demonstrated a role for the gut microbiome in modulating this inflammatory process in vivo and in priming alveolar macrophages.

Methods

We used an in vivo model of left pulmonary artery occlusion to examine the inflammation generated in mice either genetically deficient or pharmacologically inhibited in IL-1β release or signaling pathways. We also challenged alveolar macrophages and endothelial cells in vitro with colonic lumen filtrates from antibiotic treated and control mice to determine whether shed LPS and metabolites are among the priming factor(s) for alveolar macrophages.

Results/Conclusions

Using knockout mice and inhibitor studies, we have determined that the inflammasome regulates lung IR-induced sterile inflammation. Specifically, the NLRP3 inflammasome, IL-1β release, and downstream IL-1β signaling are important factors in the generation of lung IR inflammation. Finally, we believe that the exponentially higher level of shed LPS in mice with a full complement of gut microbiota, as well as levels of short chain fatty acids, such as butyrate, may intriguingly explain the priming of alveolar macrophages that results in IL-1β production. Together this may constitute a gut-lung axis of communication that modulates the lung IR sterile inflammatory process.
iNOS over expression reduces K562 cell proliferation and promotes neutrophilic differentiation
Authors: Awasthi Deepika , Singh Abhishek Kumar , Dubey Megha , Nagarkoti Sheela , Barthwal Manoj Kumar , Dikshit Madhu
Published: January 2023

Nitric oxide (NO) is low molecular weight gaseous signalling molecule which modulates various immune functions, vasodilation, proliferation, differentiation and apoptosis. NO at low concentrations protects cells from apoptosis or induced proliferation, whereas at high concentration it inhibits cell growth and induces apoptotic cell death. Increasing evidences highlight the pro- and anti-tumorigenic effect of iNOS-derived NO depending on its concentration, redox environment, NO flux and the cell type. The present study was undertaken to assess the role of iNOS derived NO on K562 cell line (human myelogenous cell line). Stable over-expression of iNOS as confirmed by enhanced iNOS mRNA/protein expression also NO donor (DETA-NO) treatment exhibited cell cycle arrest, enhanced apoptosis. Apoptosis gene expression profiling indicated that iNOS over-expressed K562 cell line generated a broad set of alterations in the gene expression: Upregulated expression of apoptosis related genes - BCL10, LRDD, Death effector domains (DEDs), caspase 4, caspase 8, tumor necrosis factor (ligand) superfamily, member10 (TNFSF10), FADD as compared to control K562 cells. Using in vitro approach we further assessed the significance of iNOS in C/EBPα induced neutrophilic differentiation of K562 cells. May-Grünwald-Giemsa and hoechst staining demonstrated that ectopic expression of C/EBPα promotes rapid neutrophilic differentiation in K562iNOS cells as compared to K562 cells. Morphologic differentiation was further accompanied by up-regulation of myeloid-specific surface markers CD11b. Altogether, these results implicate that iNOS play a crucial role in cell cycle arrest, apoptosis and neutrophilic differentiation of K562 cells.
Trim9 mediates macrophage chemotaxis in a RING-dependent manner
Authors: Tokarz Debra A , Yoder Jeffrey A.
Published: January 2023

Tissue infiltration by macrophages and neutrophils is an early and critical step in the innate immune response, yet excessive infiltration by these leukocytes can contribute to disease pathology. Defining intracellular mediators of neutrophil and macrophage chemotaxis will reveal novel targets for modulating inflammation. The tripartite motif (Trim) proteins are a family of E3 ubiquitin ligases, of which several members have defined roles in innate immune responses. Although previously considered to be brain-specific, we have identified trim9 as a gene with increased transcript levels within macrophages and neutrophils of larval zebrafish and of humans following immune stimulation with multiple toll-like receptor agonists. Prior to our studies, Trim9 had no reported immune function, however, its published role in control of axon growth guidance suggests it is important in cell migration. To investigate the role of Trim9 in leukocyte chemotaxis, we disrupted Trim9 function in zebrafish macrophages through expression of a truncated form of Trim9 that lacks the RING domain required for ubiquitin ligase activity (ΔRING-Trim9). Expression of ΔRING-Trim9 results in significantly reduced in vivo chemotaxis of zebrafish macrophages, while overexpression of full-length Trim9 under the same conditions does not. Further, macrophages expressing ΔRING-Trim9 exhibit altered morphology with reduced pseudopod formation. We propose that Trim9, through its ubiquitin ligase activity, may mediate cytoskeletal dynamics necessary for macrophage chemotaxis. Identifying Trim9 substrates and binding partners within macrophages may provide new therapeutic strategies for modulating inflammation.
Intracellular glycogen reserves fuel early glycolytic metabolism associated with dendritic cell maturation and immune function.
Authors: Amiel Eyal , Thwe Phyu M.
Published: January 2023

Dendritic cells (DCs) serve as professional antigen presenting cells of the immune system. DCs activated by toll-like receptor (TLR) ligands undergo a process of maturation that is coupled with an increase in energy demand fulfilled by a TLR-driven burst in glycolytic metabolism. Up-regulation of glycolysis in activated DCs provides these cells with molecular building blocks and cellular energy required for DC effector function. Inhibition of glycolysis impairs both the survival and effector function of activated DCs. According to the prevailing model in the field, TLR-driven glycolysis in DCs is sustained primarily by increases in glucose uptake. We have characterized that DCs store glucose in the form of glycogen in the immature state and that glycogen functions as an intracellular energy reserve that is critical for early effector responses by these cells. Our data show that both human and mouse DCs express enzymes essential for glycogen metabolism and that glycogen synthesis is upregulated by inflammatory signals. In addition, we have observed that inhibition of glycogen metabolism significantly impairs key maturation parameters of these cells. Our findings indicate that glycogen metabolism in DCs plays an important role in supporting the effector function of these cells. This work uncovers a novel pathway for metabolic homeostasis in DCs and provides a new paradigm for how the metabolic requirements of DC activation are regulated.
Mild acidosis delays neutrophil apoptosis and enhances pro-survival signals from inflammatory mediators
Authors: Filep Janos G , de Oliveira Lima dos Santos Everton , Mansouri Soukaina , El Kebir Driss
Published: January 2023

Emerging evidence indicates that local acidosis associated with infection and tissue injury triggers innate and adaptive immunity. Activation of infiltrating neutrophils contributes to transient drop in pH. We investigated the impact of extracellular acidosis on neutrophil apoptosis, one of the check points in the outcome of the inflammatory response, and on the survival signals generated by bacterial DNA and serum amyloid A. Culture of human isolated neutrophils under mild acidosis (pH 6.5–7.0) resulted in concurrent activation of NF-κB, adenyl cyclase and the ERK and PI3K/Akt signaling pathways, leading to preservation of Mcl-1. Consequently, extracellular acidosis prevented disruption of mitochondrial transmembrane potential and translocation of cytochrome c, endonuclease G and apoptosis-inducing factor (AIF) from the mitochondria to cytoplasm and nuclei, respectively and attenuated activation of caspase-3. Pharmacological inhibition of ERK, PI3K, NF-κB and adenylcyclase partially reversed the anti-apoptotic action of acidosis. Furthermore, mild acidosis enhanced the pro-survival signal from bacterial DNA and serum amyloid A by enhancing ERK and PI3K-mediated inhibition of Mcl-1 degradation. Our results identify mild acidosis as a survival signal for neutrophils by suppressing the constitutive apoptotic machinery and suggest that transient decreases in local pH could contribute to amplification of inflammation.

Funding Support: Supported by grants from CIHR (MOP-97742).
Hepatocyte death induces TRIF-mediated sterile inflammation in the liver
Authors: Brempelis Katherine J , Yuen Sebastian , Mohar Isaac , Crispe Ian Nicholas
Published: January 2023

Hepatocyte death occurs as a result of infection, such as with hepatitis B and C viruses, and as a result of non-pathogen insults, such as chronic alcohol abuse, ischemia reperfusion injury, and acetaminophen overdose. The manner in which liver-specific cell populations—hepatocytes, resident macrophage Kupffer cells (KCs), and liver sinusoidal endothelial cells (LSECs)—respond to this death is critical to understand as the resulting immune response often results not in resolution, but in enhanced liver inflammation that can ultimately lead to fibrosis and cirrhosis. Using an adeno-associated virus vector (AAV) encoding the human diphtheria toxin receptor (hDTR), we have created a murine model of hepatocyte-specific death. After administration of diphtheria toxin (DT), we see peak liver damage at 48 hours, as measured by serum alanine aminotransferase (ALT) levels. This response is largely controlled by TIR-domain-containing adapter-inducing interferon-β (TRIF), independent of both Toll-like receptor 4 (TLR4) and Interferon alpha/beta receptor (IFNAR), and is characterized by the up-regulation of Ifn-beta in hepatocytes by 24 hours and the up-regulation of both monocyte- and neutrophil-recruiting chemokines from hepatocytes, LSECs, and KCs most strongly by 48 hours. We see TRIF-dependent up-regulation of adhesion molecules (Vcam1 and Icam1) on hepatocytes and LSECs and down-regulation of MHC-II on the surface of KCs. Understanding how the TRIF pathway controls responses to hepatocyte death provides an intriguing counterpoint when compared to other models of liver death—such as nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and ischemia reperfusion injury—which occur via MyD88-dependent routes.
Roles of innate immune regulator TAPE in RIG-I signaling and antiviral defenses
Authors: Chen Kuan Ru , Yang Chia-Yu , Lin Chun-Yang , Lin Wan-Ying , Lo Yin-Chiu , Chuang Huai-Chia , Tan Tse-Hua , Ling Pin
Published: January 2023

Pattern-recognition receptors detect pathogen-associated molecular patterns to trigger downstream pathways leading to type I IFN and inflammatory cytokines production to defend pathogen infection. RIG-I-like receptors (RLRs) are key cytosolic sensors for recognizing viral RNA to trigger antiviral immunity. The underlying mechanisms linking RLR-mediated viral recognition to antiviral immunity remain to be further explored. Through our previous work, we uncovered an innate immune regulator termed TAPE (TBK1-Associated Protein in Endolysosomes), also known as CC2D1A, which is implicated in the viral RNA senor TLR3 and RLR pathways. Yet, the in vivo role of TAPE in antiviral defenses and the mechanistic mechanisms of how TAPE regulates cytosolic RIG-I signaling still remain to be established. TAPE conditional knockout mice were generated for our study. Results from in vivo studies showed that TAPEf/f CD11c-Cre mice exhibited a more severe mortality than WT mice upon influenza A virus (IAV) infection. Ex vivo studies also showed that TAPE-deficient mouse embryonic fibroblasts and macrophages were defective in type-I interferon induction upon RLR ligand stimulation. In addition, our biochemical analyses showed that the N-terminal region of TAPE was critical for interacting with the CARD domain of RIG-I while the C-terminal region of TAPE contributed most to the interaction with MAVS/IPS-1, a RLR downstream mediator. Together, our results suggest a crucial role for TAPE in linking RIG-I to type I IFN-mediated antiviral responses. Future work will further determine the in vivo role of TAPE in IAV and other RNA virus infection, and explore the mechanistic mechanisms of how TAPE regulates RIG-I signaling.
Innate immune signaling inhibition via disruption of adapter interactions.
Authors: Brown Lindsey J , Huyhn Kimberly , Satyarthi Divyam , Edwards Erin , Toshchakov Vladimir Y. , Snyder Greg A
Published: January 2023

Toll-like receptors (TLR) form homo- or heterodimers in response to pathogen-associated molecular patterns. The TLR homo- or heterodimers establish an intracellular signaling platform that recruits the signaling adapter protein Toll-Interleukin 1 Receptor (TIR) domain containing adaptor protein (TIRAP). This receptor-mediated platform initiates a signaling cascade which leads to NF-kB activation, cytokine production, and inflammation. Inflammation leads to pathogen clearance; however, excessive stimulation of the innate immune pathway can lead to injury and death. We are investigating mechanisms to disrupt TIRAP-mediated TLR signaling via two mechanisms: the bacterial TIR domain protein, TcpB from Brucella sp., and the decoy peptide, 2R9. We have previously determined the structures of TIRAP and TcpB. Presumably via molecular interactions with TIRAP, TcpB inhibited NF-kB activation. We now report surface plasma resonance binding of TIRAP with TcpB. The decoy peptide, 2R9, was derived from the TLR2 TIR domain D-helix. We have previously described interactions between 2R9 and TIRAP via surface plasma resonance. Based on the ability of 2R9 peptide to protect against lethality in response to acute influenza infection, 2R9 peptide could highlight a novel drug target to prevent injury and death caused by innate immune over activation. Using recombinant TIRAP with TcpB and 2R9 peptide, we are performing structural studies to characterize molecular interactions to identify putative sites for therapeutic intervention.
Keratinocyte Release of Alarmin HMGN1 Is triggered by Elevation of Intracellular Calcium
Authors: Yang De , Li Yana , Nie Yingjie , Trivett Anna , Oppenheim Joost J
Published: January 2023

Intra-nuclear high-mobility group nucleosome-binding 1 (HMGN1) functions as a chromatin modulator to regulate gene expression, whereas extracellular HMGN1 acts as an alarmin and contributes to the induction of antigen-specific immune responses. However, aside from passive release resulting from cell injury, it is unclear whether HMGN1 is released extracellularly by viable cells. In this study, we studied the cell type(s) capable of releasing HMGN1 and the underlying mechanism(s). Using a keratinocyte cell line KERTr, we found that ionomycin could induce the release of HMGN1 without changing its expression at the mRNA level. This release occurred in the absence of cell death. Other stimulants capable of elevating intracellular calcium such as ATP or SDF-1α also induced HMGN1 release. HMGN1 release was dependent on calcium/calmodulin and JAK2. The capacity of ionomycin and ATP to induce HMGN1 release was confirmed using cultured primary keratinocytes. Interestingly, macrophages could not release HMGN1 in response to these diverse stimuli. Thus, HMGN1 is preferentially released by keratinocytes, but not by leukocytes such as macrophages, in response to elevation of intracellular calcium.
Uncovering the mechanism of E1A antagonism of the cGAS-STING pathway
Authors: Burleigh Katelyn , Stetson Daniel B
Published: January 2023

The cGAS-STING pathway is critical for antiviral defense. Cytosolic DNA is detected by cGAS, which synthesizes the second messenger cGAMP that activates the signaling adaptor STING. Like the RNA sensing pathway, the cGAS-STING pathway converges on kinase TBK-1, which phosphorylates IRF-3, allowing it to translocate to the nucleus to induce production of type I interferons. Many viruses encode inhibitors of this pathway to avoid the ensuing antiviral state. Previously, we discovered that oncoproteins E7 and E1A encoded by human papillomavirus and adenovirus, respectively, specifically block the cGAS-STING pathway and that these proteins bind to STING. However, the mechanism underlying the antagonism of the cGAS-STING pathway is unknown. To dissect the mechanism for E1A, we used HEK 293 cells, which express E1A, to assess known events in the pathway: TBK-1 and IRF-3 phosphorylation, IRF-3 dimerization and IRF-3 nuclear translocation. Consistent with a specific block in the DNA-activated interferon response in HEK 293 cells, we found that IRF-3 dimerization and nuclear translocation were prevented in response to DNA, but not to RNA stimulation. Surprisingly, we found that early events were intact in response to DNA, including robust phosphorylation of IRF-3 on serine 386, which is required for dimerization. However, phosphorylation of serine 396, an event with unclear importance for IRF-3 activation, was impaired after DNA stimulation. This reveals that E1A blocks a potentially unappreciated late step in the pathway, preventing complete IRF-3 phosphorylation and subsequent dimerization. Ongoing work will address the temporal and spatial regulation of this novel step in STING-dependent IRF-3 activation and its antagonism by E1A.
MyD88-associated Toll-like receptor ligands prime macrophage metabolism for infection
Authors: Fensterheim Benjamin , Bohannon Julia , Guo Yin , Sherwood Edward
Published: January 2023

Toll-like receptor (TLR) ligands protect mice against an upcoming infection if administered prophylactically, however the mechanism of protection remains undefined. Here, we probed how TLR ligands alter the metabolic response of macrophages to lipopolysaccharide (LPS) challenge or infection. Wild type (WT), MyD88 knockout (KO), and TRIF KO murine bone marrow-derived macrophages (BMDMs) were pretreated with LPS, monophosphoryl lipid A (MPLA), CpG DNA, or poly(I:C), and, after a washout period, challenged with LPS. Additionally, WT mice were treated with TLR ligands and infected with intraperitoneal P. aeruginosa 3 days after priming. We found that all ligands that activate MyD88, including LPS, MPLA, and CpG DNA, primed macrophages to have an augmented glycolytic response to LPS challenge, whereas TRIF-specific poly(I:C) did not. Moreover, MyD88 was essential for this augmented glycolytic response, whereas TRIF was dispensable. LPS, MPLA, and CpG DNA significantly improved clearance and survival from a P. aeruginosa intraperitoneal infection, whereas poly(I:C) did not. In conclusion, TLR ligands prime the macrophage metabolic response to LPS via MyD88, while having variable effects on pro-inflammatory cytokine responses. Finally, MyD88-specific TLR ligands, but not TRIF-specific TLR ligands, improve the host resistance to infection.
CLEC18 family are novel C-type lectins with differential binding specificity to glycans and TLR ligands
Authors: Hsieh Shie-Liang
Published: January 2023

The human C-type lectin 18 (clec18) gene cluster, which contains clec18a, clec18b and clec18c three loci, is located in human chromosome 16q22. Even though the amino acid sequences of CLEC18A, CLEC18B, and CLEC18C are almost identical, several amino acid residues located in the C-type lectin-like domain (CTLD) and the Sperm-Coating Protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain, also known as the cysteine-rich secretory proteins/antigen 5/pathogenesis-related 1 proteins (CAP) domain, are distinct from each other. Genotyping by real-time PCR and sequencing further shows the presence of multiple alleles in clec18a/b/c loci. Flow cytometry analysis demonstrates that CLEC18 (CLEC18A, B and C) are expressed abundantly in human peripheral blood cells. Moreover, CLEC18 expression is further upregulated when monocytes differentiate into macrophages and dendritic cells (DCs). Immunofluorescence staining reveals that CLEC18 are localized in the endoplasmic reticulum (ER), Golgi apparatus, and endosome. Interestingly, CLEC18 are also detectable in human sera and culture supernatants from primary cells and 293T cells overexpressing CLEC18. Moreover, CLEC18 bind polysaccharide in Ca2+–independent manner, and the amino acid residues S/R339 and D/N421 in CTLD domain contribute to their differential binding abilities to polysaccharides isolated from Ganoderma lucidum (GLPS-F3). The S339 (CLEC18A) and R339 (CLEC18A-1) displayed differential binding affinity to TLR ligands. Their roles to determine host responses to virus infection are under intensive investigation now.
RIG-I-Like Receptors control unique innate immune responses following West Nile Virus infection
Authors: Stone Amy EL , Wilkins Courtney , Green Richard , Gale Michael J
Published: January 2023

RIG-I-like Receptors (RLRs), RIG-I, MDA5 and LGP2, play critical roles in innate immune defense against RNA viruses and are central to host immunity. RIG-I and MDA5 signal through CARDs to induce innate immune activation while LGP2 lacks these signaling domains but is thought to function as an RLR cofactor. During West Nile Virus (WNV) infection RIG-I and MDA5 work in tandem to initiate innate immune responses but the role of LGP2 in WNV innate immunity is not well-defined. Here we applied virologic, genetic, and bioinformatics approaches to assess innate immune signaling and global gene expression response to WNV infection in primary mouse embryonic fibroblasts (MEFs) and bone marrow derived macrophages (BMMs) from mice lacking RIG-I, MDA5, or LGP2, as well as from mice lacking both RIG-I and MDA5 (DKO). RNAseq analysis defined the distinct roles of RIG-I and MDA5 in induction of innate immune gene expression against WNV. Importantly, we found that LGP2 uniquely controls genes involved in innate immunity when compared to RIG-I and MDA5. We identify an LGP2-specific gene signature that reveals a unique, non-redundant role in innate immunity against WNV. Further, we used a multiplex cytokine assay to determine unique cytokine profiles for each RLR-deficient cell type, suggesting that these distinct transcriptomes lead to functional consequences in response to WNV. A model of unique actions of RLRs in WNV infection will be presented.
CD28 induces mitochondrial respiration through Slp-76 in Long-Lived Plasma Cells for ROS-dependent survival.
Authors: Utley Adam , Carlson Louise , Ventro Daniela , Dupuis Megan Murray , Mahpour Amin , Maharaj Shivana , Collins Katherine , Chavel Colin , Lee Kelvin P
Published: January 2023

Sustained humoral immunity is dependent upon bone marrow (BM) resident long-lived plasma cells (LLPCs). We have shown that CD28 signaling in LLPCs is necessary for their survival and antibody production; however, the mechanistic basis for this is unclear. In T cells, CD28 induces glycolysis at the expense of mitochondrial respiration. To our great surprise, CD28 induces mitochondrial respiration and increases the spare respiratory capacity in LLPCs, but not in short lived plasma cells (SLPCs), through Slp-76. While not affecting glycolysis, CD28 does increase the glycolytic capacity and glycolytic reserve specifically in LLPCs. Paradoxically, the mitochondrial respiration dependent production of reactive oxygen species (ROS) is required for CD28 mediated survival. Mechanistically, ROS dependent activation of NFkB drives Irf4 expression, which itself upregulates myc, both of which have been described as central regulators of metabolism in T cells. In addition, CD28 drives myc-dependent mitochondrial biogenesis in LLPCs, thereby sustaining mitochondrial respiration-dependent ROS. This leads to a self-reinforcing model wherein CD28 induces mitochondrial respiration for ROS-dependent activation of Irf4 and myc, which then drive further mitochondrial biogenesis and enhanced metabolic fitness for LLPC survival. Our work suggests that CD28, by increasing metabolic fitness, may provide a qualitatively distinct program of longevity that allows greater competitive advantage for survival in the BM microenvironment. CD28 may therefore regulate LLPC induction, survival, and function, making it an attractive target for the augmentation of vaccine design as well as in the alleviation of antibody mediated autoimmunity.
miRNAs are critical for the regulation of RAG expression and secondary Ig rearrangement in peripheral B lymphocytes
Authors: Koralov Sergei B , Coffre Maryaline , Benhamou David , Blumenberg Lili , Riess David , Snetkova Valentina , Hines Marcus , Chakraborty Tirtha , Jensen Kari , Chong Marc , Blelloch Robert , Littman Dan R , Melamed Doron , Skok Jane A , Rajewsky Klaus
Published: January 2023

The differential expression of miRNAs throughout B cell development suggests that these ncRNAs contribute to stage-specific regulation of the intricate transcriptional program during B cell development. Conditional ablation of Dicer or deletion of the miR-17~92 cluster in early pro-B cells, revealed a critical role of miRNAs in B cell differentiation.

In the present study we compare the phenotypes of mice in which enzymes critical for miRNA biogenesis, Dicer, Drosha and DGCR8 are conditionally ablated in B lymphocytes, allowing us to definitively explore the role of miRNAs in B cell development and function. Global ablation of miRNAs in B lymphocytes lead to an early block in B cell development. Rescue of B cell surviva by overexpression of the anti-apopotic factor Bcl2 revealed that in the absence of miRNAs, B cells in the periphery expressed low levels of Ig heavy chain without expressing light chain. We demonstrate that miRNA-deficient B cells fail to regulate recombination machinery in the periphery, resulting in ongoing Ig light chain gene rearrangement. In addition to the upregulation of RAG1/2 in peripheral B cells, we demonstrated ongoing DNA double strand breaks at Ig light chain loci and upregulation of surrogate light chain components. We show that these events occur downstream of deregulated PI3K signaling and we recapitulate many of these defects in wild-type B lymphocytes by targeting individual components of PI3K signaling network. Furthermore, we achieve complete rescue of miRNA deficient B cells when we introduce a pro-survival Bcl2 transgene along with an Ig transgene resistant to light chain editing. Our data highlight an important and novel role for miRNAs in the maintenance of a mature phenotype in peripheral B cells.
Distinct functions of RIP1 in immune homeostasis revealed using novel animal models
Authors: Dowling John P , Zhang Jianke
Published: January 2023

RIP1 was originally identified as a protein associated with Fas and TNFR1, which can trigger apoptosis. However, initial studies indicated that RIP1 is dispensable for apoptotic signaling. Instead, cells lacking RIP1 are defective in TNFR1-induced activation of NF-kB, a critical player involved in pro-survival signaling. Recent studies indicate that a certain form of necrosis-like death, necroptosis, requires RIP1. In contrast, the Fas-associated death domain (FADD) protein appears to play an obligatory role in apoptosis induced by Fas and TNFR1. However, the embryonic lethality phenotype of fadd−/− mice had remained a long-standing enigma, until analysis was performed to induce simultaneous deletion of both FADD and RIP1. The resulting fadd−/−rip1−/− double mutant mice displayed normal embryogenesis, indicating that fadd−/− embryos die of RIP1-dependent necroptosis. On the other hand, absence of RIP1 blocks postnatal development. Most recently, studies including ours demonstrated that only when both FADD and RIP3 were deleted will rip1−/− mice survive to adulthood. This result indicates perinatal lethality in RIP1-deficient mice is due to not only FADD-mediated apoptosis but also RIP3-dependent necrosis. Interestingly, we found that lack of RIP1 greatly diminished lymphoaccumulation disease in fadd−/−rip3−/− mice. To investigate RIP1 functions in adult immune cells, we performed conditional deletion of RIP1 in lineage committed lymphoid cells. Viable hypomorphic mutant mice with selective loss of function were employed to delineate the complex signaling pathways mediated by RIP1 in vivo. The results revealed a novel mechanism in the adult lymphoid system, which is distinct from the role of RIP1 in neonatal cells.
Characterization of notch and toll-like receptor interactions in macrophages against Paracoccidioides brasiliensis
Authors: Romera Lavínia Dal’Mas , Jannuzzi Grasielle Pereira , Almeida Sandro Rogério
Published: January 2023

Paracoccidioidomycosis (PCM) is a systemic mycosis of deep nature and granulomatosis that preferably affects the lung tissue and can disseminate to other organs and tissues, and is caused by Paracoccidioides brasiliensis, a thermal dimorphic fungus. Macrophages, cells that have an important role in regulation of immune response, recognize this fungus through pattern recognition receptors (PRR), such as Toll-like receptor (TLR). Recently it has been demonstrated the importance of Notch receptors in regulation the activity of macrophages in immune response, and also that TLR acts together with Notch receptor. Given that Notch and TLR signaling are involved in modulating macrophage function, we evaluated the ability of the fungus modulate the activation of these pathway. Our data demonstrated that activation of TLR4 in macrophages is dependent of Notch 1 receptor, and promotes an increase of IL-6, this cytokine is important to the establishment of PCM. We also observed that the increased level of IL-6 is associated with decrease of TNF-alfa, and phagocitosys. Moreover, when Notch receptor was inhibited, we observed a decreased activation of TLR4 and TLR2, decreased of IL-6 and fungal load, but an increase of TNF-alfa and phagocitosys. We also observed a decrease of fungal load in bone marrow macrophages from TLR4 knockout mice. Similar results are seen in vivo with Notch inhibition, with a decrease of fungal load. This results suggest that P. brasiliensis utilizes Notch-TLR signaling as an escape mechanism.
Profiling CD8+ CD103+ T cells in the Toxoplasma gondii infected brain
Authors: Landrith Tyler , Noor Shahani , Wilson Emma H
Published: January 2023

Toxoplasma gondii is an obligate intracellular protozoan parasite. The parasite evades clearance by the immune system and instead persists in an encysted state within the brain parenchyma. The unique environment of the brain requires a tightly regulated immune response in which T cells are essential. Nonetheless, the mechanistic details of the chronic T cell response to the parasite remain incomplete. Flow cytometry analysis reveals phenotypic diversity among brain infiltrating T cells. CD8+ CD103+ T cells form a notable proportion of this group, comprising nearly 40% of the CD8+ T cell population at the late chronic stage. Expression of CD103 can define a tissue resident memory T cell (Trm). Whether Trm could play a role in control of a chronic infection has not yet been defined. Using flow cytometry and in situ immunostaining, we show that this population is specific to the parasite and can be found throughout the infected brain including the E-cadherin positive regions of the choroid plexus, the perivascular space, and within the parenchyma in proximity to the parasite cyst. Furthermore, using RNAseq analysis, we present preliminary data establishing that this population exhibits a unique transcriptional profile relative to other CD8+ T cells in the brain and periphery. This data suggests that CD8+ CD103+ T cells in the brain are indeed a functionally distinct population, laying the groundwork for further analysis.
Aspergillus fumigatus strain-specific host response is regulated by glucose sensing.
Authors: Caffrey Alayna Katherine , Beattie Sarah R , Blaseg Nate , Hilmer Kimberly M , Zickovich Julianne M , Cramer Robert A , Obar Joshua J
Published: January 2023

Our knowledge of how Aspergillus fumigatus growth is controlled within the respiratory tract is developing, but still enigmatic. Recruitment of neutrophils and inflammatory monocytes is critical to control fungal germination. We have recently found that the CEA10 and Af293 strains of A. fumigatus induce unique inflammatory leukocyte responses. Here, we aim to understand why these A. fumigatus strains induce different immune responses and how this affects fungal-induced immunopathology and disease outcome. We used an immunocompetent murine model of invasive pulmonary aspergillosis to evaluate the immune response induced by CEA10 and Af293. We observed that CEA10 undergoes significantly greater germination than Af293, which was regulated by its ability to sense glucose through CreA-mediated signaling. Moreover, the CEA10 strain induced greater lung damage, vascular leakage, and inflammation. These findings suggest the inflammatory response to A. fumigatus might be regulated in a stepwise manner in response to the threat posed by the specific A. fumigatus strain. Specifically, the Af293 strain, which swells but fails to germinate, activates the inflammasome leading to IL-1beta-dependent neutrophil recruitment. In contrast, the CEA10 strain is able to germinate efficiently within the lung, leading to necrotic host cell death, IL-1alpha release, and subsequent IL-1alpha dependent neutrophil recruitment. Clinically, our data support the idea that A. fumigatus strain phenotypic variation may significantly contribute to disease outcomes. Understanding why different A. fumigatus strains induce distinct immune pathology can reveal novel immunotherapeutic targets for the treatment of invasive aspergillosis.
Apicidin mediated attenuation of MRSA virulence corresponds with quorum sensing inhibition and enhanced immune effector responses.
Authors: Parlet Corey P , Kavanaugh Jeffrey S , Crosby Heidi A , Cech Nadja B , Oberlies Nicholas H , Horswill Alexander R
Published: January 2023

As the leading cause of infectious mortality in the United States, S. aureus-induced disease represents a major healthcare problem. The alarming rise of infections caused by virulent, antibiotic resistant strains, such as emerging methicillin-resistant S. aureus (MRSA) isolates, highlight the need for new interventions that inhibit MRSA pathogenicity and potentiate host defense responses. The expression of MRSA virulence factors is a function of agr-driven quorum sensing, making this system a promising target for anti-MRSA therapies. Through agr-reporter-based screens, a class of compounds, called apicidins, were found to inhibit quorum-sensing activity across MRSA isolates. To test the efficacy of apicidin in vivo, mice were challenged intradermally with 2×107 MRSA (+/−) 5 mg apicidin. The abatement MRSA virulence in the apicidin-treated group was demonstrated by reduced: weight loss, dermonecrosis and cutaneous bacterial burden. By challenging mice with an agr-reporter strain, we also found that the apicidin-mediated attenuation of MRSA pathogenesis corresponded with reduced quorum sensing activity in vivo. To evaluate apicidin’s impact upon anti-MRSA effector responses, we assessed polymorphonuclear neutrophil (PMN) accumulation and function at cutaneous sites of infection. Flow cytometric analysis revealed that apicidin increased the density of PMNs within infected wounds 24 hours post infection. In addition, we found that the number of PMNs that phagocytosed MRSA organisms in vivo was increased in lesional skin preparations from apicidin treated mice. Together, these results indicate that apicidin-mediated quorum quenching represents a novel strategy to limit MRSA virulence and promote host defense.
Identification and characterization of CD1-restricted T cells
Authors: Souter M N , Nguyen-Robertson C V , Ross F J , Reddiex S J. J , Waddington J , Van Rhijn I , Eckle S B. G , McCluskey J , Aldrich A P , Rossjohn J , Moody B , Godfrey D I , Pellicci D G
Published: January 2023

Most studies of T cells have focused on those that respond to foreign peptides. However, other specialized populations of T cells exist that recognize lipid antigens and make up a substantial component of the human immune system. These lipid reactive T cells recognize antigens presented by antigen presentation molecules from the CD1 family. Four CD1 molecules exist (CD1a, CD1b, CD1c and CD1d), and each is capable of presenting a unique repertoire of lipids antigens to T cells. Much of what we have learned about lipid reactive T cells stems from studies of CD1d restricted NKT cells as these are present is both mice and humans and can be detected using CD1d/α-GalCer tetramers. In contrast, our understanding of the biology of CD1a, CD1b, CD1c restricted T cells is relatively limited. However, the recent generation of CD1a, CD1b and CD1c tetramers is helping with the identification and characterisation of these CD1-restricted T cells.

We have produced CD1 tetramers loaded with mammalian self-lipids or lipid antigens from Mycobacterium tuberculosis. In conjunction, with a tetramer-based enrichment method, we have successfully identified both autoreactive and microbial lipid antigen specific T cells from healthy human blood. We reveal the phenotypic characteristics of these CD1-restricted T cells and used CD1 mutagenesis to provide new insight into TCR recognition of CD1-lipid antigen complexes. Collectively, these studies will serve as a basis for future studies of lipid reactive T cells in health and disease.
Mother’s ‘genetic’ little helpers: microchimeric maternal cells promote reproductive fitness and survival of non-inherited traits
Authors: Kinder Jeremy Michael , Jiang Tony , Ertelt James , Xin Lijun , Strong Beverly , Shaaban Aimen F. , Way Sing Sing
Published: January 2023

Reproductive success among outbred placental mammalian species requires active tolerance in mothers to foreign paternal antigens expressed by the developing fetus. Although this essential necessity for immunological tolerance has primarily been examined from the perspective of maternal responsiveness to foreign paternal antigens expressed by the developing fetus, exposure to maternal tissue during in utero development also imprints tolerance to genetically foreign non-inherited maternal antigens (NIMA) that persists into adulthood. Nonetheless, until now the biological advantages reinforcing conservation of tolerance to NIMA across eutherian mammals remained unclear. We show vertically transferred maternal cells that establish microchimerism in offspring promote systemic accumulation of regulatory T cells with NIMA specificity. In females, NIMA-specific regulatory T cells expand during pregnancies sired by males expressing alloantigens with overlapping NIMA specificity, thereby averting fetal wastage normally triggered by disruptions of fetal tolerance. Thus, reproductive fitness among females carrying embryos expressing paternally inherited antigens overlapping with NIMA is selectively enhanced. These findings demonstrate that genetic fitness, canonically thought to be restricted to Mendelian inheritance, is enhanced in female placental mammals through vertically transferred maternal cells that promote conservation of NIMA and enforce cross-generational reproductive benefits. Future studies will investigate the cellular and molecular phenotype of the microchimeric maternal cells to elucidate specific mechanisms that drive NIMA-specific regulatory T cell accumulation.
Intestinal IL-17R signaling modulates commensal microbiota by regulating expression of Nox1 and Pigr
Authors: Kumar Pawan , Monin Leticia , Castillo Paticia , Elsegeiny Waleed Abdel Wahab , Horne William , Eddens Taylor John , Good Misty , Bibby Kyle , Montelaro Ronald , Metzger Dennis W , Gulati Ajay , Kolls Jay K
Published: January 2023

Despite recent advances in understanding the role of IL-17 in host immunity, its role in regulating enteric immune responses as well as its impact on the commensal microbiome has not been well studied. To further understand this, we have generated intestinal epithelial cell specific IL-17R knockout mice.

Our data shows that global and intestinal epithelial cell specific Il17r−/− mice have overgrowth of segmented filamentous bacteria (SFB), suggesting a critical role of IL-17 signaling in SFB colonization. Higher SFB colonization in Il17rafl/fl x villin cre+ mice resulted in expansion of IL-17A and IL-22 producing Th17 cells. The expansion of SFB was also confirmed by 16S rRNA. Furthermore this analysis also showed higher abundance of S24-7 and the Clostridiales family in Il17rafl/fl x villin cre+ mice. RNA sequencing data from the distal small intestine of SFB-colonized Il17ra−/− and Il17rafl/fl x villin cre+ mice revealed substantial reduction of Nox1 (an apical NADPH oxidase) and Pigr genes. Mouse and human primary intestinal organoid culture further confirmed a direct role of IL-17 in regulating Nox1 and Pigr expression. Reduced Nox1 expression correlated with significant reduction in H202 levels in the terminal ileum of Il17rcfl/fl x villin cre+ mice. Furthermore, Nox1−/− and IgA−/− mice showed a higher degree of SFB colonization in the feces and terminal ileum as compared to cohoused control WT mice. We have evidence that Il17rafl/fl x villin cre+ mice are more susceptible to autoimmune inflammation.

Collectively, our data indicate IL-17R-dependent intestinal signaling controls commensal bacteria by regulating the expression of Nox1 and Pigr which regulates lumenal H202 and sIgA concentrations respectively.
Short chain fatty acids regulation of neutrophil production of IL-10
Authors: Chen Feidi , Wu Wei , Cong Yingzi
Published: January 2023

Despite questions regarding the role of microbiota in regulation of host immune responses within the intestines, evidence suggests that in the context of disease and autoimmunity bacterial metabolites might impact the systemic immune response. Otherwise indigestible carbohydrates are metabolized into short-chain fatty acids (SCFAs) by gut bacteria. SCFAs have been shown to inhibit intestinal inflammation in experimental colitis through regulation of mucosal T cells and macrophages. Recently, neutrophils have been shown to regulate intestinal homeostasis and the pathogenesis of IBD, however, how SCFAs may regulate neutrophil function is unclear. In this study, we demonstrate that SCFA differentially regulate neutrophil cytokine production. Of the three major SCFAs butyrate, acetate, and propionate, only butyrate down-regulated neutrophil IL-10 production. We further showed that butyrate-inhibition of neutrophil IL-10 is independent of GPR43, in that butyrate also inhibited IL-10 production in GPR43 deficient neutrophils. We recently showed that IL-23 induced neutrophil production of IL-17 and IL-22 in a mTOR dependent manner; however, butyrate did not affect IL-23-induced IL-17 and IL-22 production in neutrophils. Taken together, our data indicates that microbiota metabolites differentially regulate neutrophil cytokine production.
Aryl hydrocarbon receptor activation fails to alter Cholera toxin-specific fecal IgA secretion after oral immunization in mice.
Authors: Dekrey Gregory K , Gawronska Elleanour , Metten Madeline
Published: January 2023

Secretory IgA plays a critical role in neutralizing enteric toxins and protecting against some intestinal pathogens. In mice, activation of the aryl hydrocarbon receptor (AhR) with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to suppress antigen-specific systemic IgM and IgG responses, whereas the impact of AhR activation on mucosal IgA responses is less clear. The goal of this study was to determine if TCDD exposure could alter fecal levels of antigen-specific IgA after oral immunization. Female C57Bl/6 mice were administered peanut oil (vehicle) or TCDD (10 μg/Kg, po), and, on the following day, each animal was immunized with whole Cholera toxin (CT, 10 μg, po) with two subsequent immunizations being given at 10 day intervals. Antibody levels were measured by ELISA in fecal extracts each week (normalized to protein) and in serum at termination (week 4). We found that total serum IgA levels were significantly decreased (P < 0.05) in TCDD-treated animals, relative to controls. In contrast, no change in total fecal IgA levels was observed in TCDD-treated mice at any time point measured (weeks 1–4). Interestingly, no change in CT-specific serum IgA levels was observed in TCDD-treated mice, whereas TCDD exposure significantly decreased (P < 0.05) CT-specific serum IgG1 and IgM levels. In Feces, as in serum, no change in CT-specific IgA levels was found in TCDD-treated mice at any time point measured (weeks 3–4). These results suggest that intestinal IgA responses are not adversely impacted by TCDD exposure, an observation that markedly contrasts with the suppression of systemic antibody responses widely seen in animals after TCDD treatment.