bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective modulation of B cell mRNA and microRNA expression by histone deacetylase inhibitor (IRM10P.615)
Authors: shen Tian , Nasrollahi Helia , Taylor Julia , Tat Connie , Zan Hong , Casali Paolo
Published: January 2023

As we suggested, epigenetic factors such as miRNAs, can interact with genetic programs to regulate B cell functions and inform antibody response. We have shown that histone deacetylase inhibitors (HDIs) valproic acid and butyrate, inhibited class-switch DNA recombination (CSR), somatic hypermutation (SHM) and plasma cell differentiation by modulating B cell intrinsic mechanisms. They repressed AID and Blimp-1 expression, which are critical for CSR/SHM and plasma cell differentiation, in mouse and human B cells by upregulating selected miRNAs that silenced AICDA/Aicda and PRDM1/Prdm-1 mRNAs, as demonstrated by qRT-PCRs. To further define the selectivity of HDI-mediated modulation of miRNA and gene expression, we performed miRNA-Seq and mRNA-Seq analysis in B cells stimulated with LPS plus IL-4 followed by VPA treatment. Consistent with our recent findings (J. Immunol. 193:5933-5950, 2014), these B cells showed reduced Aicda and Prdm1 expression, and increased expression of miR-155, miR-181b and miR-361, which target Aicda, and miR-23b, miR-30a and miR-125b, which target Prdm1,. The HDI-mediated modulation of mRNA was very selective. Among over 22,000 mRNAs analyzed, less than 20 mRNAs, including Aicda and Prdm1, significantly expressed in activated B cells were reduced by over two fold. Our findings indicate that, in B cells, HDI selectively modulate mRNAs, possibly through miRNA upregulation and as a result of HDACs existing in unique contexts of HDAC/co-factor complexes,.
B cell Rad52 deficiency enhances immunoglobulin class switching through increased recruitment of Ku70/Ku86 to S region double-strand DNA breaks (IRM10P.618)
Authors: Tat Connie , Shen Tian , Taylor Julia , Guzman Meghan , Zan Hong , Casali Paolo
Published: January 2023

Class switch DNA recombination (CSR) diversifies the biological effector functions of antibodies and is critical for the maturation of the immune response. It is initiated by AID-mediated generation of DSBs in the switch (S) regions that will undergo recombination, mainly through Ku70/Ku86-mediated nonhomologous end-joining. It has been shown, however, that Ku-deficient B cells can undergo CSR, suggesting that other DNA repair mechanisms are at work. Herein, we show that deficiency of Rad52, an important component of the homologous recombination DNA repair pathway, resulted in significantly increased CSR and is associated with increased recruitment of Ku70/Ku86 to the S region DSB ends. In Rad52-/- B cells, the increased role of Ku70/Ku86 in S region DNA recombination was further emphasized by decreased occurrence of microhomologies in S-S junctions and inter-chromosomal c-Myc/IgH translocations. Conversely, enforced expression of Rad52 in B cells impaired CSR and decreased Ku70/Ku86 recruitment to S region DNA, as revealed by ChIP assays. Accordingly, EMSAs using Sμ probes with blunt-ends or short overhangs, revealed that in CSR, Ku70/Ku86 and Rad52 bind to different types of DSB ends. Our findings show that Rad52 modulates CSR by binding to S region DSB ends. This may facilitate a Ku-independent repair pathway, which would resolve AID-mediated S region DSBs, thereby explaining the residual CSR that has been shown to occur in a conditional Ku70/Ku86 B cell knockout.
B cell Rab7 mediates induction of AID expression and class-switching in T-dependent and T-independent antibody responses (IRM10P.605)
Authors: Lou Zheng , Pone Egest , Lam Tonika , Wang Rui , Chen Yuhui , Liu Dongfang , Edinger Aimee , Xu Zhenming , Casali Paolo
Published: January 2023

IgH class switch DNA recombination (CSR) is central to the maturation of the antibody response, as it diversifies antibody biologic effector functions. By constructing conditional KO Igh+/Cγ1-creRab7fl/fl mice, we have previously identified a B cell-intrinsic role for Rab7, a small GTPase that regulates endosome maturation, in CSR and antibody responses. In Igh+/Cγ1-creRab7fl/fl B cells, Rab7 gene is ablated within 48 h of stimulation by IL-4 plus CD154 or LPS, resulting in reduced CSR to IgG1 and IgE. Here, we have addressed the mechanisms underlying the role of Rab7 in CSR. Once Rab7 was ablated, B cells were also defective in CSR to IgA, indicating that Rab7 regulates the central CSR machinery in an Ig isotype-independent manner. Indeed, expression of AID, as essential to CSR, was reduced in Rab7 KO B cells and enforced AID expression rescued CSR. Finally, super-resolution imaging of CD154-stimulated B cells revealed that Rab7 formed foci, within which CD40 was enriched. These findings, together with our demonstration that Rab7 mediated canonical NF-κB activation, as critical to AID induction, outline a novel role of Rab7 in specific signaling pathways that lead to AID expression and CSR, likely by promoting assembly of signaling complexes along intracellular membranes.
Type I IFNs are master regulators of susceptibility to post-influenza MRSA pneumonia (IRM11P.632)
Authors: Rynda-Apple Agnieszka , Shepardson Kelly , Larson Kyle , Morton Rachelle
Published: January 2023

Bacterial superinfections are common and often life-threatening complications of influenza infection. Others reported that increased susceptibility to superinfection occurs 7 days after influenza infection, and we demonstrated that it was due to decreased IL-13 and concomitant increase in IL13Ra2, which allowed for increased IFN-g, while early IL-13 production at day 3 provided protection from MRSA superinfection. However, mechanisms regulating IL-13 and IFN-g during influenza disease are unknown. We hypothesized that type I IFNs, induced by many respiratory infections, including those with influenza and MRSA are master regulators of susceptibility to superinfection. We determined that similar to the increase in IL-13, levels of IFN-b peaked at days 2-3 after influenza infection and were replaced by IFN-a by day 7, when mice are known to be susceptible to superinfection. At day 3 after influenza infection Ifnar-/- mice were more susceptible to MRSA superinfection, did not produce IL-13, but had increased IL13Ra2 and IFN-g compared to wild-type mice, and administration of rIL-13 to the Ifnar-/- mice partially restored protection from MRSA superinfection. Using bone marrow chimeric mice and Itgax-Cre mice we determined that early protection to superinfection required type I IFN signaling from the CD11c+ cell. These results support a role for type I IFNs in the early protective response to post-influenza superinfection and a role for their regulation of IL-13 and IFN-g.
Molecular regulation of autoimmunity by O-GlcNAcylation of NF-kappaB c-Rel in T lymphocytes (IRM11P.633)
Authors: Ramakrishnan Parameswaran , Tomalka Jeffrey , Jesus Tristan
Published: January 2023

The NF-kappaB protein c-Rel plays a critical role in controlling autoimmunity through regulating T cell function and T regulatory cell development. Type 1 diabetes is an autoimmune disease associated with hyperglycemia and chronic inflammatory complications. We found that hyperglycemia induces O-GlcNAcylation of c-Rel at serine residue 350 and activates the transcription of c-Rel-dependent pro-autoimmune cytokines interleukin-2 (IL-2), interferon gamma (IFNG) and granulocyte macrophage colony-stimulating factor (GM-CSF). Our recent results show that c-Rel O-GlcNAcylation decreases FOXP3 expression in T cells. These results suggest that the glucose metabolism-dependent c-Rel O-GlcNAcylation serves as a key regulatory switch with dual roles in controlling T cell and T regulatory cell function in autoimmune diabetes. Despite decades of research, drugs based on molecular targets to treat type 1 diabetes have remained elusive. This study reveals a novel molecular mechanism that regulates NF-kappaB c-Rel and autoimmune diabetes, with potential to develop novel therapeutics targeting c-Rel O-GlcNAcylation. Moreover, this study serves as the basis to explore the role of c-Rel O-GlcNAcylation in other autoimmune diseases such as celiac disease, lupus and arthritis, where c-Rel function have been implicated.
Posttranscriptional control of allergic airway inflammation by HuR (IRM11P.638)
Authors: Atasoy Ulus , Techasintana Patsharaporn , Gubin Matthew , Magee Joseph , Whitney marlyn
Published: January 2023

RNA binding protein HuR regulates Th2 differentiation by controlling mRNA stability of IL-4, IL-13 and GATA-3. We hypothesized HuR is required for initiation of allergic airway inflammation. We generated a distal lck-Cre ROSA HuRfl/fl mouse model to conditionally ablate HuR prior to T cell activation. HuR KO mice had 50% knockdown efficiency of YFP+ CD4+ T cells. We used ova challenge model of airway inflammation to ascertain effects of HuR KO in T cells. YFP+ HuR KO T cells migrated normally to lungs during ova challenge and had significant reductions in BAL IL-13, cellular inflammation and serum IgE. Additionally, they had completely abolished lung cellular inflammation to levels similar to non-immunized mice. We conducted in vitro activation experiments to determine mechanisms of suppression of airway inflammation. HuR KO CD4+ T cells were sorted into YFP+ (HuR lo) and YFP- (HuR WT) pools and activated. YFP+ T cells had profound decreases in IL-4, IL-5, IL-13 but not IFNλ at mRNA and protein levels. Gata-3 and IL-4 transcription were significantly reduced, as was expression of IL-2Ra (CD25). YFP+ KO T cells have reductions in p-stat5 signaling which is required for robust Th2 differentiation by activation of the IL-4 gene locus. Remarkably, though only 50% of CD4+ T cells have ablated HuR, this was sufficient to completely suppress Th2 differentiation in airway inflammation. These data reveal a pivotal role for HuR in controlling Th2 cytokines and airway inflammation.
Mitochondrial damage-associated molecular patterns exacerbate acute lung injury following burn (IRM11P.640)
Authors: Dunn Julia , Kartchner Laurel , Maile Robert , Cairns Bruce
Published: January 2023

Acute lung injury (ALI) is an inflammatory condition that can occur following infection or trauma and is accompanied by a 40-50% risk of mortality. Burn patients suffer from ALI even without smoke inhalation, rendering them susceptible to life-threatening pneumonia. While models exist to simulate ALI following sepsis, a model is needed to study the mechanisms that drive ALI after trauma. We hypothesize that damage associated molecular patterns (DAMPs) enter circulation following trauma and induce ALI, characterized by cellular infiltration into the lung accompanied by systemic inflammation. Because our 20% total body surface area burn model induces systemic inflammation but not ALI, we administered fragmented mitochondria (MTD) i.v. following the burn. Compared with unburned, vehicle-treated control mice, cellular recovery from lung washes doubled 3h post burn+MTD (mean 2.5± SD 0.5E5 vs. 5.0±0.7E5, *p<0.005), and remained elevated at 24h (6.2±1.7E5*) and 48h (8.2±3.5E5, p<0.05). While serum IL-6 was undetectable in control mice, it was elevated at 3h (348±49 pg/mL*) and 24h (168±47*) following burn+MTD. Our data indicates that i.v. administration of MTD in conjunction with burn injury leads to cellular infiltration into the lung characteristic of ALI that is prolonged up to 48h. Burn with MTD treatment also affected systemic inflammation, evidenced by serum IL-6. Using this model we can investigate immunotherapeutic strategies to attenuate ALI in trauma patients.
Type I interferon regulates GITRL on infiltrating monocyte-derived inflammatory APC to establish early viral control during chronic LCMV infection
Authors: Chang Yu-Han , Zhou Angela , Abdul-Sater Ali , Watts Tania H
Published: January 2023

Disease outcome in chronic viral infections correlates with early viral set-point established by the initial T cell response. Previous studies showed that GITR, an NFκB-activating TNFR family member, sustains CD4 T cell accumulation and help for CD8 T cells at the onset of chronic LCMV infection. While the endogenous effect of GITR on CD8 T cells is largely attributed to enhanced early CD4 T cell help, CD8 T cells are directly responsive to exogenous GITR agonist. How endogenous GITR co-stimulation selectively impacts CD4 T cells early post-LCMV infection (p.i.), however, remains elusive. We hypothesize that CD4 and CD8 T cells interact with distinct antigen presenting cells (APC) and that the availability of GITRL underscores the regulation of GITR co-stimulation. Herein, we identify inflammatory monocyte-derived DC and macrophages (infMΦ) as the dominant GITRL-expressing APC with approximately 5 times higher levels compared to classical DC during LCMV infection. A preliminary experiment showed that deleting exon 2 of GITRL using MΦ-specific Lys-M-Cre recapitulates the marked reduction in Th1 response against LCMV observed in the whole-body GITR knockout. GITRL exhibits similar expression kinetics to type I interferon (IFN-I) with an early but transient peak at 24–48 hours p.i. IFN-I is a potent inducer of GITRL on thioglycollate-elicited peritoneal macrophages (TG-MΦ) ex vivo. Moreover, blockade of IFN-I receptor abrogates up-regulation of GITRL during LCMV infection of TG-MΦ in vitro, as well as on infMΦ in vivo. Together, the current study suggests a critical role for infMΦ in GITR-dependent immunity to LCMV and identifies IFN-I as a key regulator of GITRL on infMΦ in vivo.
PI3K/mTOR-dependent IL-22 production modulates polyfunctional T cell responses in viral hepatitis.
Authors: Yi Panpan , Liang Yuejin , Yuan Denley , Jie Zuliang , Kwota Zakari , Cong Yingzi , Sun Jiaren
Published: January 2023

IL-22 is an important cytokine in modulating the consequences of inflammation in lesions, including the liver. However, the role of IL-22 in Lymphocytic Choriomeningitis Virus (LCMV)-induced viral hepatitis remains obscure. Here, we report that LCMV-induced IL-23 release promoted IL-22 production. And gdT cells and double negative T cells (DNT, CD3+ CD4− CD8− γδTCR− NK1.1−) were the main sources of IL-22 and IL-17 productions following LCMV infection in the liver. Interestingly, we also found that IL-23-stimulated IL-17 production in γδ T cells, and DNT cells were totally dependent on RORγt and PI3K/mTOR, but independent in AhR activities. Although IL-22 production in these two lymphocyte subsets depended on PI3K/mTOR, unlike IL-17 production, IL-22 production required additional signals from RORγt and AhR. Furthermore, IL-22 appeared to act as an important mediator in T cell responses in LCMV-induced viral hepatitis, despite the fact that the IL-22 receptor is restricted to being expressed on non-hematopoitic cells. IL-22−/− mice increased the recruitment of activated T cells to the liver and enhanced polyfunctional cytokine productions in effector CD4+ and CD8+ T cells in LCMV-induced acute or persistent viral hepatitis. Thus, our data revealed a novel connection between IL-23-PI3K/mTOR signaling pathway and the regulation of IL-22/IL-17 productions in the liver, and showed a novel role of IL-22 in modulating the plurifunctionality of anti-viral T cell responses during acute and persistent LCMV infection.
IL-17A negatively regulates lymphatic vessel formation
Authors: Park Hyeung Ju , Kim Min-Gyo , Lee Seung-Hyo
Published: January 2023

Lymphatic vessels (LVs), well-known conduits of immune cells, enable antigen presenting cells (APCs) to enter draining lymph node resulting in the induction of adaptive immune responses against various antigens. During inflammation, LVs are increased and enlarged by the effect of pro-lymphangiogenic factors including vascular endothelial growth factor -A, C, and D secreted by APCs and stromal cells, and facilitate the migration of activated immune cells. However, after antigen clearance, these expanded LVs should be shrunk similar to apoptotic cell death of activated immune cells to maintain homeostasis. It is known that, failure of inflammation resolution causes chronic inflammation in many diseases. Chronic inflammation often accompanies increased LVs which enable continuous antigen delivery and immune cell trafficking from infection sites to draining lymph node. Previously, it was discovered that several cytokines secreted by type 1 and 2 helper T (Th) cells inhibit lymphangiogenesis during resolution phase but less is known about the effect of Th17 cytokines. Here, we show that interleukin-17A (IL-17A) secreted from Th17 cells is a putative negative regulator of lymphangiogenesis in Th17-mediated immune responses, especially during resolution phase. IL-17A suppressed gene expression of lymphatic specific markers in lymphatic endothelial cells. Also, we utilized cholera toxin mixed with chicken egg ovalbumin peptide (CTO) challenge model which induces Th17 dominant inflammation to examine the in vivo effect of IL-17A. Blockade of IL-17A increased LVs and enhanced their function. Thus, this study will provide expanded knowledge on unidentified role of IL-17A as a negative regulator of lymphangiogenesis.
DOCK8 regulates fitness and function of regulatory T cells through modulation of IL-2 signaling
Authors: Singh Akhilesh K , Hagin David , Komal Khushbu , Eken Ahmet , Bhise Gauri , Hubbard Nicholas , Torgerson Troy R , Oukka Mohamed
Published: January 2023

Foxp3+ regulatory T cells (Treg) possess potent immunosuppressive activity critical for the maintaining immune homeostasis and self-tolerance. Defect in Treg cells development or function result in inadvertent immune activation and autoimmunity. Despite of recent advances in Treg cells biology, we still do not completely understand the molecular and cellular mechanisms governing the development and suppressive function of these cells. Here, we have demonstrated an essential role of the Dedicator of cytokinesis 8 (DOCK8), guanine nucleotide exchange factor required for cytoskeleton rearrangement, cell migration and immune cell survival, in controlling Treg cells fitness and their suppressive function. Treg cells-specific DOCK8-deletion led to the spontaneous multi-organ inflammation in mice due to uncontrolled T cells activation and production of pro-inflammatory cytokines. DOCK8-deficient Treg cells are defective in fitness and suppressive function. Furthermore, DOCK8 control IL-2 signaling, crucial for maintenance and competitive fitness of Treg cells, via STAT5 dependent manner. DOCK8 deficient patients are predisposed to autoimmunity and develop colitis, autoimmune hemolytic aremia and vasculitis. Thus, our study provides insight into essential function of DOCK8 in Treg cells and immune regulation, and explains the autoimmune phenotype of DOCK8 deficient patients.
IL-21 regulates the innate immune response to Staphylococcus aureus pulmonary infection.
Authors: Spolski Rosanne , West Erin E. , Li Peng , Kazemian Majid , Leonard Warren
Published: January 2023

Methicillin-resistant Staphylococcus aureus (MRSA) infection is a major complication of viral respiratory infections and immunodeficient states. Although the innate immune response mediated by neutrophils plays a major role in the clearance of MRSA, the cytokine regulation of this process is poorly understood. Here, we investigated the role of IL-21 in the host response to pulmonary MRSA infection. In experiments using a mouse model, Il21r-deficient mice more efficiently cleared pulmonary MRSA infection, suggesting a negative role for IL-21 in MRSA clearance. Interestingly, Il21r KO mice displayed an enhanced “interferon signature” response after pulmonary MRSA infection as compared to WT mice, and blocking the type I IFN response eliminated the ability of Il21r KO mice to more efficiently clear the infection. Despite the more efficient clearing of pulmonary MRSA in Il21r KO than in wild-type mice, when IL-21 was administered to wild-type mice, there was enhanced clearance of MRSA, and this clearance was accompanied by the induction of cytolytic proteases and other components of pulmonary defense. Correspondingly, human peripheral blood neutrophils responded directly to IL-21 with the induction of cytolytic proteases including granzymes, and they displayed enhanced IL-21-induced killing of MRSA that was granzyme-dependent. These data demonstrate that although the chronic absence of IL-21 signaling in Il21r KO leads to the dominant action of type I IFN responses that result in MRSA clearance, IL-21 directly induces a cytolytic program in both human and mouse neutrophils that results in MRSA killing. These data suggest that manipulation of IL-21 signaling may be a strategy for controlling bacterial infections.
IL-4 alters mast cell activation by FcγR
Authors: Haque Tamara , Ryan John J
Published: January 2023

Mast cells are involved in many inflammatory diseases, partly by their production of cytokines that promote inflammation. IgG signaling through FcγRIII/IIb in mast cells is linked to autoimmune disease such as rheumatoid arthritis, but factors controlling this signaling remain unclear. Our group has previously shown that IL-4 augments IgG- mediated TNF production by murine mast cells, but other cytokine secretion was not examined. This may be critical, since we recently found that TNF is not always co-regulated with other cytokines during mast cell activation. In fact, our current data show that IL-4 suppresses IgG-mediated MCP-1 production. Current work is examining a broader range of cytokines and chemokines. These studies suggest that mast cell responses are altered by cytokines in their microenvironment, with distinct and even opposite effects on specific cytokines and chemokines.
The gut-liver axis plays a crucial role in murine immune tolerance in the liver
Authors: NAKAMOTO NOBUHIRO , Ebinuma Hirotoshi , Amiya Takeru , Aoki Ryo , Hayashi Atsushi , Taniki Nobuhito , Chu Po-Sung , Yamaguchi Akihiro , Saito Hidetsugu , Kanai Takanori
Published: January 2023

Hepatic APCs express TLRs that recognize PAMPs or DAMPs and are involved in immune activation and tolerance. We recently showed repetitive Concanavalin A (Con A) injection, a mitogen that preferentially activates T cells, induced immune tolerance in the liver. Here, we investigated the underlying mechanisms of immune tolerance by the gut-liver axis.

METHODS

Male C57BL/6 mice were administered an initial and subsequent sub-lethal dose of ConA to induce immunological tolerance. Liver mononuclear cells were separated 12 h after the final ConA injection and analyzed by flow cytometry. Immune cell subset cytokine production stimulated with TLR ligands was measured. The composition of intestinal bacterial flora was evaluated by comprehensive metagenomics and intestinal permeability was measured by FITC-dextran.

RESULTS

A single ConA injection induced severe liver inflammation, while ConA administration 7 days after initial injection (tolerant phase) increased immunosuppresive CD11c+ DCs (D7-cDC) in the liver and promoted immune tolerance. D7-cDC had regulatory characteristics and produced IL-10 and TGF-β upon TLR9 ligand stimulation. The composition of intestinal bacterial flora along with intestinal permeability changed following ConA injection, and a species of Lactobacillus dramatically increased at tolerant phase. Finally, transplantation of a specific species of Lactobacillus to gut sterilized mice induced immunosuppressive CD11c+ cDCs and reduced liver injury by ConA.

CONCLUSIONS

Dysbiosis with altered intestinal permeability promotes the migration of immunosuppressive cells in the inflamed liver. This study identifies a novel homeostasis pathway that regulates immune tolerance through gut-liver axis.
The cellular composition of the human immune system is shaped by age and cohabitation
Authors: Liston Adrian
Published: January 2023

Detailed population-level description of the human immune system has recently become achievable. We used a “systems-level” approach to establish a resource of cellular immune profiles of 670 healthy individuals. We report a high level of inter-individual variation, with low longitudinal variation, at the level of cellular subset composition of the immune system. Despite the profound effects of antigen exposure on individual antigen-specific clones, the cellular subset structure proved highly elastic, with transient vaccination-induced changes being followed by a return to the unique baseline of the individual. Strikingly, the largest influence on immunological variation identified was cohabitation, with a 50% reduction in immunological variation between individuals who share an environment (parents) compared to the wider population. These results identify local environmental conditions are a key shaper of the human immune system.
AMPKα1 regulates T follicular helper cell metabolism and homeostasis
Authors: Li Xudong , Liang Yuqiong , Shaw Reuben J , Zheng Ye
Published: January 2023

T follicular helper (TFH) cells provide key help to germinal center (GC) B cells during humoral immune response. How TFH cells are metabolically adapted to this unique task is not clear. Here we report that TFH cells have increased AMPK activation and T-cell-specific deletion of Prkaa1, which encodes AMPKα1, results in impaired TFH response at a late time point when TFH cells move into GCs and interact with GC B cells. AMPK activation requires SAP and CD84, which are essential for T-B-interaction, and CAMKK2, which is activated by calcium signaling. AMPKα1 helps sustain TFH response by promoting autophagy, which is upregulated in TFH cells in an AMPK-dependent manner and is required for optimal TFH response. Our results suggest that AMPK plays a critical role in promoting homeostasis of TFH cells by linking TFH-B-interaction and calcium signaling to TFH-cell metabolic programing.
Therapeutic efficacy of statins and AICAR in mitochondrial dysfunction in EAE
Authors: Singh Inderjit , Samuvel Devadoss J. , Singh Avtar K. , Won Jeseong
Published: January 2023

Studies from various laboratories have provided evidence of mitochondrial dysfunction as a cause of the CNS pathology in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). Recently, we described that RhoA-ROCK-PPAR mediated activity of statins as well as activation of AMP activated protein kinase (AMPK) protect oligodendrocytes against inflammatory insults in EAE. EAE disease decreased the activation of AMPK whereas combination of lovastatin and AICAR, a specific activator of AMPK, improved the efficacy against clinical disease of EAE. As expected, mitochondrial activities of electron transport chain complexes and thus levels of ATP were decreased in the CNS of EAE as compared to control animals whereas treatment with combination of lovastatin and AICAR corrected the EAE induced mitochondrial dysfunctions while individual these drugs were partially effective. The loss of mitochondrial functions in EAE paralleled the decrease in decreased biogenesis of mitochondria as decreased in mitochondrial biogenesis proteins (PGC1α, SIRT1, and NRF1). Treatment of EAE animals with combination of AICAR and lovastatin corrected the expression of these proteins as well as increased the expression of PPARα/γ. These corrections of mitochondrial dysfunctions paralleled the levels of myelin proteins. These finding suggest that simultaneous targeting of RhoA-ROCK-PPAR signaling mechanisms by lovastatin and AMPK/PGC1α signaling mechanisms with AMPK activator AICAR provides greater neuroprotection, neurorepair, and hence improved efficacy. These findings highly relevant to improve the efficacy of statins observed in a recent study of patient with progressive MS (Chataway et al., Lancet 383:2213, 2014).
Manganese-enhanced magnetic resonance imaging detects VIPR2-mediated immune transformation in a model of Parkinson’s disease
Authors: Olson Katherine E , Schutt Charles , Bade Aditya , Dong Jingdong , Boska Michael , Shandler Scott , Mosley R. Lee , Gendelman Howard E , Liu Yutong
Published: January 2023

Progressive nigrostriatal degeneration and neuroinflammation underlie the pathobiology of Parkinson’s disease (PD). Biomarkers to detect such pathogenic events remain in development. We report that manganese-enhanced magnetic resonance imaging (MEMRI) can be used to assess inflammatory activities related to deficits in neural connectivity in an experimental PD model. Vasoactive intestinal peptide (VIP), an endogenous neuropeptide that binds to VIPR1 and VIPR2 inducing a phenotypic shift of neurotoxic effector memory to neuroprotective regulatory T cells was modified (referred to as LBT-3627) to improve its half-life and limit proteolytic cleavage. LBT-3627 was used to investigate its ability to elicit neuroprotection in mice treated with the nigrostriatal toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Treatment with LBT-3627 lead to neuronal sparing when compared to MPTP-treated mice. Seven days post MPTP intoxication, mice were scanned using MEMRI, and images were registered to an MEMRI-based brain atlas for comparison. Manganese-induced MRI signal enhancement was calculated. MEMRI detected MPTP-induced inflammation as well as a corresponding reduction in signal with low and high doses of LBT-3627. MPTP-treated mice showed a significantly higher signal enhancement within the hippocampus (p = 0.04), with a trend towards significance in both the substantia nigra and striatum when compared to saline controls. The higher signal enhancement was reversed by LBT-3627 treatment. These findings indicate the ability of MEMRI to detect MPTP-induced brain pathology and its reversal following LBT-3627 treatment, supporting MEMRI as a method to assess changes in nigrostriatal-induced neuroinflammation.
TLR and CD40 signalings synergize to induce negative regulators that inhibit B cell class-switching and plasma cell differentiation: relevance to antibody responses
Authors: Wang Rui , Lou Zheng , Casali Paolo , Xu Zhenming
Published: January 2023

During host immune responses to microbial pathogens and allergens, a B cell is exposed to T-independent (e.g. TLR ligands) and T-dependent (e.g., CD40 ligand, CD154) stimuli and integrates different signals for effective antibody responses. We have previously shown that engagement of TLRs (e.g., TLR4 by LPS and TLR9 by CpG) or CD40 induces Ig class switch DNA recombination (CSR) and B cell differentiation into plasma cells, albeit to different degrees. Here, we showed that combined stimulation of B cells through TLR and CD40 (by LPS plus CD154 or CpG plus CD154) abrogated CSR to IgG1, IgG2b, IgG3, IgA and IgE as well as plasma cell differentiation. Combined TLR/CD40 stimulation, however, did not significantly affect IgH germline IH-CH transcription or AID expression, both of which are critical for CSR, or expression of Blimp-1, a master transcription factor in plasma cell differentiation. Rather, LPS and CD154 synergized to upregulate (up to 3000-fold) expression of genes encoding EBI3 and p28, the two subunits of the cytokine IL-27. An anti–p28 blocking antibody could partially restore CSR and plasma cell differentiation in TLR/CD40 co-stimulated B cells. This together with CSR rescue by transduction of replication-defective lentiviral particles, which could inhibit IL-27 functions, indicates a negatively regulating role of IL-27 in B cell differentiation. It also suggests that engagement of TLRs and CD40 by microbial components and T helper cells may shape the antibody response by modulating B cell-intrinsic functions.

Supported by NIH grants AI 079705 and AI 105813 grants to P.C.
Asynchronous digital activation of the germline Iγ1 and Iɛ promoters in single cells reveals that temporal control determines the fate of switching to IgE in B cells
Authors: Wu Yee Ling , Stubbington Mike , Teichmann Sarah , Rada Cristina
Published: January 2023

Immunoglobulin IgE is a key effector in the adaptive immune response to parasitic infestations and a potent mediator of allergy and asthma. Switching to IgE is linked to the activation of the germline promoter (Iɛ) that drives transcription through the region (Sɛ) immediately upstream of the ɛ constant region and the recruitment of Activation Induced Deaminase (AID) to induce double strand breaks. The molecular basis of AID recruitment to Sɛ and thus the specificity of switching to IgE remains elusive given that the same stimuli that activate Iɛ also initiate transcription from the Iγ1 promoter upstream of the γ1 constant region.

We have used knock-in fluorescent reporter mice for the endogenous Iγ1 and Iɛ promoters to monitor the kinetics of in vitro I-promoter activation by cytokines and isotype switching. While the activation of I-promoters is biallelic, a significant number of activated cells (2/3) displays asynchronous activation, with activation of a single Iγ1 allele followed by activation of the second allele over a period of 12–24 hrs in vitro. Intriguingly, early activation of the Iγ1 promoter is unaffected by the rearrangement status of the cis VDJ region.

Single-cell transcriptomic and qPCR analyses on activated B cells reveal enormous heterogeneity in I-promoter activation within individual cells, identifying a distinct early population of cells in which only the Iɛ promoter becomes active, which is correlated with enhanced switching to IgE. Heterogeneity of allele activation in single cells is emerging as a general property of transcriptional programming. In B cells, the intrinsic temporal heterogeneity of I-promoter activation in individual cells is crucial for the control of AID targeting and the levels of IgE switching.
Control of B cell activation and migration by PI 3-kinase: role of inositol polyphosphate 4-phosphatases
Authors: Marshall Aaron J , Hou Sen , Wu Xun , Li Hongzhao
Published: January 2023

Balanced activation of the PI3K pathway by antigen and other stimuli is required for effective humoral immunity while avoiding autoimmunity and lymphoproliferative disease. The critical regulatory phosphatase SHIP dephosphorylates the PIP3 generated by PI3K to produce a distinct molecule PI(3,4)P2. A number of key signaling molecules bind to PIP3, including the protein kinase Btk; however much less is known about the function of PI(3,4)P2. The inositol phosphatase INPP4A can specifically hydrolyze PI(3,4)P2; however the functions of this phosphatase in B cell activation are unknown. We hypothesized that INPP4A can provide a tool to deplete PI(3,4)P2 and determine its range of functions. Human B lymphocytes over-expressing active or phosphatase-dead INPP4A were generated, and the cells expressing active enzyme were found to generate lower levels of PI(3,4)P2 upon chemokine or BCR stimulation. Chemokine-induced migration responses were inhibited by active, but not inactive INPP4A. An assessment of BCR signaling was carried out that compared BCR-stimulated cells expressing active versus inactive INPP4A in a screen encompassing over 800 protein phosphorylation sites. The results indicate that active INPP4A can suppress phosphorylation of Akt and known Akt targets. We are currently assessing whether PDK1, a major kinase upstream of Akt which also directly binds PI(3,4)P2, is regulated by INPP4A. Interestingly, other kinases known to interact with PDK1 (PKCdelta, RSK) or to be directly phosphorylated by PDK1 (PKCgamma) showed decreased phosphorylation in cells expressing active INPP4A. We are currently testing the hypothesis that PDK1/Akt and Btk/PLCγ2 signal osomes are differentially regulated by INPP4A.
Toll-like receptor 9 signaling antagonizes the B cell receptor-dependent ability of B cells to process and present antigen to helper T cells.
Authors: Akkaya Munir , Kim Ann S , Miozzo Pietro , Sohn Hae-Won , Akkaya Billur , Pena Mirna , Pierce Susan K
Published: January 2023

B cells express both an adaptive antigen-specific B cell receptor (BCR) and innate Toll-like receptors (TLRs) allowing the functional outcome of the B cell’s engagement with antigen to be modulated in response to pathogen-derived TLR ligands. In T cell-dependent responses, the BCR both signals for B cell proliferation and differentiation and also internalizes bound antigen for processing and presentation to helper T cells. Here we show that although the presence of the TLR9 ligand, CpG, does not affect early BCR mediated events following antigen binding including phosphorylation of key components of the signaling cascade and the formation of BCR clusters on cell surface, CpG alters the outcome of BCR signaling, resulting in unique transcriptional profiles, enhanced proliferation and differentiation to antibody secreting cells. In contrast, CpG dramatically limits the ability of B cells to process and present antigen to helper T cells. In the presence of CpG, BCR-induced upregulation of CD86 and MHC class II expressions are antagonized and antigen internalized by the BCR is not properly trafficked to antigen processing compartments resulting in reduced numbers of peptide-MHC class II complexes on the B cell surface. Indeed, CpG treated antigen-specific B cells show a reduced ability to maintain contact with antigen-specific T cells and to activate antigen-specific helper T cell proliferation in vitro. These results suggest that TLR9 activation in T cell dependent responses would drive B cell toward proliferation and antibody secretion and away from events that are highly dependent on the ability of B cells to present antigen to helper T cells, such as entry into germinal centers and affinity selection.
Immune modulation by butyrophilin 1A1 (BTN1A1)
Authors: Wang Jinghua , Manick Brian , Wu Guoping , Kalabokis Vassili
Published: January 2023

The butyrophilins are B7-like T cell co-regulatory molecules. These molecules are of increasing interest in cancer immunotherapy as they may represent a novel subset of immune check-point regulators. In this study, we expressed the recombinant human butyrophilin 1A1 extracellular domain with a C terminal 6-His tag (BTN1A1) in NSO mouse myeloma cell line, and investigated the biological functions of the purified recombinant BTN1A1 protein in vitro. Human T cells were treated with plate-bound anti-CD3 and BTN1A1. The levels of multiple cytokines were measured in cell culture supernatants using R&D Systems Proteome Profiler™ Human Cytokine Array and confirmed by measuring individual cytokines using cytokine-specific Quantikine® ELISA kits. BTN1A1 significantly decreased T-cell derived cytokines such as IL-2, IL-21, IL-25, CD40L, and C5a, but not IFN-gamma. BTN1A1 also markedly inhibited anti-CD3-induced human T cell proliferation in a dose dependent manner. Furthermore, BTN1A1 showed binding to anti-CD3 activated, but not rest T cells. Moreover, recombinant human BTN1A1 inhibited p38 MAPK, CREB, and TOR Phosphorylation, suggesting BTN1A1 modulates TCR signaling through the p38 MAP kinase, CREB, and TOR pathways. Taken together, our data suggests that BTN1A1 acts as a co-inhibitory molecule to modulate T cells through an unknown receptor on the surface of T cells. BTN1A1 may be a potential target of immune check-point molecules for therapeutic purposes.
Characterization of a new lymphoid organs-specific endothelial cells subset with high expression of VISTA negative co-stimulator protein
Authors: Salehi Maryam , Radichev Ilain , Savinov Alexei
Published: January 2023

Peripheral tolerance is regulated through a network of co-stimulatory and co-inhibitory ligands and receptors. Targeting these molecules or their pathways has been promising in clinic in both oncoimmunology and autoimmunity. V-set domain Ig Suppressor of T cell Activation (VISTA) is a recently identified co-inhibitory member of the B7 family. VISTA suppresses T cell immune responses; interference with its function results in susceptibility to autoimmunity. Investigating expression pattern of VISTA in various cell types and tissues in mice, we identified a sub-population of endothelial cells present only within secondary lymphoid organs, which express high levels of VISTA; a finding not previously reported. Characterization of this subset revealed that VISTA-expressing endothelial cells (VEECs) are predominantly of lymphatic endothelial cells (LECs) origin, however, only less than half of LECs in lymphoid organs are VEECs. As LECs have been shown to induce tolerance via co-inhibitory molecules such as PD-L1, we studied the role of VEECs in regard to immune tolerance. FACS-sorted VEECs cells from secondary lymphoid organs of NOD and DBA mice were evaluated for the expression levels of various peripheral tissue antigens associated with immune tolerance. Our results show that in NOD VEECs the AIRE transcription factor, as well as some diabetic autoimmunity-associated peripheral tissue antigens of pancreatic origin (insulin-2 and Rrad) are significantly up-regulated. To further investigate the regulatory role of VEECs, we applied co-culture assays and evaluated the effects of VISTA expression by endothelial cells on T cell function.
Defining the phenotype and transcriptome of IgG3+ B cells
Authors: Sadanand Saheli , Yu Wen-Han , Alter Galit
Published: January 2023

Recent studies suggest that highly polyfunctional non-neutralizing HIV-specific IgG3 antibodies are associated with protection from HIV infection in the RV144 HIV vaccine trial. While it is well established that IgG3 Abs can drive a wide range of Fc-mediated innate effector functions, it remains unclear whether intrinsic factors are important for selection and maintenance of IgG3-secreting B cells and what extrinsic signals preferentially induce IgG3 class-switch. We hypothesized that (1) IgG3+ B cells will generally express enhanced levels of activation-associated surface proteins and markers important for interactions with T cells as compared to IgG1+ B cells and (2) IgG3+ B cells will have a unique transcriptome that helps prevent downstream class switch recombination. Our initial flow cytometry and RNA-sequencing (RNA-seq) analyses have focused on comparing IgG3+ memory B cells to IgM+ and IgG1+ memory B cells in HIV negative donors. We have found that IgG3+ B cells have higher expression of CD80 and CD95, but lower expression of CD40, than IgG1+ B cells. Preliminary bulk RNA-seq analyses similarly suggest that IgG3+ B cells have a unique transcriptome, with distinct expression of genes involved in transcriptional regulation, nucleosome assembly and chromatin organization. Interestingly, IgM+ and IgG1+ memory B cells share an overlapping transcriptional regulation cluster that may exist to prevent class-switch to IgG3 (from IgM) and induce class-switch to IgG1 (from IgG3). We are currently investigating the heterogeneity of resting and stimulated IgG3+ memory B cells at the single-cell level to better understand why some, but not all, IgG3+ memory B cells retain expression of IgG3.
HSP90B1 Regulates TLR-dependent Monocyte Signaling and its Common Variants are Associated with BCG-specific T-cell Responses and Protection from Pediatric TB Disease
Authors: Graustein Andrew , Misch Elizabeth A , Musvosvi Munyaradzi , Shey Muki , Shah Javeed , Wells Rick , Hanekom Willem , Hatherill Mark , Scriba Thomas , Hawn Thomas
Published: January 2023

Introduction

HSP90B1 is a chaperone for multiple Toll-like receptors (TLRs), regulates innate immune signaling in murine myeloid cells and is expressed in T-cells. We hypothesized human HSP90B1 regulates monocyte responses to Mycobacterium tuberculosis (Mtb) and that its variants are associated with T-cell responses to the BCG vaccine and susceptibility to TB disease.

Methods & Results

We screened 17 haplotype-tagging HSP90B1 gene region SNPs for association with BCG-induced immune responses in South African infants vaccinated with BCG at birth. We stimulated peripheral blood drawn at 10 weeks of age with BCG in a whole blood cytokine assay. Three HSP90B1 SNPs (rs10507172, 10507173, and 2164747) were associated with increased IL-2 secretion (P<0.05, generalized linear model). SNPrs10507173 was associated with increased IL-2 production in the CD4+ T-cell subset (p=0.028, ANOVA) based on intracellular cytokine staining and flow cytometry. The same three alleles were associated with protection from pediatric TB disease in a gene association study of 226 children who contracted tuberculosis compared with 626 controls who remained healthy (dominant model, unadjusted p<0.05, OR 0.58–0.72; after adjusting for ethnicity, p<0.05 for 2/3 SNPs). We used CRISPR/Cas9 gene editing to reduce HSP90B1 expression in U937 monocyte cells. Relative to controls, HSP90B1-deficient cells secreted less TNF when exposed to TLR1/2/6 (p=0.02) and TLR4 (p=0.01) ligands and Mtb whole cell lysate (p=0.001).

Conclusion

These data suggest HSP90B1 deficiency is associated with an impaired monocyte response to Mtb ligands. In infants, HSP90B1 variants are associated with increased BCG-specific IL-2 responses T-cells and protection from TB disease.
Aspirin Triggered Lipoxin A4 regulates NF-κB and induces antimicrobial peptides expression in airway epithelial cells to limit mucosal inflammation to enhance host response in pneumonia
Authors: Sham Ho Pan , Abdulnour Raja-Elie E , Krishnamoorthy Nandini , Douda David N , Colby Jennifer K , Levy Bruce D
Published: January 2023

Bacteria induced pneumonia remains a major global threat to health. Even with effective antibiotic treatment, the host response can be exuberant, resulting in tissue damage and morbidity. Resolution of inflammation is an active process governed in part by specialized pro-resolving mediators (SPMs). We hypothesized that the SPM, aspirin triggered lipoxin A4 (ATL) engaged airway mucosal epithelial cells to enhance resolution. In a murine model of bacterial aspiration pneumonia for E. coli, ATL (100 ng, iv) decreased the lung bacterial burden in an additive manner with ciprofloxacin (0.2 mg/kg, ip). Further, ATL also significantly limited neutrophils recruitment and decreased pro-inflammatory cytokine (IL-1α) and chemokine production (KC). These ATL mediated protective responses were mediated in part via regulation of NF-kB activation. In response to LPS and E. coli in vitro, ATL selectively influenced airway epithelial cells expression of NF-kB regulators A20, SIGIRR and ST2. In an in vitro bacteria killing assay by airway epithelial cells, ATL directly limited bacterial growth by increasing select antimicrobial peptides expression (LL37 and BPI). mCRAMP deficient mice (human homolog of LL37) failed to limit lung bacterial growth in E. coli pneumonia and ATL treatment was significantly less efficient in promoting host defense in these mice. In conclusion, the SPM ATL can limit pathogen mediated mucosal inflammation and enhance host defense in pneumonia via the selective regulation of NF-κB and antimicrobial peptides in airway epithelial cells. These findings with ATL provide evidence for SPM targeted treatment of the host response to pneumonia that has the potential for therapeutic benefit in combination with antibiotics.