bluuebunny/crossref_metadata_2025 huggingface dataset:
SphK1/S1P Axis Regulate PPARγ Levels to Program Metabolically Fit Anti-Tumor T Cells
Authors: Chakraborty Paramita , Thyagarajan Krishnamurthy , Chatterjee Shilpak , Selvam Shanmugam Panneer , Ogretmen Besim , Mehrotra Shikhar
Published: January 2023

Metabolic competition with highly glycolytic tumor and overcoming tumor-induced suppression are prerequisites for achieving prolonged tumor control by adoptive T cell therapy (ACT). Given the importance of sphingosine-1-phosphate (S1P) signaling that are independent of immune cell trafficking, we evaluated if altering sphingosine kinase (SphK) mediated S1P level have benefits for anti-tumor T cell response. A comparison of melanoma epitope gp100 reactive T cell receptor transgenic pMel vs. pMel-SphK1−/− mice derived splenic T cells showed better control of subcutaneously established murine B16-F10 melanoma, increased infiltration at the tumor site with activated pMel-SphK1−/− T cells. Additionally, pMel-Sphk1−/−T cells also exhibited enhanced recall response tracked in different organs after secondary antigen exposure. Mechanistically, we found that attenuation of SphK1/S1P signaling results in higher Sirt1 (an NAD+ dependent protein deacetylase) activity, lower PPARγ activity and higher retention of FOXO1 in the nucleus. While higher nuclear localization of FOXO1 regulates higher migration of SphK1−/− CD8+ T cells, lower PPARγ activity endows them with increased ability to use their stored lipid content even in starved condition and decreased iTreg generation. Owing to their efficacious utilization of lipid as fuel, Sphk1−/− T cells persist longer in nutrient deprived tumor microenvironment. In reciprocal studies, endogenous production of S1P in Sphk1−/− T cells (transfected with WT Sphk1 vector) enhanced PPARγ expression and thereby reversed their phenotype in terms of iTreg generation and lipid utilization. Overall, these data highlight that strategies targeting SphK/S1P axis improves efficacy of ACT.
Homologous and heterologous protection against influenza B virus following live attenuated influenza virus (LAIV) immunization
Authors: Santos Jefferson , Finch Courtney , Sutton Troy , Obadan Adebimpe , Aguirre Isabel , Wan Zhimin , Lopez Diego , Geiger Ginger , Gonzalez-Reiche Ana Silvia , Ferreri Lucas , Perez Daniel R.
Published: January 2023

Influenza B virus (IBV) is considered a major human pathogen, responsible for seasonal epidemics of acute respiratory illness. Two antigenically distinct IBV hemagglutinin (HA) lineages co-circulate worldwide with limited antigenic cross-reactivity between each other. Licensed seasonal influenza vaccines are available as inactivated, recombinant, or live attenuated influenza virus (LAIV) vaccines. Although LAIV vaccines mimic a natural infection that is thought to provide better cross-protection than inactivated vaccines, the former are not currently recommended by the CDC for use in the US due to the sub-optimal efficacy. In order to improve LAIV vaccines and following the footsteps of similar work performed by our laboratory using influenza A viruses, we sought to develop an IBV LAIV vaccine. Thus, we created an IBV LAIV using a two-strategy combination that incorporates temperature sensitive (ts) mutations and an epitope tag in the PB1 gene segment. Such modifications resulted in a LAIV candidate with ts characteristics in vitro. We assessed vaccine safety following intranasal immunization and found that IBV LAIV displayed an attenuated (att) phenotype in vivo with virus replication restricted to the upper respiratory tract. Immunization experiments further showed that a single dose of IBV LAIV candidate conferred sterilizing immunity against lethal homologous challenge. Single immunization with IBV LAIV also provided complete protection against heterologous challenge as evidenced by reduced weight loss and lung viral titers. The ability of LAIV to induce cross-protective immune responses may have important implications for the development of universal influenza vaccine approaches.
Synergistic anti-tumor efficacy of combined TLR3 and CD40 neoantigen vaccine requires Batf3-dependent dendritic cells
Authors: Hoki Toshifumi , Yamauchi Takayoshi , Odunsi Kunle , Ito Fumito
Published: January 2023

Therapeutic cancer vaccine therapy targeting neoantigens has yielded encouraging results, yet overall clinical responses to the treatment have thus far limited. A better understanding of the mechanisms that regulate priming of neoantigen-specific T cells is required to improve vaccine strategy. To this end, we used murine MC38 colon adenocarcinoma cells that harbor a single-epitope mutation within the Adpgk protein with the neo-epitope presented in MHC-I H-2Db molecules. MC38 tumor-bearing mice were treated with Adpgk mutant epitope in combination with toll-like receptor (TLR) 3 agonist, anti-CD40 agonist, or both. Therapeutic vaccination with the Adpgk mutant peptide combined with TLR3 and anti-CD40 agonists (TLR3/CD40) significantly improved survival compared with either TLR3 or CD40 stimulation alone. This was associated with expansion and differentiation of CD8+ T cells in the periphery as well as in the tumor microenvironment. Significantly increased terminally differentiated neoantigen-specific CD8+ T cells in blood and spleen were identified using the tetramer staining assay. Interestingly, frequency of neoantigen-specific CD8+ T cells in blood was inversely correlated with the tumor volume, suggesting epitope spreading and broadening T-cell clonality induced by TLR3/CD40 stimulation. Importantly, therapeutic efficacy of neoantigen/TLR3/CD40 vaccination was abrogated in Batf3 knockout mouse, suggesting Batf3-dependent dendritic cells play a critical role in priming adaptive immunity. These studies provide the rational basis for the use of combined TLR3 and CD40 agonists together as essential adjuvants to optimize vaccines designed to elicit therapeutic immunity against cancer neoantigens.
Ectromelia-encoded protein B22 restricts CD4+ T cell activation.
Authors: Forsyth Katherine S. , Fisher Devin , Hersperger Adam , Eisenlohr Laurence Crane
Published: January 2023

Orthopoxviruses encode many immunomodulatory proteins that profoundly interfere with various aspects of the immune system. Ectromelia (ECTV) is the orthopoxvirus that naturally infects mice, affording the opportunity to study viral-host interactions that have co-evolved. Importantly, disease progression closely mimics that of monkeypox and smallpox in humans. CD4+ T cells are one immune cell type crucial for the control of ECTV and have important roles in both direct cytolytic killing of infected cells and helping B and CD8+ T cell activation. Here we show that ECTV infection of antigen presenting cells inhibits their ability to activate CD4+ T cells directly. However, the inhibitory mechanism is not straightforward as neither down-regulation of surface MHC-II nor soluble factors are responsible. A promising candidate protein for this inhibition is B22, which is located on the plasma membrane and has homologs in most virulent strains including smallpox and monkeypox, but not vaccine strains. Indeed, we have determined that B22 contributes substantially to ECTV virulence. Here we show that B22 is both necessary and sufficient to restrict CD4+ T cell activation, with experiments ongoing to elucidate the specific mechanism of inhibition.
Primary human B cells and plasma cells exhibit different sensitivities to vaccinia virus binding and infection
Authors: Shepherd Nicole , Lan Jie , Li Wei , Xing Yanyan , Rane Sushmita , Yu Qigui
Published: January 2023

Previous reports from our lab and others have shown differential vaccinia virus (VACV) binding to and infection of primary human leukocyte subsets, and that VACV preferentially binds and infects antigen-presenting cells (APCs). Although various groups have examined VACV infection in monocytes, macrophages, and dendritic cells, few studies have evaluated VACV binding and infection in primary B cells, a main type of APCs. In this study, we evaluated VACV binding to and infection of primary human B cells at various stages of differentiation. We observed plasmablasts were resistant to VACV binding, while B cells in other differentiation stages exhibited no defects in VACV binding. However, VACV preferentially infected memory and activated B cells whereas plasma and mature-naïve B cells were resistant to VACV infection. Additionally, VACV infection in total B cells was abortive, halted at the late viral gene expression stage. Furthermore, B cell function, measured by cytokine production, was not significantly affected by VACV infection within 24 h post-infection, but revealed the trend of decreased and increased cytokine production in 8 h and 24 h post-infection, respectively, when comparing uninfected and infected cells. Understanding VACV infection in primary human B cells at various stages of differentiation is important for the development of a safer smallpox vaccine and better vectors for vaccines against cancers and other infectious diseases.
Mechanisms of Zika virus antibody neutralization
Authors: Zhao Haiyan , Crowe James E. , Diamond Michael S. , Fremont Daved H.
Published: January 2023

Zika virus (ZIKV) infection during pregnancy has emerged as a global public health problem because of its ability to cause severe congenital disease. To gain insights into the humoral immune response against ZIKV, we set out to characterize a panel of neutralizing monoclonal antibodies (mAbs) with the goal of correlating B-cell epitopes with neutralizing potency. We developed six mouse mAbs against ZIKV including four that are ZIKV specific and neutralized infection of African, Asian, and American strains to varying degrees. The crystal structures of four of these mAbs in complex with DIII domain of the viral E protein revealed three spatially distinct epitopes, corresponding to the lateral ridge, C-C′ loop, and ABDE sheet regions. Of these, antibodies that target the lateral ridge epitope can confer protection in a lethal mouse model of ZIKV infection. Currently, we are focusing on a subset of human mAbs from healthy subjects with prior ZIKV infection. One of these human neutralizing mAbs binds an epitope in domain II, exhibits broad inhibitory activity in vitro, and protects pregnant and non-pregnant mice from ZIKV infection. Other neutralizing mAbs target DIII and show significant neutralizing activity in vitro. Strikingly, mechanistic studies indicate that the most potent murine and human mAbs in our panels are all capable of blocking pH triggered membrane fusion, and confocal microscopy studies indicate that they can traffic into target cells in complex with virions. These studies provide a path for developing prophylactic or therapeutic antibodies for use in pregnancy and designing epitope-specific vaccines against ZIKV.
Mature anti-insulin B cells survive and present antigen in the absence of Bruton’s tyrosine kinase.
Authors: Nyhoff Lindsay E. , Clark Emily S. , Khan Wasif N. , Kendall Peggy L.
Published: January 2023

Bruton’s tyrosine kinase (Btk) is a tec-family kinase present in B lymphocytes and innate immune cells. Btk is an important regulator of autoreactive B cells. In the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D), Btk-deficiency is protective against disease development and results in significant loss of anti-insulin IgG, even as total IgG is preserved. Anti-insulin B cells drive T1D by presenting antigen to autoreactive T cells. In a transgenic model, conventional Btk-deficiency reduces anti-insulin B cells by 95%, effectively blocking their development. However, the ability of mature anti-insulin B cells to survive or present antigen without Btk was unknown. We induced deletion of Btk using a loxP-flanked Btk mouse model paired with tamoxifen-inducible Cre. Surprisingly, these anergic anti-insulin B cells survive without Btk, as normal numbers of mature B cells were maintained in transgenic Btkflox/Cre-ERT2 animals after tamoxifen treatment. Btk-negative anti-insulin B cells also remained able to internalize and present antigen to cognate T cells and to phosphorylate phospholipase C γ2 in response to anti-IgM. These findings show that though anti-insulin B cells require Btk for their development, it is not required for mature anti-insulin B cell survival. In addition, our finding that Btk-negative anti-insulin B cells can present antigen may have implications for the use of Btk-inhibition in autoimmunity driven by this mechanism.
Ikaros regulates B-cell tolerance
Authors: Schjerven Hilde , Rodriguez Princess , Hagen Danielle , Rasmussen Marit , Ayongaba Etapong F. , Frietze Seth
Published: January 2023

B-cell development and activation are tightly regulated at multiple steps to ensure a protective immune response, and at the same time avoiding harmful self-reactivity and autoimmunity. Ikaros is a transcription factor that is critical for B-cell development, as demonstrated by the complete lack of B-cells in Ikaros-null mice. Furthermore, Ikaros is shown to play important roles also at later stages of B-cell development, but the precise roles of Ikaros at different stages of B-cell development is still not fully understood. In recent years, Ikaros (encoded by the IKZF1 gene) has been linked to autoimmune disease in humans through both genome-wide association studies (GWAS) as well as recent reports of germline IKZF1 mutations in patients with autoimmune disease. However, the mechanisms underlying altered Ikaros function and the development of autoimmunity are not known. We previously developed Ikaros-mutant mouse models with targeted deletions of the exons encoding the DNA-binding zinc finger 1 (ZnF1) or ZnF4, and found that both mutants have B cells, but display selective partial defects at different stages of B-cell development. We recently found that the Ikzf1-ZnF4-mutant strain displays very high levels of serum Anti-Nuclear Antibodies (ANA) at young age, a hallmark of autoimmune disease. This indicates that Ikaros (and specifically exon 6 encoding ZnF4) is required to regulate B-cell tolerance, and we hypothesize that Ikaros regulates this, at least in part, by establishing a chromatin structure that sets restrictions on B-cell responses to limit autoreactive B cells. We are using our Ikaros-mutant mice to further study the role of Ikaros in B-cell development and tolerance, and will present our results to-date at the meeting.
TLR7 and IFNγ signaling converge at STAT1 in B cells to control germinal center mediated autoimmunity
Authors: Chodisetti Sathi Babu , Domeier Phillip P. , Choi Nicholas , Rahman Ziaur S.M.
Published: January 2023

Toll like receptor 7 (TLR7) is required for the development of spontaneous germinal center (Spt-GC) mediated autoimmunity. Accordingly, autoimmune-prone B6.Sle1b mice carrying an extra copy of TLR7 in the Yaa locus (named B6.Sle1bYaa mice) exhibit significantly elevated Spt-GC responses that strongly correlate with exacerbation of lupus disease. However, the underlying molecular mechanisms in TLR7 driven autoimmunity are not clearly defined. Here we report that overexpression of TLR7 or stimulation with its agonist, imiquimod increases IFNγR and STAT1 expression, and the strength of IFNγR-STAT1 signaling in B cells. B6.Sle1bYaa mice deficient in IFNγR (Sle1bYaa.IFNγR−/−) fail to develop Spt-GCs, resulting in markedly reduced serum autoantibody (autoAb) titers and renal pathology. Further, B cell-intrinsic IFNgR or STAT1 deficient mice fail to generate Spt-GC responses upon imiquimod treatment. Interestingly, TLR7 stimulation induces STAT1 phosphorylation at serine-727 in B cells. Consistently, lupus-prone B6.Sle1b mice with alanine substitution for serine-727 in STAT1 (Sle1b.STAT1-S727A) show significantly reduced Spt-GC responses, resulting in decreased number of ANA-producing antibody forming cells and serum autoAb titers. Finally, using mixed bone marrow chimeras, we find that B cell-intrinsic STAT1-S727 phosphorylation is required for increased autoAb titers in B6.Sle1b mice. Mice with S727A mutation in STAT1, however, respond normally to T-dependent protein immunization. Together these data highlight the importance of a crosstalk between IFNγ and TLR7 signaling in B cells at STAT1 in Spt-GC responses and lupus-like autoimmunity.
Anti-inflammatory Activities and Component Change of Processed and Fermented Gastrodiae Rhizoma
Authors: Park Shin Young , Kim Eun Ju , Song Bit Na , Jeong Da Som , Kim So Young , Park Bo Ram , Choi Han Seok
Published: January 2023

Gastrodiae Rhizoma (GR) is a traditional herbal medicine in Korea, China, and Japan, which has been used for the treatment of headaches, hypertension, oxidative stress, mental disorders, inflammation, and cardiovascular diseases. This study investigated the anti-infammatory activity and main component content of processed (PGR) and fermented Gastrodiae Rhizoma (FPGR). Processed method was soaked in vinegar solution until completely absorbed and steaming for 2 hours. Fermentation was carried out using Lactobacillus brevis and incubated at 37□ for 1~3 day. The anti-inflammatory activites were determine pro-inflammatory factors such as nuclear factor κB (NF-κB), nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-induced RAW 264.7 cell line and major functional components (gastrodin, gastrodigenin, vanillylalcohol, p-hydroxybenzaldehyde) were also measured. The results showed that the FPGR reduced NO, PGE2, iNOS, COX-2, NF-κB production without cytotoxicity and improved major components. These results suggest that FPGR extracts may be a developed the functional food related to anti-inflammation due to the significant effects on inflammatory facters. Therefore, fermented and processed Gastrodiae Rhizoma can be useful methods which can help applied as functional resources in the industrial area.
Identification of cytokine-specific sensory neural signals in murine vagus nerve activity recordings
Authors: Zanos Theodoros , Silverman Harold , Levy Todd , Tsaava Tea , Battinelli Emily , Lorraine Peter , Ashe Jeffrey , Chavan Sangeeta S. , Bouton Chad , Tracey Kevin J.
Published: January 2023

The nervous system maintains physiological homeostasis through reflex pathways that modulate organ function. This process begins when changes in the internal milieu (e.g. blood pressure, temperature, or pH) activate visceral sensory neurons that transmit action potentials along the vagus nerve to the brain stem. IL-1β and TNF, inflammatory cytokines produced by immune cells during infection and injury, and other inflammatory mediators have been implicated in activating sensory action potentials in the vagus nerve. However, it remains unclear whether neural responses encode cytokine-specific information. Here we develop methods to isolate and decode specific neural signals to discriminate between two different cytokines. Nerve impulses recorded from the vagus nerve of mice exposed to IL-1β and TNF were sorted into groups based on their shape and amplitude and their respective firing rates were computed. This revealed sensory neural groups responding specifically to TNF and IL-1β. These cytokine-mediated responses were subsequently decoded using a Naïve Bayes algorithm that discriminated between no exposure and exposures to IL-1β and TNF (mean successful identification rate 82.9±17.8%, chance level 33%). Recordings obtained in IL-1 receptor knockout mice were devoid of IL-1β related signals, but retained their responses to TNF. Genetic ablation of TRPV1 neurons attenuated the vagus neural signals mediated by IL-1β and distal lidocaine nerve block attenuated all vagus neural signals recorded. Using a novel methodological framework, the results obtained in this study suggest cytokine-specific information is present in sensory neural signals within the vagus nerve.
Gpr109a limits microbiota-induced IL-23 production to constrain ILC3-mediated colonic inflammation and carcinogenesis.
Authors: Bhatt Brinda , Zeng Peng , Zhu Huabin , Sivaprakasam Sathish , Xiao Haiyan , Dong Lixin , Shiao Pamela , Kolhe Ravindra , Li Honglin , Ganapathy Vadivel , Singh Nagendra
Published: January 2023

A set of coordinated interactions between gut microbiota and the immune cells surveilling the intestine play a key role in shaping local immune responses and intestinal health. There are several immunoregulatory mechanisms in place that prevent the needless activation of immune cells against harmless gut bacteria. The dysregulation of interleukin-23 (IL-23) plays a critical role in the induction of several inflammatory diseases including inflammatory bowel disease (IBD). However, the molecular mechanisms regulating IL-23 production are poorly characterized. Gpr109a is a G-protein coupled receptor that is expressed at a very high level on innate immune cells and has been previously shown to play a key role in the induction of colonic Tregs. We show that Gpr109a−/−Rag1−/− mice exhibit spontaneous rectal prolapse and colonic inflammation, as characterized by the presence of an elevated number of IL-17-producing Rorγt+ innate lymphoid cells (ILC3). The genetic deletion of Rorγt ameliorated the spontaneous colonic inflammation in Gpr109a−/−Rag1−/− mice. Gpr109a-deficient colonic dendritic cells promote ILC3 by producing higher amounts of IL-23. Antibiotics treatment to deplete gut microbiota decreased IL-23 production, ILC3, and colonic inflammation in Gpr109a−/−Rag1−/− mice. Administration of niacin, a Gpr109a agonist, suppressed both IL-23 production by colonic DCs and ILC3 count in a Gpr109a-dependent manner. Collectively, our data presents a model suggesting that targeting Gpr109a will be potentially beneficial in the suppression of IL-23 mediated immunopathologies.
Validation and discovery of mechanisms that promote vitiligo pathogenesis using single-cell RNA-sequencing of cells isolated from skin interstitial fluid
Authors: Strassner James P. , Gellatly Kyle J. , Rashighi Mehdi , Ahmed Maggi , McDonel Patrick , Garber Manuel , Harris John E.
Published: January 2023

Vitiligo is an autoimmune disease in which CD8+ T cells destroy melanocytes, leading to patchy, depigmented lesions of the skin. To investigate possible immune processes in the skin that could explain the patchy appearance of vitiligo inflammation, we performed single-cell RNA-sequencing on cells isolated from the skin using a modified suction blistering technique. We compared the transcriptomes of cells isolated from lesional and non-lesional skin of six vitiligo subjects with active, spreading lesions as well as normal skin from six healthy subjects. Using differential gene analysis to cluster more than 18,000 cell transcriptomes in an unbiased fashion, we were able to identify the cellular components of the skin: melanocytes, keratinocytes, and Langerhans cells, as well as subgroups of immune cells. Consistent with previous findings by our group and others, this approach revealed an IFNγ signature in vitiligo lesions. This method identified 87 ligand-receptor pairs that may promote vitiligo pathogenesis, including CXCR6 and CXCL16 that were just recently implicated in vitiligo. The data set also revealed that Langerhans cells actively express many genes which have known risk alleles for vitiligo, highlighting a potential pivotal role in the development of vitiligo. Overall, single-cell RNA-sequencing of the skin infiltrate in vitiligo lesions is providing robust data that confirms many previous findings, but adds additional layers of nuance missed by conventional studies of bulk material. This level of resolution will yield powerful insight into the identification of novel drug targets in previously unknown signaling pathways in vitiligo and other diseases of the skin.
Immune mechanisms in the pathogenesis of idiopathic anterior uveitis.
Authors: Bora Nalini S. , Matta Bharati , Bora Puran S.
Published: January 2023

Idiopathic anterior uveitis (IAU) is a blinding disease characterized by inflammation of the iris and the ciliary body (CB) of the eye. IAU is treated symptomatically only using non-specific therapies such as steroid and immunosuppressive agents. These treatment modalities do not address the underlying mechanisms and are associated with adverse side effects. Experimental autoimmune anterior uveitis (EAAU) is a clinically relevant animal model of human IAU. In the current study we used EAAU animal model to investigate underlying mechanisms involved in regulation of inflammation in IAU. EAAU was induced in Lewis rats by immunization with melanin associated antigen (MAA). We observed that Th17 cells (IL-17 producing CD4+ cells and CD4+RORγt+ cells) accumulated in rat eyes at the onset and the peak of the disease. During the resolution of EAAU, Th17 cells declined while CD4+CD25+FoxP3+ T regulatory (Tregs) cells accumulated in the eyes. Administration of recombinant TGF-β2 during the early stages of EAAU prevented the induction of uveitis by inhibiting CD4+T proliferation and increasing the number of CD4+CD25+FoxP3+ Tregs. Severity of active (on-going) EAAU was reduced only for the time of active treatment with TGF-β2. Adoptive transfer of CD4+CD25+ T regulatory cells after the onset of EAAU resulted in sharp decline in disease activity and early resolution of EAAU. In conclusion, our results demonstrate that TGF-β2 plays a pivotal role in regulation of intraocular inflammation in EAAU. Collectively, these results raise the possibility that human IAU can be treated perhaps by enhancing the generation of Tregs by TGF-β2. Additionally, Treg cell therapy is potentially a promising strategy in the treatment of established human IAU.
Novel insights into the puberty switch mechanisms of the sex bias in human asthma
Authors: Coden Mackenzie , Jeong Brian M. , Chiarella Sergio , Rodriguez Raul , Berdnikovs Sergejs
Published: January 2023

The gender bias in human asthma prevalence changes with puberty, with males having a higher asthma incidence before puberty and females having higher rates after. The mechanisms involved in this switch are unknown. Our previous research showed that multiple hormonal systems are dysregulated in allergic disease. Because sex hormones interact with all endocrine system axes, we hypothesized that non-sex hormones and their downstream functions in allergic disease are driven by sex related changes in puberty. Measuring non-sex hormone protein levels from human serum samples by multiplex ELISAs, we found a striking sexual dimorphism in endocrine regulation. Pre-puberty asthmatic males had higher T3, T4, and GH levels compared to healthy controls, and pre-puberty asthmatic females had lower C-peptide and insulin levels. Interestingly, we saw the opposite pattern post-puberty. In a kinetic murine ovalbumin asthma model, adult male mice had higher C-peptide and insulin baseline levels, which were maintained during inflammation. Males also had higher GIP levels in the acute phase and higher PP, PYY, and resistin levels in the model’s late phase. Since hormones regulate tissue homeostasis, we examined the puberty switch in key epithelial remodeling markers in the inflamed mouse lung. Thbs2, Nes, Postn, Tnc, and Wnt5a expression was different relative to baseline in both sexes pre-puberty, but only females had altered expression of these markers post-puberty. Interestingly, though the Th2 immune response was higher in females post-puberty, there was no sexual dimorphism pre-puberty. These results implicate that sex hormones exert systemic level regulation determining the age and gender dependent progression of asthma pathogenesis.
AKT targets CSK to regulate proximal BCR signaling in germinal center B cells
Authors: Luo Wei , Hawse William F. , Trivedi Nikita , Weisel Florian , Shlomchik Mark J.
Published: January 2023

We have recently shown (Luo et al., Immunity, in press) that, compared to naïve B cells (NBC), both B cell receptor (BCR) and CD40 signaling are rewired in germinal center (GC) B cells (GCBC). BCR signaling in GCBC induces only transient activation of Syk, leading to partial activation of the PI3K-AKT pathway, generating p-Foxo1 but not p-S6. There is minimal BCR-directed activation of other downstream pathways such as NF-κB. Conversely for CD40 ligation, only NF-κB but not PI3K is activated in GCBC. Initially we found that Lyn, the most upstream kinase in the BCR cascade, has more inhibitory vs activating phosphorylation in GCBC. To understand how this occurs, and why AKT targets only selected substrates in GCBC, we used an AKT-substrate-specific mAb to immunoprecipitate AKT targets in GCBC. We analyzed these by mass spectrometry to identify potential GCBC-specific AKT targets. This analysis revealed that AKT itself was retargeted in GCBC compared to NBC. In GCBC, AKT appeared to preferentially target negative regulators, including CSK, a kinase of the inhibitory Tyr of Lyn. In vitro kinase and functional studies confirmed that AKT could phosphorylate CSK. We then demonstrated that this phosphorylation results in markedly increased CSK enzymatic activity to phosphorylate Lyn. Consistent with this, we found Lyn activity in GC B cells is inhibited by CSK catalyzed phosphorylation. Critically, inhibiting AKT substantially enhanced activation vs inhibitory Tyr phosphorylation of Lyn, and enhanced Syk phosphorylation and downstream signals upon BCR stimulation in GCBC. Taken together, our findings identify and document a unique AKT negative feedback loop that dampens proximal BCR signaling in GC B cells.
Identification and functional analysis of NKp46+ natural killer cells from peripheral blood of squirrel (Saimiri sps.) and owl monkey (Aotus sps.)
Authors: Nehete Pramod N. , Chitta Sriram , Nehete Bharti Pramod , Vanchiere John A. , Abee Christian R.
Published: January 2023

Natural Killer (NK) cells as minor subsets of peripheral blood lymphocytes (PBL) are innate in nature to clear invading pathogens and tumor cells. Small rodent species to nonhuman primates and humans, immune systems consist of a spectrum of NK cell subsets with phenotype and functional diversity. This study examined NK cell subsets and expression of NKp46 in squirrel and owl monkeys, which are both New World primate species. Squirrel and owl monkeys are recommended by the World Health Organization as excellent experimental models for studies of malaria infection due to their susceptibility to some of the same strains that cause disease in humans and endemic viruses that are analogues of opportunistic human viruses. NK cells were identified from CD3 negative lymphocytes and sub-phenotyped by CD16 and CD8 expression, to classify CD8+ and CD8− NK cells and absolute numbers in peripheral blood were compared. Analysis of expression of NCR (Natural Cytotoxicity Receptors) proteins, NKp30 and NKp46 showed that NK cells from NHP had preferential expression of NKp46 over NKp30 and expression is mostly restricted to subset of CD8+ NK cells. Peripheral blood lymphocytes, enriched based on NKp46 expression, were further phenotyped as classic CD3−CD16+CD8+NK cells. Functional analysis of enriched cells was focused to verify whether subsets differ in cytolitic and/or “innate” in nature and to correlate with NCR protein expression. Enriched NK cells were co-cultured for stimulation with standard target cells including tumor cells and analyzed for cytokine production. The potential plasticity of peripheral blood NK cells in NHP based on steady state and/or inflammatory/pathogenic status of species will be analyzed and discussed.
Bisphenol-A diminishes neutrophil recruitment in zebrafish
Authors: Carson Sandra , Ohashi Sarah , Gerber Drew , Tieu Tam , Walser-Kuntz Evan , Walser-Kuntz Debby
Published: January 2023

The prevalent use of bisphenol-A (BPA) in food containers, thermal paper, and other products provides an environmental challenge to the immune system of both humans and wildlife as BPA disrupts estrogen activity and alters signaling. We are exploring the impact of in vivo BPA exposure on innate immunity in both mice and zebrafish. Our earlier work in C57BL/6J mice showed that low dose, in vivo BPA exposure significantly decreases LPS-induced expression of CXCL-1, a chemokine that recruits neutrophils to the site of infection and the mouse homolog of human IL-8, in spleen cells. To determine if reduced chemokine secretion has a functional outcome, we expanded our studies to the zebrafish embryo model. Real time PCR analysis indicates that 4 to 6 day post fertilization (dpf) zebrafish exposed to environmentally relevant doses of BPA show reduced background expression of CXCL-8, the zebrafish homolog of mouse CXCL-1, and a reduction in the total number of neutrophils (4 dpf). BPA exposure also reduces the upregulation of CXCL-8 induced by a tail cut injury in 4 dpf zebrafish. MPO::GFP zebrafish expressing GFP under a neutrophil-specific myeloperoxidase promoter were used to track neutrophil movement for six hours following a tail cut injury. 4 dpf zebrafish exposed to BPA show a significantly reduced neutrophil recruitment to the site of injury compared to controls (p=0.004), a finding which correlates with the reduction in injury-induced CXCL-8 expression. Together our findings demonstrate that BPA exposure during early zebrafish development diminishes two key innate immune responses, thus potentially increasing susceptibility to infection.
CD38: biomarker and functional role in human macrophages
Authors: Guerau-De-Arellano Mireia , Amici Stephanie A. , Young Nicholas , Papenfuss Tracey L. , Torrelles Jordi , Jarjour Wael
Published: January 2023

Macrophages mediate innate immune responses via a spectrum of phenotypes that range from inflammatory to resolving. We previously identified CD38 as a robust marker of inflammatory macrophages useful in both murine in vitro and in vivo models. We hypothesized that CD38 plays a similar biomarker and/or functional role in human macrophages and autoimmune inflammatory disease. Using human macrophages of primary or monocytic cell line origin, we found that CD38 is robustly induced in human macrophages exposed to LPS and IFN-g inflammatory stimuli, but not with the alternative stimulus, IL-4. Analyses in the systemic autoimmune disease Systemic Lupus Erythematosus (SLE) revealed that CD38hi classical and non-classical monocyte subpopulations are associated with disease activity. In addition, genetic or pharmacologic CD38 loss-of-function in human primary macrophages resulted in suppressed inflammatory cytokines IL-6 and IL-12p40 secretion in human primary macrophages. Taken together, these findings support a biomarker and functional role for CD38 in inflammatory macrophages/monocytes and inflammatory disease.
Glutathione Reductase Promotes Fungal Clearance and Suppresses Inflammation During Systemic Candida albicans Infection
Authors: Liu Yusen , Kim Victoria Y. , Li Jinhui , Batty Abel , Crowell Sara A. , Jin Yi , Zhang Jian , Nelin Leif D.
Published: January 2023

Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide (GSSG) to glutathione (GSH), a major cellular antioxidant. We have previously shown that Gsr facilitates neutrophil bactericidal activities and is pivotal for host defense against bacterial pathogens. However, it is unclear whether Gsr is required for immune defense against fungal pathogens. It is also unclear whether Gsr plays a role in immunological functions outside of neutrophils. To address these questions, we studied the effect of Gsr knockout on immune defense against C. albicans. Upon infection by C. albicans, Gsr−/− mice displayed dramatically increased fungal burden in the kidneys, cytokine and chemokine storm, striking neutrophil infiltration and histological abnormalities in both the kidneys and hearts, and substantially elevated mortality. Large fungal foci surrounded by massive numbers of neutrophils were detected outside of glomerulus in the kidneys of Gsr−/− mice but were not found in wildtype mice. Examination of the neutrophils and macrophages of Gsr−/− mice also revealed several defects, including compromised phagocytosis, attenuated respiratory burst, and impaired fungicidal activity in Gsr−/− neutrophils in vitro. Moreover, upon C. albicans stimulation, Gsr−/− macrophages produced increased levels of inflammatory cytokines and exhibited elevated p38 and JNK activities, at least in part due to lower mitogen-activated protein kinase phosphatase (Mkp)-1 activity and greater Syk activity. Thus, Gsr-mediated redox regulation was found to be crucial for fungal clearance by neutrophils and the proper control of the inflammatory response by macrophages during host defense against fungal challenge.
Metabolic barriers underlie interferon gamma-mediated restriction of intracellular bacterial pathogenesis
Authors: Price Jordan , DiPeso Lucian , Nomura Daniel , Vance Russell E.
Published: January 2023

Interferon gamma (IFNγ) plays a critical role in controlling Legionella pneumophila (L. pneumophila) infection in vivo and in vitro. IFNγ and IFNγ receptor-deficient mice fail to clear L. pneumophila and eventually succumb to infection. In vitro, L. pneumophila replication is restricted in IFNγ-stimulated macrophages; however, the mechanism of IFNγ-dependent restriction of L. pneumophila remains mysterious and does not require most known IFNγ-driven antimicrobial pathways, including induction of autophagy and production of reactive nitrogen. As enhanced glycolysis is a notable metabolic characteristic of IFNγ and toll-like receptor-stimulated macrophages, we hypothesized that changes in macrophage metabolism are required for IFNγ-mediated restriction of L. pneumophila. Using a strain of L. pneumophila that is resistant to the glycolysis inhibitor 2-deoxyglucose (2DG), we were able to rescue bacterial growth in IFNγ-stimulated macrophages treated with 2DG. Interestingly, we observed that 2DG-mediated induction of the unfolded protein response appears to be more important than glycolysis inhibition in terms of interfering with IFNγ-mediated restriction of L. pneumophila. Our data suggest that metabolic shifts in IFNγ-stimulated macrophages could play a role in restriction of intracellular bacterial pathogen replication by altering the pool of available intracellular metabolites.
RAGE inhibitor prevents cognitive dysfunction and reduces inflammatory mediators in experimental pneumococcal meningitis
Authors: Generoso Jaqueline S. , Collodel Allan , Simões Lutiana Roque , Dagostin Caroline , Dominguini Diogo , Lodetti Bruna França , Giridharan Vijayasree V. , Michels Monique , Vieira Andriele , Dal-Pizzol Felipe , Barichello Tatiana
Published: January 2023

The inflammatory processes during pneumococcal meningitis increase the production of inflammatory mediators culminating with increased production and deposition of amyloid proteins. Our hypothesis is that the interaction of the RAGE-Aβ can stimulate multiple intracellular signaling pathways which leads to the inflammatory mediator’s production. This can lead to neuronal damage and impairment of the integrity of the blood-brain barrier (BBB) causing memory impairment and learning. Therapeutic strategies suggest that RAGE is an important target for the treatment of inflammatory diseases, since inflammation increases Aβ synthesis and regulates RAGE positively, and the activation of this pathway induces long-term cognitive dysfunction. The aim of this study was evaluated cytokines levels; BBB integrity; behavioral response and Aβ deposition, RAGE and high mobility group box-1 (HMGB-1) expression in adult Wistar rats after meningitis induction treated with RAGE-antagonist (FPS-ZM1). To evaluate the levels of tumor necrosis factor α (TNF-α), interleukin (IL) 1β, and IL-6 the animals were killed at 24 h; for BBB integrity were killed at 12, 18 and 24 h; for the behavioral and expression of RAGE, HMGB1 and Aβ the animals received treatment with ceftriaxone and were killed within 10 days after the induction of meningitis. In the tests the inhibition of RAGE with FPS-ZM1 decreased the cytokines production only prefrontal cortex, prevented the BBB break in 12, 18 and 24 h in the hippocampus and cerebral cortex, and decreased cognitive damage 10 days after meningitis induction. We demonstrate that bacterial meningitis may trigger cognitive impairment through up-regulation RAGE axis and RAGE-antagonist prevented cognitive impairment.
The effects of ROS on neutrophil containment of the intracellular bacteria, Listeria monocytogenes
Authors: Okunnu Busola , Berg Rance E.
Published: January 2023

Extracellular superoxide dismutase (ecSOD) is commonly regarded as having a protective function during ROS induced inflammation. However, using ecSOD congenic mice expressing differing levels of ecSOD activity (HI, WT & KO), we found that high ecSOD activity is not conducive to host survival during Listeria monocytogenes (Lm) infection. Interestingly, although the infected ecSOD HI mice had a higher percentage of neutrophils present in the target organs in comparison to the ecSOD KO mice, they also had a higher bacterial burden. To better understand the function of neutrophils during infection with Lm, we studied phagosomal containment in the context of varying levels of ecSOD activity. A higher percentage of KO neutrophils took up and allowed for phagosomal escape of Lm. However, although there was no difference in the relative amount of Lm that escaped per neutrophil in the context of ecSOD activity, the KO neutrophils took up more Lm than the HI neutrophils. Therefore, neutrophils from the KO mice were more effective at keeping Lm contained in comparison to the HI neutrophils. The effect of ecSOD on the ability of monocytes to contain Lm was also analyzed. It was observed that ecSOD had no effect on monocyte association and phagosomal containment of Lm. However, independent of ecSOD, a lower percentage of monocytes associate with and allow for phagosomal escape of Lm, in comparison to neutrophils. In conclusion neutrophils are more effective than monocytes at phagocytosis of Lm and ecSOD activity hinders neutrophil phagosomal containment of Lm. Future studies focused on how ecSOD activity modulates downstream neutrophil functions, such as Lm killing, will lead to a better understanding of the overall effects of ROS on neutrophil functions.
Trogocytosis: a novel mechanism neutrophils use to kill a large, motile extracellular parasite
Authors: Mercer Frances , Ng Shek Hang , Brown Taylor M. , Boatman Grace , Johnson Patricia J.
Published: January 2023

Trichomonas vaginalis (Tv), a human-infective parasite, causes the most common non-viral sexually transmitted infection worldwide and contributes to adverse inflammatory disorders. The immune response to Tv is poorly understood. Neutrophils (PMN) are the major immune cell present at the Tv-host interface and are thought to clear Tv. However, the mechanism of PMN clearance of Tv has not been characterized. We demonstrate that human PMN rapidly kill Tv in a dose dependent, contact-dependent and NET-independent manner. In contrast to phagocytosis, we observed that PMN killing of Tv involves taking “bites” of Tv prior to parasite death, using trogocytosis to achieve pathogen killing. Both trogocytosis and parasite killing are dependent on the presence of PMN serine proteases and human serum factors, consistent with a model that opsonins mediate initial tight contact, but that serine protease activity is required for “nibbling.” Trogocytosis has previously been described as a way for immune cells to exchange membrane proteins, as a mechanism to spread intracellular bacteria from one cell to another and for E. histolytica to injure or kill host cells. Our analyses are the first to demonstrate the use of trogocytosis by a mammalian phagocyte for pathogen clearance and reveal a novel mechanism used by PMN to kill a large, highly motile target.
Food allergy is associated with altered epithelial gene expression driven by early life dysbiosis
Authors: Plunkett Catherine H. , Feehley Taylor J. , Bao Riyue , Belda-Ferre Pedro , Hong Sung Min Choi , Campbell Evelyn , Aitoro Rosita , Nocerino Rita , Paparo Lorella , Antonopoulos Dionysios A. , Andrade Jorge , Canani Roberto Berni , Nagler Cathryn
Published: January 2023

A marked increase in disease prevalence has made food allergies a major public health concern. One hypothesis for this rise is that recent lifestyle factors have altered the composition of the intestinal microbiota, increasing susceptibility to allergic disease. Host-microbiota interactions are essential for immune homeostasis, and perturbations of naturally-selected bacterial populations, a condition called dysbiosis, are linked to many different pathologies. To investigate the role of the microbiota in regulating food allergies, we colonized germ free mice with bacteria from healthy or cow’s milk allergic (CMA) infants. We found that colonization with bacteria from healthy infants is sufficient to protect against sensitization to the milk allergen β-lactoglobulin (BLG). In contrast, colonization with bacteria from CMA infants fails to protect, resulting in high concentrations of BLG-specific IgE and anaphylactic responses following BLG challenge. Analysis of bacterial taxa in feces from multiple healthy and CMA infants identified significant differences in bacterial composition that distinguished health status in both the human donors and the colonized mice, emphasizing the clinical relevance of our gnotobiotic model. RNAseq of ileal epithelial cells revealed differentially expressed genes (DEGs) that separate healthy- and CMA-colonized mice. Integration of ileal bacteria and ileal DEGs identified taxa that were significantly correlated with gene expression changes. Together, these results demonstrate that the composition of the intestinal microbiota regulates systemic responses to dietary antigen and suggest that modulation of these communities may be useful in treatment or prevention of food allergy.
Engineered immunomodulatory protein tyrosine phosphatase ameliorates inflammatory skin diseases
Authors: Kim Won-Ju , Koo Ja-Hyun , Cho Hyun-Jung , Lee Jae-Ung , Kim Ji Yun , Lee Sohee , Lee Hong-Gyun , Kim Jong Hoon , Oh Mi Seon , Suh Minah , Shin Eui-Cheol , Ko Joo Yeon , Sohn Myung Hyun , Choi Je-Min
Published: January 2023

Atopic dermatitis and psoriasis are the two most common chronic inflammatory skin diseases with unmet needs. There are still no effective and safe topical therapeutics for inflammatory skin diseases and current developing biologics have limitation of administration methods. Here, we identified a novel transdermal delivery peptide (AP) and generated engineered immunomodulatory protein to inhibit dermatitis. AP-conjugated proteins exhibited significant intracellular transduction efficacy in keratinocytes, fibroblasts, and immune cells. Transdermal delivery of AP-dTomato protein showed localization into the dermis and epidermis in both mouse and human skin. Next, we generated AP-conjugated phosphatase domain of TC-PTP protein (AP-rPTP) for regulating cytokine and T cell receptor signaling. AP-rPTP inhibited pSTAT1, pSTAT3, pSTAT5 and pSTAT6 upon cytokine stimulation in mouse splenocytes. AP-rPTP also regulated T cell activation, proliferation and Th1, Th2 differentiation upon CD3/28 stimulation. The transdermal paper-patch of the AP-rPTP protein significantly ameliorated ear thickness, tissue inflammation, and cytokine expression in allergic dermatitis as well as in psoriasis-like mice models. These results collectively demonstrate the feasibility of utilizing AP peptide for biologic drug delivery via paper-patch and suggest AP-rPTP as a novel immune modulatory drug candidate for inflammatory skin diseases.