bluuebunny/crossref_metadata_2025 huggingface dataset:
Morphine dysregulates Paneth cell antimicrobial peptide secretion in a TLR2 dependent manner.
Authors: Jalodia Richa , Meng Jingjing , Sharma Madhulika , Ramakrishnan Sundaram , Roy Sabita
Published: January 2023

Opioids are the gold standard for moderate to severe pain management. Although, the co-morbidities associated with chronic morphine use on systemic immune function has been well demonstrated by our lab and others its effect on mucosal immunity and gut homeostasis are less well defined. We have recently demonstrated that morphine treatment disrupts intestinal epithelial barrier function and suppresses systemic immune function leading to altered gut microbial dysbiosis and inflammation. We have also shown in vivo that morphine can alter the immune response by APCs leading to reduced host immune response. However, the cross talk between the gut microbiome and host intestinal epithelium and immune cells has never been explored. Paneth cells are one of the main secretory component of intestinal epithelial layer that secrete antimicrobial peptides which maintains gut homeostasis between pathogenic and commensal microbes. They also secrete pro-inflammatory cytokines which regulate the functions of mucosal immune cells. In both a murine in vivo model and in an in vitro organoid epithelial culture model we show a direct effect of morphine on paneth cells. Intraperitonial morphine treatment significantly increases the transcription level of antimicrobial peptides, a-defensins, lysozyme, Regg as well as intestinal pro-inflammatory cytokine IL1b, IL6 and TNFa levels in C57/B6 mice model. These results are also corroborated in in vitro organoid cultures. Surprisingly, these effects were completely abolished in a Toll-like receptor-2 (TLR2) knock out mice suggesting that the effect is mediated through TLR2. The role of TLR 2 and the cell type that mediates morphine’s effects will be further investigated using TLR2 floxed mice.
Lactic acid suppresses LPS-induced cytokine production in mast cells by limiting glycolysis and ATP availability
Authors: Caslin Heather , Abebayehu Daniel , Qayum Amina Abdul , Hoeferlin Alexis , Chalfant Charles
Published: January 2023

Despite a mortality rate of 30–50%, there are no targeted molecular treatments for sepsis. Our understanding of the secondary immunosuppressive phase is recent, and it is unclear whether many mediators are helpful or detrimental to patient survival. Elevated blood lactate and slow lactate clearance are associated with mortality, however, any regulatory roles are currently unknown. Because mast cells can be activated systemically and contribute to sepsis, this project aims to elucidate lactic acid effects on mast cell activation and signaling. Lactic acid significantly suppressed LPS-induced cytokine and chemokine production as well as NFκB transcription in bone marrow derived and peritoneal mast cells. These effects were dependent upon pH and MCT-1 transporter activity. Further, lactic acid significantly suppressed cytokine and chemokine induction following LPS-induced septic shock in vivo. We also examined the role of lactic acid as a feedback regulator of glucose metabolism. Lactic acid treatment reduced glucose uptake, lactate export, and hexokinase 2 expression following LPS activation. The glycolytic inhibitors 2-deoxyglucose, sodium oxamate, or dichloracetate mimicked lactic acid effects, suppressing LPS-induced cytokine production and NFκB transcription. Furthermore, adding ATP to the culture media at the time of LPS activation reversed lactic acid effects. These results indicate that suppressing glycolysis and ATP production are necessary and sufficient for lactic acid effects. Future studies should target enhancing glycolysis and ATP production to improve immune function and counteract lactic acid effects in sepsis.
Vagal tonic activity modulates immune suppression in sepsis survivors
Authors: Rana Minakshi , Son Myoungsun , Fei Yurong , La Bella Andrea , Ochani Mahendar , Chiu Pui Yan , Sherry Barbara , Diamond Betty
Published: January 2023

Sepsis survivors exhibit persistent immune dysfunction and compromised quality of life with premature mortality due to increased vulnerability to infections. Previously we demonstrated in a cecal ligation and puncture (CLP) model, sepsis survivors had a reduced TNFα response to LPS-challenge in vivo. The vagus nerve is known to modulate TNFα production. To evaluate the role of the vagus nerve in the diminished TNFα production, sepsis-surviving mice were subjected to bilateral subdiaphragmatic vagotomy 2 weeks post-CLP. CLP-surviving vagotomized mice exhibit increased (p<0.05) TNFα levels in response to LPS-challenge 4 weeks post surgery compared to CLP. Moreover, vagus nerve stimulation in sham mice dampened (p<0.05) the LPS-induced TNFα response while it had no effect in CLP-surviving mice; suggesting that vagus nerve signaling is constitutively active in CLP-sepsis survivors. To identify whether there was an alteration in the number of acetylcholine-producing T cells that relay the vagus signal to splenic macrophages, we used ChAT-EGFP mice. Our findings show that the frequency of splenic ChAT-EGFP+ cells among memory CD4+CD44highCD62LlowT cells is increased (p<0.001) in CLP-surviving mice compared to sham, and vagotomy in these mice resulted in decreased (p<0.01) frequency of ChAT-EGFP+ cells, buttressing the conclusion that the vagus nerve is constitutively firing action potentials to activate more ChAT+ T cells. Methyllycaconitine, the α7 nicotinic acetylcholine receptor antagonist, increased (p<0.01) LPS-induced TNFα secretion in CLP-surviving mice compared to saline treated-CLP. In summary, our study demonstrates that altered vagus nerve activity contributes to the immune impairment in sepsis survivors.
Resistance of macrophages to pore-forming toxins might depend on MEK1/2 and MLK3 activity
Authors: Ray Sucharit , Keyel Peter A.
Published: January 2023

Necrotizing soft tissue infections (NSTIs) are lethal bacterial infections that quickly spread through soft tissues. Virulence factors in NSTIs include pore-forming toxins (PFTs) like Streptolysin O and Perfringolysin O secreted by Streptococcus pyogenes and Clostridium perfringens. These PFTs form large transmembrane pores on host cells leading to cell death. Cells resist death through several poorly understood membrane repair mechanisms, including ceramide generation. Membrane repair is necessary for macrophages to survive and to eliminate PFT-secreting bacteria. Interestingly, macrophages are ~10–20 times more resistant to PFTs than most cells, but this mechanism of increased resistance is unknown. One possibility is that MAP kinase signaling enhances repair because ceramide can signal in other systems through a MAP kinase cascade involving Mixed-Lineage Protein Kinase 3 (MLK3), Mitogen-Activated Protein Kinase Kinase 1/2 (MEK1/2) and Jun amino-terminal kinases (JNK). We tested if MLK3 and downstream kinases MEK1/2, JNK, and Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) contribute to the repair mechanism in cells. We treated immortalized cell lines or murine bone marrow derived macrophages with either the MEK1/2 inhibitor U0126, the JNK inhibitor SP600125, or the MLK3 inhibitor URMC099, challenged cells with Streptolysin O or Perfringolysin O and measured cell death by flow cytometry. Inhibition of MEK1/2 or MLK3, but not JNK, made cells ~2-fold more sensitive to killing by toxins. Overall, these findings suggest that MEK1/2 activation contributes to a general mechanism of membrane repair. Enhancing this mechanism could provide new tools for protecting cells from PFTs during NSTIs.
Neutrophil cathepsin G inhibits hapten-presenting dendritic cell production of IL-12 and skewing of hapten-reactive CD4 T Cell development to IFN-γ- and IL-17-producing effector cells in allergic contact sensitivity
Authors: Kish Danielle , Min Susie , Fairchild Robert L.
Published: January 2023

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten skin sensitization and challenge. Sensitization of WT mice induces hapten-reactive CD8 cells producing CHS effector cytokines IFN-γ/IL-17 and IL-4-producing CD4 cells that cannot mediate CHS. Although CXCR2-dependent Gr-1+ cell recruitment into hapten challenged skin is required to direct effector CD8 T cell infiltration to elicit CHS, sensitization of CXCR2−/− mice or neutrophil-depleted WT mice induced both hapten-reactive CD4 and CD8 cells producing IFN-γ and IL-17. CD4 T cell-mediated CHS responses were not generated during hapten sensitization of neutrophil depleted WT mice treated with anti-IL-12 mAb or neutrophil depleted IL-12−/−mice. Neutrophil depletion during hapten sensitization of WT mice markedly increased numbers of IL-12-producing hapten-primed dendritic cells (DC), DC-derived p40, and T-bet-expressing CD4 T cells in the skin-draining lymph nodes. Sensitization of mice lacking the neutrophil serine protease cathepsin G induced hapten-reactive CD4 and CD8 T cells producing IFN-γ and IL-17 with elevated and elongated CHS responses to challenge. Few Gr-1+ cells were detected in the skin draining lymph nodes following senstization suggesting this regulation may not be mediated through direct Gr-1-DC interactions. In support of this, induction of CHS effector CD4 T cells producing IFN-γ in neutrophil-depleted WT mice was eliminated by subcutaneous injection of active, but not inactivated, cathepsin G during sensitization. Thus, hapten skin sensitization induces neutrophil release of cathepsin G that systemically inhibits hapten-presenting dendritic cell production of IL-12 and the development of CHS effector CD4 T cells.
A double edge function of type 17 immunity in Streptococcus pneumoniae pathogenesis during a co-infection with Influenza A Virus
Authors: Khan Nadeem , Sohail Ifrah , Schmit Taylor , Jacobson Brenden
Published: January 2023

Streptococcus pneumoniae (Spn) is a respiratory pathogen responsible for significant global disease burden (otitis media, pneumonia, sepsis). Despite success with current Pneumococcal Conjugate Vaccines, a significant proportion of humans remain colonized with Spn, rendering them at risk for developing Spn infections. Upper respiratory nasopharyngeal (NP) colonization by Spn is a precursor to disease, and viral induced inflammation is a key risk factor for the transition of commensal colonization to disease pathogenesis.

We utilized a mouse co-infection model of Spn and influenza virus to determine the role of virus induced acute inflammation in the transition of Spn colonization to disease pathogenesis. We show that early Spn NP colonization is allowed by an immune quiescence state, maintained by the lack of IL-17 expression. Introduction of influenza virus in Spn colonized mice elicits a robust IL-17 driven inflammatory response, which induces robust neutrophilia and tissue damage in the NP. The IL-17 mediated inflammatory response was associated with a significant increase in Spn NP bacterial density, leading to Spn invasion and bacteremia. Antibody mediated neutralization of IL-17 suppressed NP inflammation and bacterial density and prevented bacteremia. IL-17 receptor expression was also detected in nasal myeloid cell infiltrate. RoRγt-KO mice remained protected from bacteremia, mimicking the reduced pathogenesis of IL-17 neutralized WT mice. Using RoRγtGFP mutant mice, we detected the recruitment of GFP expressing RoRγt cell population in NP lavages and nasal associated lymphoid tissues.

We conclude that IL-17 blockade could contain influenza induced NP pathology and protect against Spn invasive pathogenesis.
AMP-activated kinase (AMPK) promotes innate immunity and antiviral defense against Zika virus induced ocular infection
Authors: Kumar Ashok , Singh Pawan Kumar , Giri Shailendra
Published: January 2023

Purpose

We recently reported that Zika Virus infected multiple retinal cell types and caused chorioretinal atrophy in mouse eyes. In this study, we investigated the potential role of AMPK, a master regulator of cellular metabolism, in pathogenesis of ocular ZIKV infection.

Methods

Primary human RPE cells were infected with ZIKV for various time points and AMPK phosphorylation was detected Western blotting (WB). AMPK activity was modulated by pharmacological activators (AICAR and Metformin) and inhibitors (Compound C) of AMPK. ZIKV replication was assessed by immunofluorscence staining for 4G2 and western blot detection of NS3 proteins. For in vivo studies B6 mice eye were challenged with ZIKV with or without AICAR treatment and disease progression were measured by fundus imaging. Activation of innate immune and antiviral response genes was assessed by qPCR.

Results

ZIKV challenge upregulated the AMPK phosphorylation in Pr. RPE cells in a time-dependent fashion. Our results indicate that AMPK deficient mouse embryonic fibroblast (MEF) cells were more permissive to ZIKV replication as compared to WT MEFs. AMPK activation using AICAR and Metformin restricted ZIKV replication in Pr. RPE cells and presence of Compound C reversed the effect of AICAR/Metformin. In vivo AICAR treatment ameliorated ZIKV-induced chorioretinal atrophy in mice with increased antiviral responses. ZIKV infection leads to activation of MEK pathways and an MEK pathways inhibitor, U0126, attenuated ZIKV replication.

Conclusion

Collectively, these findings indicate the AMPK activation evokes antiviral innate responses and restricts ZIKV replication. Hence, targeting AMPK signaling can be explored to develop anti-viral therapy against ZIKV infection.
Glycyrrhizin (GL) modulates HMGB1/miR-222-associated M2bMφ polarization and improves host antibacterial resistance of γ-irradiated mice against gut bacteria-associated sepsis
Authors: Ito Ichiaki , Loucas Bradford D. , Suzuki Sumihiro , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

M2bMφ demonstrated in the mesenteric lymph nodes (MLNs) of whole body γ-irradiated mice (WBI-mice) have been characterized as responsible cells for the increased susceptibility of them to gut bacteria-associated sepsis. In our previous studies, MLN-Mφ from WBI-mice treated with GL have been characterized as non-M2bMφ. In this study, we determined how GL suppresses the γ-irradiation-associated M2bMφ polarization. WBI-mice were treated with GL (10 mg/kg, i.p., twice a day) 8 and 9 days post-irradiation. The antibacterial resistance of WBI-mice to Enterococcus faecalis oral infection (a model of sepsis caused by bacterial translocation) was evaluated by a bacterial growth in organs. F4/80+ cells (Mφ) were isolated from NMLs of GL-treated WBI-mice 10 days post-irradiation, and analyzed for the expression of miR-222 and GAS5 by real-time PCR. Serum HMGB1 level was measured by ELISA. In the results, the pathogen vigorously grew in various organs of WBI-mice, while the bacteria did not grow in organs of the same mice treated with GL. HMGB1, increased in sera of WBI-mice 2–15 days post-irradiation, stimulated the expression of miR-222 with a function to degrade GAS5 RNA (a factor to control the M2bMφ polarization). However, miR-222 was not expressed in the same Mφ cultures supplemented with GL. GL has been shown to bind to HMGB1 directly and inhibit its cytokine activity. The reduction of GAS5 and the M2bMφ polarization were not demonstrated in Mφ from WBI-mice treated with GL. These results suggest that GL improves host antibacterial resistance of WBI-mice against gut bacteria-associated sepsis through the modulation of HMGB1/miR-222-associated M2bMφ polarization.
Heterogeneous ribonucleoprotein M influences specific cytokine outcomes upon Salmonella Typhimurium infection
Authors: West Kelsi , Patrick Kristin L. , Watson Robert O.
Published: January 2023

A complex and dynamic regulatory system is needed to fight infection while also preventing induction of inflammatory disease. In macrophages, much is known about how pathogens modulate the innate immune transcriptional response. However, the significance of alternative splicing and manipulation of splicing factors upon infection remains unclear. We have identified several splicing factors that are differentially phosphorylated upon Salmonella Typhimurium bacterial infection, strongly suggesting an important role for RNA processing in the regulation of innate immune responses. A major family of identified phosphorylated splicing factors was the hnRNP (heterogeneous ribonucleoprotein) RNA binding factors, which contribute to multiple aspects of nucleic acid metabolism. Upon LPS stimulation and Salmonella Typhimurium infection, we have found that one of these proteins, hnRNPM, modulates expression of an inflammatory cytokine, IL-6. We have also identified hnRNPM binding sites within the introns of IL-6. We are currently investigating how hnRNPM localizes to chromatin, how hnRNPM intron recognition regulates IL-6 transcript levels, and how the phosphorylation of hnRNPM influences these processes.
Therapeutic potential of staphylococcal superantigen-like protein 7 for acute complement activation mediated diseases
Authors: Li Yan , Lin Feng
Published: January 2023

Previous studies have demonstrated that staphylococcal superantigen-like protein 7 (SSL7), a protein produced by the opportunistic bacterial pathogen Staphylococcus aureus, potently inhibits the formation of the complement membrane attack complex and release of C5a by binding to complement component 5 (C5). However, because of the predicted immunogenicity of SSL7 in humans, its potential as a new complement inhibitor for treating Complement-Mediated Diseases were uncertain. In this study, we found that administration of SSL7 efficiently protected red blood cells from complement-mediated lysis and reduced hemoglobinuria in a mouse model of paroxysmal nocturnal hemoglobinuria (PNH).

Interestingly, at the dose required for effective complement inhibition in vivo, although repetitive administrations of SSL7 once every other week for six weeks elicited anti-SSL7 antibody production, SSL7 was still able to attenuate acute complement activation mediated Hemolysis and hemoglobinuria significantly in these mice. In addition, anterior chamber administration of SSL7 before repeatedly treating mice with SSL7 suppresses the production of SSL7-reactive antibodies and retain the treatment efficacy of SSL7. These results suggest that SSL7 could thus be developed as an economical alternative to the existing C5-targeted drug eculizumab, especially for controlling acute complement activation in catastrophic conditions such as drug-induced immune hemolytic anemia, ABO-incompatible red blood cell, transfusion, and cardiopulmonary bypass surgery.
cGAS-STING activation in Ly6Chi monocytes induces IFNg during pulmonary pneumococcal infection
Authors: Patel Seema , Tucker Heidi R. , Mansouri Samira , Blaauboer Steven M. , Jin Lei
Published: January 2023

Streptococcus pneumoniae is the most common cause of pneumonia, resulting in high morbidity and mortality worldwide despite advanced antimicrobial therapies and vaccines. Human individuals with constitutively active STING mutations have recurrent pneumococcal infections. Moreover, we found that mice lacking STING expression are more resistant to pulmonary S. pneumoniae infection compared to their wild-type controls. Here we investigated the role STING in the pathogenesis of pulmonary pneumococcal infection. We discovered two waves of IFNg production in the lung and IFNg at 48hrs, but not at 24hrs post-intra-nasal S. pneumoniae infection (hpi) is STING dependent. Using STING conditional knockouts, CCR2−/− and GREAT (interferon-Gamma Reporter with Endogenous polyA Transcript) mice, we found Ly6Chi monocytes to be the major contributors of lung IFNg production at 48hpi. Further, IFNg production requires cGAS-STING signaling pathway in Ly6Chi monocytes. IFNg stimulates inflammatory M1 macrophage polarization. We found STING−/− lung macrophages exhibit anti-inflammatory M2 like phenotype. We propose that cGAS-STING induces late stage IFNg in Ly6Chi monocytes to promote M1-like macrophage differentiation and exacerbate the pulmonary pneumococcal infection. Our novel findings a) Two distinct waves of IFNg production, b) cGAS-STING pathway in Ly6Chimonocytes but not macrophages induce lung IFNg production at 48hpi. How cGAS-STING signaling pathway induces IFNg production in monocytes will be discussed. The findings of this project will add substantial information to the role of IFNg in the host defense mechanism against S. pneumoniae and help design more effective treatment options.
Eomesodermin promotes a central memory phenotype in CD8+ T cells through the induction of IL-10.
Authors: Reiser John , Gerber Allison , Sadashivaiah Kavitha , Furusawa Aki , Banerjee Arnob , Singh Nevil J.
Published: January 2023

Following an immune response, a fraction of antigen-specific T cells persist as memory cells. Of these, central memory CD8+ T cells (TCM), which express secondary lymphoid organ-homing molecules and self-renew efficiently, are critical for maintaining long term memory. High levels of the T-box transcription factor Eomesodermin (Eomes) are associated with the TCM phenotype, but the mechanisms by which Eomes controls different aspects of TCM function are poorly understood. Using Eomes-knockout (EKO) CD8+ T cells, we demonstrate that Eomes regulates the expression of a number of TCM-associated markers including Bcl6, Ly6C and CD62L. In addition, we found that Eomes promotes the expression of IL-10 in activated CD8+ T cells. Interestingly, the addition of exogenous IL-10 was sufficient to upregulate CD62L expression in WT cells. Taken together, our data suggest that Eomes uses IL-10 to promote at least part of the central memory phenotype in CD8+ T cells. Consistent with this model, we find that TCM are secreting IL-10 in vivo, without additional re-stimulation. These findings warrant a modified model of TCM differentiation which includes distinct roles for Eomes and IL-10 in a T cell-intrinsic program driving TCM formation.
Immune impact of tick-borne co-infections on canine leishmaniosis
Authors: Scorza Breanna , Toepp Angela , Mahachi Kurayi , Petersen Christine
Published: January 2023

Visceral leishmaniasis (VL), a zoonotic protozoal disease, kills an estimated 20,000 people annually (WHO, 2012). Dogs are the primary reservoir for parasite transmission to humans and clinical status correlates with infectiousness to sand fly vectors. Like humans, progression from asymptomatic to symptomatic CanL disease is accompanied by loss of CD4+ T cell responsiveness to Leishmania antigen. The molecular signals driving exhaustion in either species are poorly understood. Intriguingly, within a naturally-infected cohort of 174 dogs with CanL, asymptomatic dogs were 9.9× more likely to progress to clinical leishmaniosis if seropositive for ≥2 tick-borne co-infections (TBCs) over a six-month study period (p=0.0071). In Brazil, where dogs are a predominant reservoir for human VL, 80% of dogs were found to be seropositive for at least one TBC. We hypothesize that TBCs cause acute systemic inflammation in Leishmania-infected asymptomatic dogs, which promotes T cell exhaustion and symptomatic CanL. To evaluate this hypothesis, Type 1 immunity (circulating CD4 and CD8+ T cell proliferation, cytokine production, and inhibitory receptor expression in response to Leishmania antigen stimulation ex vivo and peripheral NK cell phenotypes) and clinical progression of CanL (complete blood count, blood chemistry panel, and clinical signs of CanL) will be compared in Leishmania infantum-infected dogs negative for TBCs or seropositive for Anaplasma spp., Ehrlichia spp., and/or Borrelia spp. These data may provide new strategies to prevent disease progression in dogs or people already infected with Leishmania and lead to less parasite transmission to humans by decreasing canine reservoir host parasitemia.
IL-12 and IL-23 dependent NK cell response is essential for protective immunity against secondary Toxoplasma gondii infection
Authors: Ivanova Daria Leonidovna , Gigley Jason P.
Published: January 2023

Natural Killer (NK) cells can develop memory-like features and contribute to long term-immunity in mice and humans in response to viral infections. NK cells are critical for protecting against acute T. gondii infection. How they impact long-term immunity in response to the parasite and mechanism(s) involved is unknown. Using a vaccine-challenge model of parasite infection we addressed these questions. After lethal reinfection of vaccinated mice with or without NK cells, we find they are required for immune control of secondary parasite infection. Unexpectedly, secondary responses of CD4 and CD8 T cells are also reduced in the absence of NK cells during reinfection of vaccinated mice. NK cells mount a secondary response noted by increased IFNγ production, however, adoptive transfer and NK cell fate mapping reveal that T. gondii-experienced cells are not intrinsically different from naïve cells in persistence and ability to protect. This suggests a cell extrinsic mechanism controls protective NK cell response during secondary infection. Although NK cell IFNg is IL-12 dependent during acute T. gondii infection, using anti-IL-12p70 antibody or IL-12p35−/− mice we find that secondary NK cell response is not fully dependent upon IL-12. IL-23 depletion with anti-IL-23p19 in vivo also significantly reduces the secondary NK cell response suggesting both IL-12 and IL-23 are involved. Anti-IL-12p40 treatment, which blocks both IL-12 and IL-23, completely eliminates protective secondary NK cell response, supporting this hypothesis. These results identify a previously unknown protective role of NK cells during secondary T. gondii infection that is not only IL-12 dependent, but also requires IL-23 and may involve an interaction with T cells.
Dynamics of the T follicular helper response in Borrelia burgdorferi infection
Authors: Hammond Elizabeth M. , Olsen Kimberly , Baumgarth Nicole
Published: January 2023

Borrelia burgdorferi (Bb), the bacterial agent of Lyme disease, has evolved numerous immune evasion mechanisms that result in Bb persistence in mice, its natural host, following infection via tick-bite. IgG antibodies control Bb-tissue loads but cannot clear the infection. We previously observed abnormalities in T-dependent B cell responses in mice following infection. Specifically, we demonstrated premature germinal center (GC) collapse, a lack of affinity maturation, memory B cell and long-lived plasma cell responses, and Ig responses that were skewed toward IgM. Due to the central role of CD4+ follicular T helper (TFH) cells in initiating and maintaining GCs, we hypothesized that Bb infection causes TFH dysfunction. Since Bb-specific CD4 T cell responses have not previously been characterized, we first identified an I-Ab restricted epitope on Arthritis related protein (Arp) of Bb, and used tetramers to characterize these Bb-specific CD4 T cells by FACS. We observed a surprisingly robust induction of Arp-specific TFH by day 14 of infection, which was followed by a precipitous decline in total and Arp-specific TFH by day 21 that preceded the observed collapse of GC. In order to explain this sharp TFH decline we studied the expression of inhibitory receptors on TFH. We also followed the induction of FOXP3+ regulatory T follicular (TFR) cells, which are known to limit GC responses. Not only were we able to demonstrate induction of a significant population of TFR, we also found that expression of the inhibitory Programmed death receptor 1 (PD-1) was significantly higher on Arp-specific TFH compared to overall TFH. Our data suggest that overshooting TFH regulatory mechanisms may inhibit T-dependent B cell responses during Bb infection.
Helminth-induced B lymphocytes mediate control of IL-17 and emphysema through their production of RELMα
Authors: Chen Fei , Wu Wenhui , millman Ariel , Palma Mark , El-Naccache Darine W. , Lothstein Katherine E. , Gause William C.
Published: January 2023

The intestinal nematode parasite, Nippostrongylus brasiliensis (Nb), can cause emphysema and the mechanisms remain poorly defined. Here we show that emphysema develops as early as 7 days after Nb inoculation and is associated with increased gene expression of Il17a, as well as increases in type 2 cytokines Nb infection of Il17a−/− mice results in a less severe emphysematous pathology, and decreased neutrophil influx to the lung, with little effect on the development of the type 2 immune response. Surprisingly, Nb infection of B cell deficient Jh−/− mice results in a more severe emphysema at day 7, and as late as day 30, and is associated with higher numbers of neutrophils and increased expression of Il17a in the lung. Most of the B cells in the lung are B2 regulatory cells and are activated through IL-4R signaling. However, IL-10 and other regulatory cytokines typically associated with B regulatory cell function did not play a major role in this response. Instead, at early stages of the response B cells produced high levels of RELMa. Transfer of B lymphocytes from WT mice or Il10−/− mice, but not from Il4Rα−/− mice, or Relmα−/− mice to recipient Jh−/−mice restored the control of emphysema, decreased the influx of neutrophils, and downregulated IL-17A. Although macrophages also expressed RELMa, it was not upregulated until 4 days after inoculation, whereas B cells upregulated RELMa as early as day 3. These studies show that elevations in IL-17 trigger emphysematous pathology and reveal a novel role for helminth-induced B cells in controlling IL-17A and the severity of emphysema through their production of RELMa.
Low affinity CD8+ T cells are a critical component in the development of experimental cerebral malaria
Authors: Jacobs Jesica R. , King Thayer P. , Lamb Tracey J. , Evavold Brian D.
Published: January 2023

Malaria is responsible for at least 500 million deaths annually. Of those who become infected with malaria, around 1% develop cerebral malaria, which is fatal in 15% – 20% of patients. Therefore, understanding the immune mechanisms contributing to the lethality of cerebral malaria are critical for developing appropriate therapies. Infection with Plasmodium berghei ANKA in C57BL/6 mice induces experimental cerebral malaria (ECM), which requires activated CD8 T cells. In these experiments, we used the highly sensitive two-dimensional (2D) micropipette adhesion frequency assay to define the affinity and frequency of the immunodominant glideosome associated protein 50 (GAP50)40–48 epitope. We found both high and low affinity GAP50 specific CD8 T cells are found in the spleen (d5) and brain (d6) of ANKA infected mice. However, the 2D micropipette assay was more sensitive than tetramer staining with respective measurements of 20% vs. 1.2% of CD8 T cells in the spleen and 30% vs. 13.2% of CD8 T cells in the brain of ANKA infected mice measured as specific for GAP50. Furthermore, 67% of the antigen specific CD8 T cells in the spleen and 50% in the brain had low affinity for GAP50. These data reveal that tetramer vastly underestimates the number of antigen specific cells responding during ANKA infection. Infection with the NK65 strain of P. bergei results in similar levels of parasitemia and frequency of CD8 T cells in the brain, but ECM does not develop. Unlike ANKA infection, micropipette analysis of CD8 T cells from the brains of NK65 revealed only high affinity CD8 T cells for GAP50. These data indicate that under appreciated low affinity CD8 T cells are a vital component of the pathogenic and lethal response observed in experimental cerebral malaria.
GM-CSF regulation of autophagy in monocytes enhances immune response to Cryptococcus gattii
Authors: Elsegeiny Waleed Abdel Wahab , Salas Antonio , Kullas Amy , Williamson Peter R.
Published: January 2023

Cryptococcal meningoencephalitis is responsible for over 15% of HIV-related deaths worldwide and is also the most common cause of non-viral meningitis in the US. The role of T-cells in cryptococcal disease is well studied however a significant portion of previously healthy non-HIV patients with cryptococcal meningoencephalitis have an intact CD4+ T-cell compartment. We observed that patients with GM-CSF autoantibodies have a decrease in autophagy within their classical monocyte compartment. This reduction in autophagy is correlated with an enhancement of IL-1β production, additionally GM-CSF synergizes with TLR2, TLR4, and TLR5 in the induction of IL-1β. The importance of GM-CSF inhibition of autophagy appears to be most dramatic when monocytes are in conditions of weak TLR stimulation, particularly TLR4 stimulation with LPS. The capsule for Cryptococcus gattii, the prominent species involved with cryptococcal meningoencephalitis in previously health adults, has been shown to have the ability to dampen or block surface recognition by the immune system, thus GM-CSF synergy may have a critical role for driving the immune response against C. gattii infection. In the murine model of myeloid-specific ATG5 deficiency, mice have enhanced survival to C. gattii infection. Additionally, this GM-CSF down-regulation of autophagy is independent of mTOR activity, and contingent on a cyclic AMP/CTLA4/DCP2 pathway.
Immune responses of channel catfish against parasite Ichthyophthirius multifiliis following theront vaccination
Authors: Xu Dehai , Zhang Qizhong , Shoemaker Craig A. , Zhang Dunhua , Moreira Gabriel S.A.
Published: January 2023

Ichthyophthirius multifiliis (Ich) is a severe fish parasite and results in heavy losses of freshwater fish. The fish surviving natural infections or vaccinated with live theronts develop strong immune responses. Little is known about how immune genes are induced or how they interact and lead to specific immunity against I. multifiliis in channel catfish Ictalurus punctatus. This study evaluated the differential expression of immune-related genes, including immunoglobulin, immune cell receptor, cytokine, complement factor and toll-like receptors in head kidney from channel catfish at different time points after vaccination with live theronts of I. multifiliis. The vaccinated fish showed significantly higher anti-Ich antibody than those of control fish. The vast majority of immunized fish (95%) survived the parasite challenge. Expression of IgM and IgD heavy chain genes exhibited a rapid increase from 4 hours (h4) to 2 days (d2) post vaccination. Expression of immune cell receptor genes (CD4, CD8-α, MHC I, MHC II β, TcR-α, and TcR-β) showed up-regulation from h4 to d6 post vaccination, indicating that different immune cells were actively involved in cellular immune response. Cytokine gene expression (IL-1βa, IL-1βb, IFN-□ and TNF-α) increased rapidly at h4 post immunization and were at an up-regulated level until d2 compared to the bovine serum albumin control. Expression of complement factor and toll-like receptor genes exhibited a rapid increase from h4 to d2 post immunization. Results of this study demonstrated differential expression of genes involved in the specific or non-specific immune response post immunization and that the vaccination against Ich resulted in protection against I. multifiliis infection.
Fasciola hepatica Glutathione S-tranferase suppresses inflammatory cytokines and chemokines in vivo against lethal doses of lipopolysaccharide on C57Bl/6 mice.
Authors: Aguayo Vasti M. , Espino Ana
Published: January 2023

The world-wide helminth parasite, Fasciola hepatica, is an excellent immunomodulator. It survives in the host by preventing immune responses against itself by using excretory-secretory products (ESPs). One of the ESPs well-known for its anti-oxidant properties is Glutathione S-transferase (GST). Although it has been studied as a vaccine, the function GST plays in immunomodulation has scarcely been studied. Previously, we reported how the use of GST prior to TLR ligands significantly decreased TLR activation in vitro and boosted the survival rate of mice injected intraperitoneally with GST before a lethal dose of LPS by 80%. In this study, we investigate whether these C57BL/6 mice that survived after a lethal dose of LPS experienced a reduction in inflammatory cytokines and/or chemokines. For this, C57BL/6 6–8 week-old mice were allotted into two groups of 12 animals each. The control group (n=12) was injected intraperitoneally with 0.1M endotoxin free PBS and allowed to rest 1 hr, while the experimental group (n=12) was injected intraperitoneally with 15 ug of GST and allowed to rest 1 hour. Afterwards, both groups were injected with a lethal dose (1 mg) of E.coli LPS (serotype 0111:B4; Sigma) and monitored during a 48 hour period. Sera from mice in the experimental group were compared to the control group by using a Cytokine 20-Plex Mouse Panel kit (Invitrogen) for a wide array of cytokines and chemokines. Our results demonstrate that injecting 15ug of GST intraperitoneally 1 hour before a lethal dose of LPS gives the surviving 80% of C57BL/6 mice significantly reduced amounts of pro-inflammatory cytokines and chemokines, such as IL-1β (p = 0.0004), IL-12p70 (p = 0.0001) and MIP-1α (p = 0.0024), after 48 hrs.
Immune-microbiome axis regulates liver inflammation.
Authors: Castillo Patricia , Kumar Pawan , Hand Timothy , Kolls Jay K.
Published: January 2023

Autoimmune hepatitis (AIH) is a liver disease characterized by circulating autoantibodies, persistent liver inflammation, and subsequent hepatocyte damage. Both Th17 cells and the intestinal microbiome have been implicated in AIH. Patients have elevated serum IL-17, an altered microbiota, and increased bacterial translocation. In a murine AIH model, concanavalin A (con A) hepatitis, Il17ra−/− mice are protected, and gentamicin has been reported to ameliorate disease. We sought to better understand the relationship between Th17 cells and the microbiome in hepatitis.

To study this, we generated intestinal epithelial-specific IL-17RA deficient mice (Il17rafl/fl × villin cre+ mice). Absence of enteric IL-17R signaling induced commensal dysbiosis, expansion of intestinal Th17 cells, and increased bacterial translocation to the liver at the naïve state. These mice were more susceptible to con A hepatitis, exhibiting increased lethality, serum ALT, and serum and liver IFNγ as compared to controls. This elevated liver IFNγ was susceptible to BET protein inhibition ex vivo, suggesting the Ifng locus is open and susceptible to epigenetic regulation. Cohousing studies eliminated ALT differences between knockouts and controls, suggesting a role of the microbiome.

We hypothesize that disrupted enteric IL-17 signaling at the naïve state promotes intestinal dysbiosis and increased bacterial translocation to the liver. This causes epigenetic changes in the liver Ifng locus, poising cells to secrete more IFNγ upon simulation and worsen disease. Understanding the mechanism underlying exacerbated disease in Il17rafl/fl × villin cre+ mice will elucidate the role of enteric Th17 cells and the microbiome in AIH and other liver pathologies.
Microbiota metabolites SCFA promote intestinal epithelial repair and wound healing through promoting epithelial cell production of milk fat globule-EGF factor 8
Authors: Bilotta Anthony James , Ma Chunyan , Huang Xiangsheng , Yang Wenjing , Chen Liang , Yao Suxia , Cong Yingzi
Published: January 2023

The mechanisms by which gut microbiota regulate intestinal homeostasis are still largely unknown. The short chain fatty acids (SCFA), mainly acetate, propionate, and butyrate, are bacterial metabolites produced from the fermentation of dietary fibers, which have been shown to regulate intestinal homeostasis. In this study, we investigated whether SCFAs promote intestinal epithelial repair and wound healing through promoting epithelial cell production of milk fat globulin E8 (MFG-E8), a known molecule promoting wound healing and cell migration via actin reorganization. Treatment with SCFAs enhanced intestinal epithelial cell (IEC) migration significantly, and promoted wound healing in both mouse and rat IEC in vitro. We then investigated how SCFAs regulated IEC gene expression by microarray. Microarray analysis showed 401 genes differentially expressed upon stimulation with butyrate, of which 196 were upregulated and 205 were downregulated. Among upregulated genes, MFG-E8 has been shown in several systems to promote IEC proliferation and tissue repair. We confirmed MFG-E8 upregulation by qRT-PCR for mRNA and Western blot for protein. Interestingly, SCFAs also upregulated several genes pertinent to actin reorganization including RhoA, Rho GTPase, and PAK1 as well as genes known to effect proliferation such as cyclin D2 and PKCδ, which are down stream of MFG-E8 signaling. Our study thus demonstrated that SCFAs promote intestinal epithelial cell tissue repair, possibly through upregulation of MFG-E8.
Microbiota metabolites derived from tryptophan modulate macrophage inflammation and migration through AMP-activated protein kinase
Authors: Ding Yufang , Krishnan Smitha , Alaniz Robert Christopher , Lee Kyongbum , Jayaraman Arul
Published: January 2023

It is established that the gut microbiota plays an important role in Non-Alcoholic Fatty Liver Disease (NAFLD) and different bacterial taxa have been correlated to disease. However, the specific metabolites through which the microbiota exert their effects and the target pathways in host cells are not fully understood. We used liquid chromatography-mass spectrometry metabolomics to identify two tryptophan-derived metabolites – tryptamine (TA) and indole-3-acetate (I3A) – that require the microbiota and are depleted in high-fat diet mice compared to low fat diet mice. Both I3A and TA reduced palmitate and LPS induced production of pro-inflammatory cytokines (TNFα, IL-1β and MCP-1) in RAW 264.7 macrophages, as well as inhibited macrophage migration toward the chemokine MCP-1. We identified that both palmitate and LPS reduced levels of p-AMPK and the addition of I3A and TA reversed p-AMPK reduction. Since the p-AMPK activator AICAR reduced palmitate and LPS induced inflammatory cytokine production, we propose that I3A and TA modulate inflammation through modulating p-AMPK levels.
The delivery of luminal substances across small intestinal epithelium via goblet cell associated antigen passages is increased in the presence of dietary gliadin.
Authors: McDonald Keely G. , Knoop Kathryn A. , Kulkarni Devesha Hrishikesh , Gustafsson Jenny K. , Miller Mark J. , Newberry Rodney D.
Published: January 2023

Celiac disease is an enteropathy caused by CD4+ T cell responses to dietary gliadin, resulting in damage to the intestinal mucosa and malabsorption. How gliadin peptides cross the epithelium to interact with the immune system is unclear. We used an in vivo imaging approach on live mice and ex vivo assays on mouse and human intestinal explants to evaluate how gliadin traversed the intestinal epithelium. We observed that gliadin crossed the epithelial barrier of mice and humans via the recently described goblet cell associated antigen passages (GAPs), and moreover, gliadin increased the formation of GAPs. The induction of GAPs by gliadin was independent of acetylcholine signaling, the pathway inducing GAP formation in the steady state. Consistent with the increase in GAPs induced by gliadin, LP-DCs isolated from mice given luminal ovalbumin (Ova) concurrently with gliadin induced enhanced antigen specific T cell responses to Ova in ex vivo LP-DC T cell co-culture assays. In vivo Ova specific T cell responses in the MLN were enhanced in mice given Ova concurrent with gliadin. Gliadin did not increase the antigen presentation capacity of the LP-DCs when added to ex vivo cultures, indicating that increased luminal antigen delivery via GAPs mediates the enhanced antigen specific T cell responses to luminal Ova. In the absence of GC’s and GAPs gliadin did not enhance the delivery of luminal antigens to the LP-DCs and did not enhance T cell responses to luminal antigens. These studies identify that gliadin uses GAPs to cross the epithelial barrier, and that gliadin induces further GAP formation in a goblet cell intrinsic manner, thus facilitating the delivery of gliadin and other antigens to the immune system.
Moving beyond microbiome-wide associations to causal microbe identification
Authors: Surana Neil , Kasper Dennis L.
Published: January 2023

Microbiome-wide association studies have established that numerous diseases are associated with changes in the microbiota. These studies typically generate a long list of commensals implicated as biomarkers of disease, with no clear relevance to disease pathogenesis. If the field is to move beyond correlations and begin to address causation, an effective system is needed for refining this catalog of differentially abundant microbes and allow for mechanistic studies. We demonstrate that triangulation of microbe–phenotype relationships is an effective method for reducing the noise in microbiota studies and enabling identification of causal microbes. We found that gnotobiotic mice harboring different microbial communities exhibited differential survival in a colitis model. Co-housing of these mice generated animals that had hybrid microbiotas and displayed intermediate susceptibility to colitis. Mapping of microbe–phenotype relationships in parental mouse strains and in mice with hybrid microbiotas identified the bacterial family Lachnospiraceae as a correlate for protection from disease. Using directed microbial culture techniques, we discovered Clostridium immunis, a novel bacterial species, that—when administered to colitis-prone mice—protected them against colitis-associated death. To demonstrate its generalizability, we used our approach to identify 2 bacteria that induce intestinal expression of an antimicrobial peptide. Thus, we have used microbe–phenotype triangulation to move beyond the standard correlative microbiome study and identify causal microbes for 2 distinct phenotypes. Identification of disease-modulating commensals by our approach may also be applicable to human microbiome studies.
Prevention of systemic autoimmunity by dietary modulation of the gut microbiota
Authors: Ruiz Daniel Fernando Zegarra , El-Beidaq Asmaa , Iniguez Alonso , di Ricco Martina Lubrano , Mubiro Derek , Vieira Silvio Manfredo , Ruff William Edwards , Sterpka John , Kriegel Martin Alexander
Published: January 2023

Diet-induced modulation of host immune-microbiota interactions could be an attractive way to ameliorate autoimmunity. Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease with no cure. We used resistant starch (RS) as a novel approach to prevent SLE. We fed RS to a spontaneous TLR7 Tg and TLR7 agonist (Imiquimod, IMQ)-inducible lupus model to modulate gut microbial communities and lupus pathogenesis.

Depletion of the gut microbiota with antibiotics reduced mortality in TLR7 Tg mice. Also, IMQ-induced lupus was ameliorated in GF compared to SPF B6 mice, supporting a role for the microbiota in the pathogenesis. In both models, RS reduced mortality, kidney involvement, hepatosplenomegaly, and type I interferons. Plasmacytoid dendritic cell (pDC) accumulation and type I interferon signaling in spleen and Peyer’s patches was also reduced. Fecal 16S rDNA sequencing and culture of internal organs revealed that Lactobacillus reuteri translocates to MLN, liver and spleen in TLR7 Tg mice, and that fecal loads and translocation were suppressed by RS. Lupus induction by IMQ in GF B6 mice was exacerbated by L. reuteri monocolonization. In addition, L. reuteri gavage in the IMQ-inducible model worsened lupus manifestations, which were prevented by RS feeding before onset of disease. RS induced short-chain fatty acids (SCFA) in ileum and blood of TLR7 Tg mice, which suppressed growth of L. reuteri in vitro.

In summary, RS exerts beneficial effects in lupus-prone hosts through suppression of pathobionts and type I interferon pathways, which are known to contribute to human SLE. This study provides novel insights into diet-host-microbiota interactions and may aid in the development of functional foods to treat autoimmune diseases.