bluuebunny/crossref_metadata_2025 huggingface dataset:
Mycoplasma superantigen enhances the severity of K/BxN autoimmune arthritis through TLR4/BCL6/TFH pathway
Authors: Mu Hong-Hua , Nourian Maziar M. , McKennon Kevin B , Wang Jingyi
Published: January 2023

Mycoplasma arthritidis mitogen (MAM), a potent superantigen (SAg) secreted by M. arthritidis, is known to contribute to mouse inflammatory/autoimmune arthritis. We previously demonstrated that C3H mouse strains differing in expression of TLR4, also differ in Th-1 versus Th-2 and Th17 cytokine profiles in response to MAM due to a differential engagement of TLR2 and TLR4. HMGB-1, a member of heat shock protein family that is abundantly expressed in many cell types upon activation, has been implicated in regulating TLR signaling pathways following its interaction with RAGE. In the present study, we established a new model of mouse arthritis induced by injection of K/BxN sera. Also, we provide evidence that HMGB1 is an essential endogenous activator of the immune response in K/BxN-sera transfer BALB/c mice following MAM immunization. Level of HMGB1 expression was promptly increased in K/BxN sera-transferred mice after MAM injection. The increase in HMGB1 was required for initiation of MAM-induced enhancement of BCL-6, type I interferon (IFN) and follicular helper T cell responses. Blockade of HMGB1 caused prolonged activation of BCL-6 pathways, reduced level of type I IFN and augmented production of IL-10. Also, MAM-immunized K/BxN mice injected with HMGB1 antibody were less susceptible to arthritis suggesting HMGB1 may accomplish this mainly through its interaction with TLR4. Thus, HMGB1, in synergy with mycoplasma superantigen MAM, functions as biologically important modulators of TLR4/BCL6/TFH, and thus the targeting of this pathway can be a useful therapeutic strategy for treating human rheumatoid arthritis.
Mechanisms of Coagulation-Complement pathway interplay in ischemia/reperfusion induced tissue injury.
Authors: Robbins Racheal , Peng Suzette , Moratz Chantal M
Published: January 2023

Impaired fibrinolysis in systemic lupus erythematosus (SLE) is a contributing factor in disease complications including hyper-coagulation, tissue ischemia/reperfusion (IR), and increased risk of thrombosis: including stroke and deep vein thrombosis. Aberrant complement activation has been identified as a major factor in tissue injury in SLE. Previous work in the laboratory focused on mechanisms of complement activation in IR induced tissue injury in B6.MRL/lpr mice, an autoimmune prone mouse strain with a propensity to develop lymphadenopathy, glomerulonephritis, and polyarthritis. This work identified a C3-independent mechanism of downstream complement C5a generation contributing to tissue injury. Evidence of interplay between the coagulation and complement pathways was identified. Current work elucidates the mechanism by which the two pathways interact. Using a superior mesenteric artery IR model, we utilized complement and specific extrinsic or intrinsic coagulation pathway inhibitors to evaluate and compare resulting tissue pathology, generation of secondary inflammatory mediators, extent of innate immune activation, and generation of breakdown products involved in regulating feedback regulatory loops after ischemic/reperfusion injury. Inhibition of the extrinsic pathway had a greater impact on downstream complement activation, tissue pathology, vascular integrity, and recruitment of innate immune components versus the inhibition of the extrinsic pathway. Understanding the mechanisms of interplay between the pathways in the autoimmune mouse has significant clinical implications – especially in autoimmune diseases in which inflammation and hypercoagulability are known manifestations.
Intestinal Bacterial Composition of Lymphopenic Mice and Susceptibility to CD45RBhigh T Cell-Induced Colitis
Authors: Webb Cynthia Vannessa Reinoso , Koboziev Iurii , Furr Kathryn L , Phillips Caleb , Kottapalli Kameswara Rao , Ostanin Dmitry V , den Bakker Hendrik C , Grisham Matthew B
Published: January 2023

Background

Following relocation to our current institution we observed a 40% reduction in the incidence of T cell-induced colitis in our well-established mouse model of IBD. The objectives of this study were to: a) quantify and compare the colonic microbiota in healthy and colitic mice obtained from our current (TTUHSC) and former insitution (LSUHSC), and b) determine whether colonization of healthy mice housed at our current institution with feces from healthy or colitic mice from LSUHSC alters the incidence and/or severity of colitis.

Methods

DNA from frozen feces was isolated using standard protocols and 16S rRNA sequencing was performed using the Illumina MiSeq platform. For some studies, RAG1−/−mice were colonized (via gastric gavage) with 40 mg donor feces 1 week prior to adoptive transfer of naïve (CD4+CD45RBhigh) T-cells.

Results

Intestinal microbial populations are markedly different between the two institutions in healthy and colitic animals. Transplant of fecal microbiota from LSUHSC mice with chronic colitis into healthy RAG−/− recipients accelerates the onset, increases the incidence (>95%) and enhances the severity of chronic colitis following T cell transfer. However, colonization of healthy WT or RAG−/− mice (in the absence of T cell transfer) with colitic feces did not induce disease over the 8 week observation period.

Conclusion

We conclude that colonization of healthy RAG−/− mice with dysbiotic but nonpathogenic microbiota obtained from colitic mice markedly increases the incidence and severity of chronic colitis in the T cell transfer model.
Characterization of the inflammatory effects of λ-carrageenan at the genomic and proteomic levels
Authors: Lewandowski Annie , Deaver Christine , Myers Michael J
Published: January 2023

λ-Carrageenan (λCGN) is often used to model local inflammation, but the mechanism by which this compound functions at the genomic and proteomic level has not been delineated. Whole blood from swine was stimulated with λCGN to determine inflammatory biomarkers. We also utilized a HEK-293 (human embryonic kidney) cell line transfected with a SEAP (secreted embryonic alkaline phosphatase)-release plasmid to determine which TLR (Toll-Like Receptor; TLR) signaling cascade was stimulated. Results indicated that Monocyte Chemotactic Protein-1 gene expression and protein were increased by λCGN stimulation while Serum Amyloid A gene expression and protein remained unchanged. λCGN stimulation increased mRNA levels of Phosphatidylinositol 3-Kinase and Alveolar Macrophage Chemotactic –II, decreased mRNA levels of Thrombospondin-2 Precursor, and had no effect on Interleukin-6 mRNA levels. λCGN stimulation increased protein levels of Interleukin-8 and had no effect on protein levels of Tumor Necrosis Factor-α. SEAP release from HEK-293 cells stimulated with λCGN indicated that both TLR2 and TLR4 signaling cascades were initiated. λCGN stimulation did not activate TLR3, TLR5, TLR7, TLR8, TLR9, NOD1, NOD2, Dectin-1a, or Dectin-1b. Experiments silencing either TLR1 or TLR6 to determine which TLR2 heterodimer is activated by λCGN are currently underway. In conclusion, while λCGN stimulation appears to signal through both the TLR2 and TLR4 pathways, significant differences exist between the genes and proteins stimulated by λCGN and pure TLR4 agonists such as E. coli-derived lipopolysaccharide.
Ron receptor tyrosine kinase plays a protective role in diet induced obesity and associated steatohepatitis
Authors: Allen Joselyn Natasha , Zhang Limin , Yu Shan , Balandaram Gayathri , Peters Jeffrey M , Patterson Andrew D , Hankey Pamela A
Published: January 2023

Obesity is one of the leading causes of preventable death in the U.S and a well-established risk factor for several pathological conditions including steatohepatitis. The activation of inflammatory tissue resident macrophages has proven to play a key role in the progression of the metabolic syndrome. Consequently, mechanisms that manipulate macrophage phenotype has become of great interest for developing therapeutic strategies for combating obesity and its associated diseases. Our lab has shown that Ron tyrosine kinase, a receptor expressed on tissue resident macrophages, limits an inflammatory response while promoting tissue repair. In this study, we first investigate the impact of Ron signaling on metabolic features preceding steatohepatitis using high fat diet (HFD) fed wild type (WT) and Ron deficient (Ron KO) mice. Ron KO mice demonstrated more severe events associated with obesity such as increased adiposity, exacerbated hyperglycemia and trend toward increased liver weights when compared to WT animals. Altered Ron signaling resulted in a shift in the plasma lipoprotein distribution from primarily VLDL and LDL to predominantly HDL in the WT control mice. To then establish the role of Ron signaling in steatohepatitis, high cholesterol diet (HCD) fed ApoE KO and Ron KO ApoE KO mice (DKO) were evaluated. We showed that DKO mice experience more severe steatohepatitis which is attributed to an increased prevalence of activated inflammatory Kupffer cells. In vitro studies ascertain that Ron manipulation of macrophage phenotype is in part, a result of downstream AMPK activation. These data indicate the role of Ron in attenuating obesity and its associated diseases and identify a novel downstream pathway involved in this process.
Localization and trafficking of pulmonary dendritic cells during early stages of airway inflammation
Authors: Lyons-Cohen Miranda R , Nakano Hideki , Whitehead Gregory S , Cook Donald N
Published: January 2023

Pulmonary dendritic cells (DCs) take up inhaled antigens, migrate to draining lymph nodes (LNs), and stimulate antigen-specific T cells. Bona fide pulmonary DCs (CD11c+CD88−) comprise CD103+ and CD11b/CD172a+ subsets, and their migration from lung to LNs is directed by the chemokine receptor, CCR7. However, the precise localization of these two different DC subsets in the lung, and how they move during the early stage of inflammation, are largely unknown. In this study, using multi-color high resolution imaging of precision cut lung slices, we found that at steady state, both CD103+ and CD172a+ DCs reside immediately adjacent to airways lined with CD324/E-cadherin-expressing epithelial cells. Interestingly, this region of the airways is also closely associated with lymphatic vessels, whereas regions lacking CD324 are not associated with either DCs or lymphatic vessels. Inhalation of LPS induced a Ccr7-gfp reporter gene within 2 h post-exposure, but the DCs remained closely associated with airways. By 6 h post- exposure, the number of airway-associated DCs decreased, while DCs associated with lymphatic vessels significantly increased. Very few DCs were detected in lung parenchyma. These observations suggest that after sensing danger signals, DCs translocate directly from the airway to lymphatics without first transiting through the parenchyma. In agreement with this rapid migration, activation of antigen-specific T cells was detected in regional LNs within 6 h of exposure.
TGF-β receptor 3 (betaglycan) regulates Th1 differentiation and T dependent B cell responses.
Authors: Raman Chander , Sestero Christine M. , Orlandella Rachael M , Boland Molly T , De Sarno Patrizia , Kesterson Robert A , Soldevila Gloria
Published: January 2023

TGFβR3, also known as betaglycan, is a co-receptor for the TGFβ receptor and is predicted to have an important function in promoting high affinity binding of TGF-β to TGFβR1 or TGFβR2. TGFβR3 also binds inhibins with high affinity and antagonizes activin dependent activation of SMADs a function independent of TGFβR1/TGFβR2 signaling. Contrary to TGFβR1 and TGFβR2, our knowledge on the role of TGFβR3 in the immune system is very sparse. Its biology has been elusive because the knock-out of Tgfbr3 exhibit embryonic lethality. We developed the first Tgfbr3f/f mouse in which Cre mediated deletion of Tgfbr3 is very efficient. We found that deletion of TGFβR3 in thymic T cells (Tgfbr3f/f.CD4Cre) had no effect on CD4 or CD8 double positive (DP) or single positive (SP) T cell populations or peripheral T cell populations. Treg numbers in the thymus and periphery were unaltered and the mice did not develop spontaneous autoimmunity as observed when Tgfbr2 is deleted during thymic development. These observations indicated to us that TGFβR3 likely had a function independent of “classical” TGF-β signaling through TGFβR1/2 receptors. To test for this possibility we generated Tgfbr3f/f.dLckCre) mice in which is Tgfbr3 is selectively deleted in mature CD4 and CD8 T cells. We found that T dependent Ab responses, particularly of the IgG2c subclass, was greatly enhanced in mice with T-cell restricted loss of TGFβR3. We further determined that the differentiation of naïve CD4 T cells to IFN-γ expressing Th1 cells under polarizing and non-polarizing conditions was enhanced when TGFβR3 was deficient. These and other data indicate that regulation of Th1 differentiation by TGFβR3 is novel and independent of TGF-β signaling.
B cells have bifurcated Toll-like receptor signaling allowing both inflammatory cytokine and type 1 interferon responses.
Authors: Akkaya Munir , Rawat Mukul , Akkaya Billur , Chaturvedi Akanksha , Pena Mirna , Miozzo Pietro , Kim Ann S , Pierce Susan K
Published: January 2023

B cells express the endosomal, DNA sensing, Toll-like receptor 9 (TLR9) that, in response to CpG oligonucleotides, signals through the adaptor protein MyD88 and activates the NF-kB pathway leading to the transcriptional activation of proinflammatory cytokines. In dendritic cells TLR9-MyD88 signaling is bifurcated into two distinct pathways, the NF-kB pathway and an IRF7-dependent pathway that results in the activation of type 1 interferon (IFN) genes. In these cells, monomeric CpG-B activates the NF-kB-dependent pathway and multimeric CpG-A or CpG-A coupled to the liposomal transfection reagent, Dotap (CpG-A-Dotap), activates the IRF-7-dependent pathway. Here we provide evidence that the B cell TLR9 signaling pathway is similarly bifurcated. Both CpG-B and CpG-B-Dotap potently activate the NF-kB-dependent pathway leading to IL-6 synthesis but neither trigger an IFN response. In contrast, CpG-A-Dotap but not CpG-A alone stimulates an IFN response as shown by increases in the levels of mRNA encoding IFN and in the amount of IFN protein produced and also by the activation of B cells from IFN-reporter mice both in vitro and in vivo. By confocal microscopy CpG-A and CpG-B appear to traffic to different intracellular vesicles. However, the cellular machinery that mediates the IL-6 and IFN pathways seems to be linked in that pretreatment of B cells with CpG-B blocks the subsequent activation of the IFN pathway by CpG-A-Dotap. It will be of interest to determine if natural DNA containing ligands might trigger the two pathways in B cells.
The transcriptional co-factor PARP14 limits severity of allergic skin inflammation
Authors: Krishnamurthy Purna , Serezani Ana PM , Kaplan Mark H
Published: January 2023

Atopic dermatitis (AD) is a chronic relapsing inflammatory condition of the skin that is characterized by pruritic skin lesions, erythema, and swelling, resulting in an impaired skin barrier. Patients with AD and mice subjected to models of AD develop an inflammatory T helper 2 (Th2) immune response and reduced expression of epidermal differentiation complex (EDC) genes, which encode proteins needed for keratinocyte maturation and barrier integrity. We are exploring the molecular controls regulating Th2 cells and keratinocytes in a mouse model of AD. Poly-ADP ribose polymerse-14 (PARP14) is a transcription co-factor that enhances Th2 differentiation. Correspondingly, PARP14-deficient mice have reduced lung inflammation in a model of allergic airway disease; however, its potential role in AD has not been explored. In keratinocytes, PARP enzymatic activity does not alter expression of EDC genes. Surprisingly, PARP14 deficiency resulted in an increase in the severity and number of skin lesions in an experimental mouse model of AD. Disease severity and increasing age corresponded with increases in Th2 cytokine secretion and antibody production (IgE). Adoptive transfer of PARP14 deficient T cells from a mouse model of AD resulted in increase in alopecia, epidermal thickening and altered EDC gene expression. Thus, PARP14 serves opposing roles in allergic disease: promoting pathogenic Th2 immune responses in the lung while potentially limiting T cell-dependent inflammatory responses during skin inflammation.
Exon 2 of Foxp3 contributes to the peripheral tolerance of the immune system
Authors: Du Jianguang , Wang Qun , Wang Lina , Zhou Baohua
Published: January 2023

Foxp3 is essential for the development and function of CD4 regulatory T cells (Tregs) that maintain peripheral immune tolerance. A major difference of Foxp3 expression between human and mouse is the alternative splicing of exon 2 in human Tregs. In vitro studies showed that the exon 2 region of Foxp3 associates and inhibits the transcription factors RORα and RORγt, which are critical for Th17 differentiation. However the in vivo role of exon 2 is still unknown. By deleting exon 2 of mouse Foxp3 gene (dE2), we show here that peripheral and mesenteric lymph nodes of dE2 mice contained about twice amount of lymphocytes when compared with wildtype mice due to the higher level of CD4 T cell activation and proliferation. Exon 2 deletion resulted in more severe air way inflammation in OVA-induced chronic asthma, characterized by higher number of lymphocytes and eosinophils in bronchoalveolar lavage, and high level of OVA-specific serum IgE. CD4 T cells in the mediastinal lymph nodes of dE2 mice expressed higher IL-4, IL-9 and IFN-γ, but surprisingly not IL-17, compared with those in wild type mice. These data indicate that Foxp3 exon 2 is important in maintaining proper functions of peripheral Tregs, and thus contributes to the peripheral tolerance to dampen allergic inflammation against harmless antigens.
Using mass cytometry to identify novel B cell subsets in red meat allergy
Authors: Cox Kelly M , Commins Scott , Capaldo Brian , Solga Mike , McSkimming Chantel , Chew Claude , Schuyler Alexander , Lannigan Joanne , McNamara Coleen , Erickson Loren
Published: January 2023

Previous studies have identified a novel food allergy driven by IgE antibodies specific for galactose-α-1,3-galactose (alpha-gal), an oligosaccharide found in red meat. While it is known that B cells play an important role in allergy as the producers of IgE antibodies that drive the allergic response, little is known about the phenotype of these B cells. The number of markers used to identify the major human B cell subsets by flow cytometry has been limited to common B cell proteins and thus precludes high dimensional immune phenotyping of B cell subsets, including unique phenotypes present in allergic individuals. We have addressed this problem by using mass cytometry (CyTOF), which enables the simultaneous analysis of up to 40 markers in a single staining panel. Here we analyzed the expression of 23 cell surface markers in PBMCs from 19 alpha-gal-allergic patients and 20 non-allergic controls by CyTOF. Additionally, we combined our CyTOF data with clinical endpoints to identify markers that may correlate with allergic disease. Our data reveals substantial heterogeneity within major B cells subsets on an individual level. Furthermore, our analysis identifies a number of markers that vary significantly in their expression in allergic versus non-allergic B cells and correlate with serum alpha-gal IgE titers. We hypothesize that B cells with this phenotype play an important role in mediating alpha-gal allergy. These findings demonstrate the power of using CyTOF and analytical tools to extract a hierarchy from high dimensional cytometry data in an unsupervised manner to identify known B cell subsets as well as to find novel B cell populations that differ between alpha-gal allergic and non-allergic individuals.
Mechanisms and consequences of Siglec-8 internalization and toxic payload delivery in human eosinophils
Authors: O’Sullivan Jeremy , Cao Yun , Bochner Bruce
Published: January 2023

Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a cell surface protein expressed selectively on human eosinophils, mast cells, and basophils that induces eosinophil apoptosis and inhibits mast cell mediator release. Hence, Siglec-8 is an ideal target for diseases involving these cells. However, the effective delivery of therapeutic agents to these cells through Siglec-8 requires an understanding of the dynamics of Siglec-8 surface expression, which have not been elucidated. We observed by flow cytometry that Siglec-8 endocytosis in eosinophils proceeds slowly and plateaus at 80% loss in response to antibody or polyvalent synthetic ligand. Surprisingly, based on sensitivity to pharmacological inhibition, Siglec-8 appears to be internalized in both a clathrin- and lipid raft-dependent manner. Actin cytoskeletal rearrangement and tyrosine kinase and PKC activity are necessary for endocytosis. Interestingly, Siglec-8 is shuttled to the cell surface in a manner dependent on actin rearrangement, microtubule polymerization, and the activities of tyrosine kinases, dynamin, and PI3K. Neither process is significantly affected by inhibitors of Src family kinases or PTPs. Internalized Siglec-8 localizes to the lysosome, and sialidase treatment of eosinophils enhances ligand binding, indicating the presence of masking sialylated cis ligands on the cell surface. Finally, conjugating the ribosome-inactivating protein saporin to an anti-Siglec-8 mAb enhanced cell death induction in unstimulated eosinophils. The dynamics of Siglec-8 surface expression therefore appear to be suitable for sustained targeting and may deliver drugs to effectively treat diseases involving these cell types, including malignancies.
Specific subsets of kinases mediate Siglec-8 engagement-induced ROS production and apoptosis in human eosinophils.
Authors: Janevska Daniela , O’Sullivan Jeremy A. , Cao Yun , Bochner Bruce S.
Published: January 2023

Siglecs are type I transmembrane proteins expressed primarily on leukocytes. Among them is Siglec-8, a CD33 subfamily member that is selectively expressed on the cell surface of human eosinophils. Siglec-8 has an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM), putatively responsible for signal transduction. In cytokine-activated eosinophils, Siglec-8 binding causes apoptosis with increased mitochondrial damage and ROS production, but exact signaling mechanisms are unknown. Using a mAb (2C4) against Siglec-8 in combination with small molecule inhibitors, we first examined Siglec-8-mediated apoptosis and ROS production by flow cytometry after 24 hr IL-5 priming (30 ng/mL) of human eosinophils. We observed that 2C4-mediated eosinophil apoptosis was inhibited by PP1 and SU6656 (Src kinase inhibitors), ibrutinib (Btk inhibitor), LY294002 (PI3K inhibitor), GF109203x (PKC inhibitor), and sodium orthovanadate (a protein phosphatase inhibitor) at IC50’s of 1.7 μM, 1.8 μM, 0.9 nM, 1.2 μM, 2.4 μM, and 22 μM respectively. Complete inhibition of ROS production occurred at expected IC90’s. Western blot analysis following Siglec-8 cross-linking with 2C4 showed increased phosphorylation of Src530, Csk, PI3Kδ, and Blk that was detectable within 15 min, and c-Abl phosphorylation that was detectable within 60 min. Additionally, co-immunoprecipitation data shows that Siglec-8 associates with SHP-2, a protein tyrosine phosphatase. While the sequence of signaling events is yet to be determined, Siglec-8 mediated apoptosis in eosinophils involves the unexpected recruitment of molecules normally associated with cell survival.
Epithelial cell IKKβ regulates eosinophil levels in the intestine and severity of allergic responses to ingested allergens
Authors: Kim Eunsoo , Lembert Melanie M. , Martin Tara L. , Rowe John C. , Steiner Haley E. , Cormet-Boyaka Estelle , Boyaka Prosper N.
Published: January 2023

Allergic sensitization to food allergens has subsequent potential to developing allergic responses in the gastrointestinal tract, but also to skin or lung. Our previous study showed that lack of IKKβ in intestinal epithelial cells regulates favors IgA responses to ingested allergen, which in turn limits the severity of allergic responses in the airway. In this study we investigated whether intestinal epithelial IKKβ also regulated allergic responses to oral antigens. Wild-type C57BL/6 and IKKβΔIEC mice, which lack IKKβ in intestinal epithelial cells, were orally sensitized to a food antigen in the presence of cholera toxin. Allergen-specific serum IgE responses and fecal IgA responses were similar between the groups. However, after oral allergen-challenge, IKKβΔIEC mice only developed minimal clinical and histological signs of allergy, including drop in body temperature and mucus in small intestinal villi and crypts. Interestingly, IKKβΔIEC mice expressed lower levels of CCL11 (eotaxin) and eosinophils than control wild-type mice and their levels were only weakly increased after oral allergen sensitization and challenge. In summary, this study reveals a new role of intestinal epithelial cells in the regulation of allergy in the GI tract through a NF-κB-CCL11 axis.
Spingosine-1-phosphate receptor 1 agonist alleviates hypersensitivity pneumonitis induced by Methanosphaera stadtmanae
Authors: Huppé Carole-Ann , Lecours Pascale Blais , Talbot Maude , Morissette Mathieu , Marsolais David
Published: January 2023

Hypersensitivity pneumonitis (HP) is characterized by the formation of B cell-rich tertiary lymphoid tissue (TLT) in the lung, and by a granulocytic response upon antigen rechallenge. Clinically, antigen avoidance is the only effective approach to manage HP symptoms and progression. Sphingosine-1-phosphate receptor 1 (S1PR1) regulates key mechanisms underlying the formation of TLTs such as lymphocyte trafficking and intercellular interactions. Given that TLTs are key to the antigen-specific response in the lung, we investigated the impact of an S1PR1 agonist on pulmonary TLTs accumulation and determined the effect of quantitative TLTs modulation on the granulocytic response to an antigen rechallenge. Experimental HP was induced in mice using intranasal instillations of an immunogenic archaea found in farm bioaerosol, Methanosphaera stadtmanae (MSS), for 3 weeks. After a 48 hours resting period, mice were treated for 7 days with the S1PR1 agonist, RP001 (1mg/kg/day), or its vehicle using osmotic pumps. Five days after treatment initiation, mice were either euthanized, rechallenged with saline or rechallenged with MSS. Mice were euthanized 48h later to quantify pulmonary TLTs and inflammatory cells. RP001 did not influence TLTs accumulation and lung inflammation prior to MSS rechallenge. After MSS rechallenge, RP001 reduced the accumulation of TLTs by 49% (p=0.02) and reduced neutrophil accumulation by 39% (p=0.002), when compared to vehicle. Thus, S1PR1 modulation, after the initiation of a HP-like disease, alleviated TLTs accumulation along with the neutrophilic response to an antigen rechallenge. Our results support the concept that S1PR1 modulation can interfere with mechanisms that sustain HP.
The ORMDL3-ceramide axis may be a novel therapeutic target for the control of allergic airway disease
Authors: Sturgill Jamie L , Conrad Daniel H , Oyeniran Clement , Spiegel Sarah
Published: January 2023

Asthma, defined as a chronic inflammatory condition characterized by episodic shortness of breath with expiratory wheezing and cough, is a serious health concern. The WHO estimates that asthma affects more than 230 million people worldwide. There is a strong genetic component to asthma and numerous genome-wide association studies have identified ORM (yeast)-like protein isoform 3 (ORMDL3) as an asthma associated gene. Surprisingly however, the mechanism by which ORMDL3 contributes to asthma pathogenesis is not well understood. The yeast ortholog of ORMDL3 is a negative regulator of serine palmitoyltransferase (SPT), the rate limiting step in de novo ceramide synthesis, yet elevations of ceramide rather than its reduction have been linked to lung inflammation. Thus, we examined the role of ORMDL3 in asthma immunopathology. Consistent with its role in yeast, we show that decreasing expression of ORMDL3 in lung epithelial cells and macrophages increases ceramide and conversely, modest increases in ORMDL3 decrease ceramide levels. In a house dust mite (HDM) mouse model of allergic airway disease, allergen challenge induced expression of ORMDL3 and resulted in a concomitant increase in lung ceramide. Intriguingly, the use of specific drugs, which inhibit ceramide synthesis, prevented HDM-induced airway hyperreactivity (AHR) and suppressed airway inflammation. Nasal administration of the orally available FDA approved prodrug FTY720/fingolimod reduced both ORMDL3 expression and ceramide production while mitigating airway inflammation, hyperreactivity, and mucus hypersecretion in HDM challenged mice. Thus the ORMDL3 -ceramide pathway may be a novel therapeutic target for the control of allergic asthma.
Sex differences in M2-polarization during allergic lung inflammation
Authors: Keselman Aleksander M , McCormick Sarah M , Fang Xi Jessie , Heller Nicola M
Published: January 2023

Asthma is a chronic airway inflammatory disease affecting over 300 million people world-wide. Asthma exhibits sex differences by affecting mostly women, suggesting that hormones like estrogen may participate in disease progression. Alveolar macrophages become activated in asthma, promoting pathology but also repairing tissue damage. Estrogen is known to enhance macrophage activation and function in a variety of systems although its role in asthma is poorly understood. We hypothesized that macrophages from female mice are intrinsically hyper-responsive to IL-4 and that estrogen signaling enhances M2-polarization. We assessed IL-4-induced M2-gene expression in bone marrow-derived macrophages (BMMs) from male and female C57BL/6 mice and found enhanced expression of two M2 genes, Ym1 and Arg1, in response to IL-4 in female compared to male BMMs. Histone H3 acetylation and K4-trimethylation was elevated in female BMMs, consistent with enhanced transcriptional activity. Pre-treatment of BMMs with an ERα-specific agonist further enhanced IL-4-induced M2-gene expression. Next, we assessed sex differences in a mouse model of allergic lung inflammation. We found that alveolar macrophages from female compared to male mice exhibited increased Ym1 expression following allergen challenge. Therefore, macrophages exhibit intrinsic sex differences in their ability to polarize to an M2-phenotype and estrogen augments IL-4-induced M2-gene expression. Together these data suggest that intrinsic and hormonal factors contribute to sex differences in macrophages in asthma. Understanding the role of estrogen signaling in M2-polarization is paramount for identifying novel therapeutic targets to better treat women with asthma.
Differences in Innate immune cell production of IFNα and IFNβ to cell activation in the Autoinflammatory Diseases CANDLE (Chronic Atypical Neutrophilic Dermatosis with Lipodystrophy and Elevated temperature) and SAVI (STING Associated Vasculopathy with Onset in Infancy)
Authors: Marrero Bernadette , Liu Yin , Biancotto Angelique , Calvo Katherine R , Huang Yan , De Jesus Adriana A , Goldbach-Mansky Raphaela
Published: January 2023

The Type I IFN mediated autoinflammatory diseases SAVI and CANDLE are caused by gain-of-function mutations in Stimulator of Interferon Genes (STING) and loss-of-function mutations in proteasome components. We evaluated the innate immune cellular origin of IFN Type I in SAVI and CANDLE.

We examined expression of IFN genes from FACS sorted T, B, NK and monocytes in SAVI (n=5) and CANDLE (n=3) normalized to healthy controls (HC). The Interquartile Range of Type I IFN expression was 78(4–151) and 481-fold(56-38,242) of IFNB1 and 60(3–116) and 0.4-fold(0.1–39) of IFNA7/IFNA17 in CANDLE and SAVI monocytes, respectively. Monocyte depletion reduced IFN message by 90% and FACS analysis confirmed that monocytes and not dendritic cells are the main source of IFNs. The overall mean ratio of IFNβ to IFNα was 989:1 in SAVI and 1.3:1 in CANDLE monocytes.

Mean IFNα plasma levels measured by pan IFNα ELISA with 31pg/ml ±58 SEM in CANDLE and 230pg/ml ±112 SEM in SAVI. IP10 serum levels were 2215pg/ml ±702 SEM and 3632pg/ml ±860 SEM in CANDLE and SAVI, respectively. STING pathway activation with cGAMP in the presence of cycloheximide (CHX), a blocker of translational elongation, led to IFNB1 but not IFNA1, 2, 7, 17, 21 while CHX + epoxomicin, a proteasome inhibitor led to transcription of IFNAs and IFNB1 suggesting secondary upregulation of IFNAs in SAVI.

Compared to healthy controls, CANDLE and SAVI had increased left-shifted immature CD13lo CD16+ neutrophils (9 and 4-fold, respectively), as well as higher expression of the activation marker CD36+ (6.9 and 6.9-fold, respectively) in total granulocytes.

In conclusion, Type I IFN is mainly produced in monocytes and analyses of the IFN signaling in CANDLE and SAVI will allow us to develop improved targeted therapies.
Reduced serum BCMA levels distinguish patients with primary antibody deficiency
Authors: Maglione Paul J. , Li Mingjie , Sanchez Eric , Radigan Lin , Chen Haiming , Berenson James R. , Cunningham-Rundles Charlotte
Published: January 2023

B-cell maturation antigen (BCMA) is a TNF family receptor that binds a proliferation inducing ligand (APRIL) and B cell activating factor (BAFF). BCMA is expressed by immunoglobulin secreting cells, but not other B cells, and supports their survival. Primary antibody deficiency (PAD) is the most common primary immunodeficiency and may lead to infection and complications including autoimmunity, enteropathy, lung disease, and malignancy. PAD patients with these complications have reduced survival, yet there are no validated tests to predict those at risk. We previously demonstrated that BCMA is present in the serum and its levels are elevated in patients with multiple myeloma. As BCMA is expressed by both normal and malignant antibody-producing cells, we examined serum levels of BCMA in PAD. Patients with PAD (n=68) had markedly reduced BCMA levels (median, 7.30) compared with healthy donors (n=119; median, 35.20; P < 0.0001). When subgrouped based upon PAD diagnosis, serum BCMA was reduced compared to controls among those with common variable immunodeficiency (CVID) (n=48; median, 7.16; P <0.0001) and X-linked agammaglobulinemia (n=8; median, 2.30; P <0.0001), but not among those with selective IgA deficiency or hyper IgM syndrome. CVID patients with any inflammatory complication, or enteropathy specifically, had significantly lower serum BCMA than CVID patients without complications (P < 0.01 for both). Serum BCMA moderately correlated with isotype-switched memory B cells (r = 0.473), but poorly correlated with IgA, IgG, or IgM levels or total B cell levels. These results demonstrate that reduced serum BCMA levels are found in PAD patients, distinguish types of PAD, and are associated with the development of complications in CVID.
Cysteine-cysteine chemokine receptor 5 (CCR5) profiling in HIV infected individuals attending Federal Medical Centre, Yenagoa, Bayelsa State.
Authors: Mirabeau Tatfeng Youtchou
Published: January 2023

Cysteine-cysteine chemokine receptor 5 (CCR5) is a protein found on the surface of CD4 cells which Human immunodeficiency virus (HIV-1) uses as a co-receptor when entering the host’s target cells. There is a variant of CCR5 known as CCR5 delta 32 that results due to a deletion mutation. CCR5 delta 32 plays an important role in the acquisition of HIV-1 and progression to AIDS. This study was done to determine the CCR5 genotype of HIV positive patients attending Heart to Heart clinic in Federal Medical Centre, Yenagoa, Bayelsa State. A total of 50 whole blood samples were collected by simple random sampling from 50 HIV positive patients attending Heart to Heart clinic in Federal Medical Centre, Yenagoa, Bayelsa State. DNA was extracted from each sample using ZR mini prep DNA extraction kit. CCR5 gene in each DNA extract was amplified by polymerase chain reaction on a 9700 ABI Thermo Cycler and then resolved on 1% agarose gel electrophoresis. Out of the 50 samples examined, 50 (100%) were homozygous for the normal CCR5 gene, 0(0%) were homozygous for the CCR5 delta 32 gene and no heterozygosity was recorded. This study revealed a very low or absence of CCR5 delta 32 deletion gene among HIV patients in Yenagoa suggestive of their rapid progression towards AIDS if other measures are not observed.
Age related changes in inflammatory and regulatory subsets within a healthy population are biased by CMV infection and oldest age.
Authors: Norton Elizabeth B
Published: January 2023

Age-related inflammation is detrimental to the host and plays a key initiator of disease. However, it is unclear how patterns of immune cells and particularly inflammatory and regulatory subsets occur within a healthy population. We investigated peripheral blood markers for immunologic subsets and inflammatory markers using samples collected from a local Blood Bank. 319 subjects between 25–81 years were included that had passed standard collection and infection status criteria. Batches of freshly collected whole blood samples were lysed and immediately processed for flow cytometry using 9-color panels of lymphocyte, myeloid, and platelet markers. Data was normalized by fold change to the young (25–35 yr) group included within batches (7 total) compared to older (36–45, 46–55, 56–64, or 65+ year) age groups. Overall donor age was significantly related to weight, blood pressure, and CMV seropositivity by Spearman’s correlation analyses. We observed few immunologic changes between age groups, except significant differences within the 56–64yr age group. These individuals expressed significantly higher levels of antigen-experienced CD45RO+ CD8 T-cells, regulatory TGF-beta+ CD4 T-cells, and plasma B-cells that correlated with CMV seropositivity. The 56–64 age group also expressed higher levels of non-classical monocytes and platelet monocyte aggregates, both related to inflammation. A lack of changes observed in the highest age group (65+) may be due to selection biases from earlier death and disease. Our data supports that responses to chronic viral infections like CMV may be a major regulator of healthy aging, and also reveals potential biases in recruited populations of older adults.
Transient antigen priming promotes B cells recruitment into humoral response or return to quiescence in vivo.
Authors: Grigorova Irina , Turner Jackson , Benet Zachary
Published: January 2023

Consistent with the Bretscher and Cohn’s two-signal model of lymphocyte activation, B cells must acquire antigen (Ag) via their B cell receptors (BCR), and then get help from cognate Th cells to initiate long-term antibody responses. Matzinger’s model then implied that for immune tolerance, BCR signaling in the absence of T cell help must lead to B cell anergy or death. This view has been supported by multiple in vivo studies of B cells continuously exposed to self-Ags. However, the fate of B cells transiently exposed to Ag in vivo is unclear. Intravital imaging suggested the transient dynamics of large particulate foreign Ags acquisition by B cells during the initial phase of immune response, but could not address whether such exposure is sufficient for B cell participation in the humoral response. In this study we show that in the presence of T cell help, transient single Ag acquisition promotes B cells’ recruitment into the germinal center, memory and plasma cell responses in vivo. We also demonstrate that transiently Ag-primed B cells do not undergo apoptosis in the absence of T cell help in vivo, but return to quiescence, and upon reacquisition of Ag and T cell help can mount a potent humoral response. Based on these findings we suggest a possibility of multiple cycles of Ag acquisition by B cells and return to quiescence. When T cell help is limiting, this mechanism may promote a greater diversity of B cell clones to enter the humoral response to foreign Ags. However, a failure of B cells to undergo anergy or apoptosis after a transient acquisition of self-Ags may contribute to breach of B cell tolerance.
NADPH oxidase-derived superoxide provides a third signal for CD4 T cell effector responses
Authors: Padgett Lindsey E , Tse Hubert Ming
Published: January 2023

Efficient T cell immune activation requires a synergistic interaction between innate immune-derived reactive oxygen species (ROS) and pro-inflammatory cytokines. Our laboratory previously demonstrated that in the absence of NADPH oxidase (NOX)-derived superoxide, autoreactive T cell responses were profoundly compromised. To examine the role of ROS deficiency on antigen-specific T cells in a non-autoimmune setting and further test the hypothesis that Th1 T cell responses would be diminished in the absence of ROS, we generated the OT-II.Ncf1m1J mouse containing superoxide-deficient, OVA323-339-specific CD4 T cells. Stimulation of OT-II.Ncf1m1J elicited a 4-fold decrease in IFN-γ (p<0.05), concomitant with 2-fold reductions in CD4 T cell-derived IL-12Rβ2 and T-bet, required for Th1 activation. Th1 responses elicited by OVA323-339 stimulation were redox-sensitive, as addition of exogenous superoxide to OT-II.Ncf1m1J CD4 T cells restored IFN-γ synthesis and IL-12 signaling to approximately OT-II CD4 T cell levels. Conversely, Th1 responses were curbed in wild type OT-II T cells via free radical dissipation with a superoxide dismutase mimetic. This data highlights the importance of NOX-derived ROS in providing a third signal for adaptive immune maturation by modulating IL-12 signaling and may represent a promising therapeutic strategy in curbing CD4 T cell autoreactivity.
Glucocorticoids enhance neutrophil survival via G-CSF
Authors: Banuelos Jesus , Cao Yun , Shin Soon Cheon , Lu Nick
Published: January 2023

Glucocorticoids promote the survival of neutrophils, the mechanism of which is not clear. We found that glucocorticoids and proinflammatory cytokines synergistically induced G-CSF in primary human neutrophils and macrophages and multiple cell lines. The induction is at the transcriptional level and mediated by NF-κB and the glucocorticoid receptor as demonstrated by site-directed mutagenesis and ChIP assays. In both an LPS-induced airway inflammation model and a chronic OVA-induced airway inflammation model, BAL G-CSF, but not KC or IL-6, was resistant to glucocorticoid suppression. Glucocorticoids together with NF-κB inhibitors, but not either agent alone, decreased BAL G-CSF and neutrophils. Neutrophils from G-CSFR null mice, in contrast to those from wild type animals, were no longer protected by glucocorticoids. Furthermore, knockdown of G-CSF abolished the ability of BEAS 2B bronchial epithelial cells to protect neutrophils from spontaneous apoptosis. These data identify G-CSF as a potential target to increase glucocorticoid sensitivity in neutrophil-driven inflammatory conditions such as chronic obstructive pulmonary disease and neutrophil-dominant endotypes of asthma.
The AIM2-like receptors are dispensable for activation of the interferon stimulatory DNA (ISD) pathway.
Authors: Gray Elizabeth , Stetson Daniel B
Published: January 2023

Detection of intracellular DNA triggers activation of the STING-dependent interferon-stimulatory DNA (ISD) pathway, which is essential for antiviral immune responses; on the other hand, inappropriate immune responses to self DNA result in autoimmunity. Multiple DNA sensors have been proposed to activate the ISD pathway, including cyclic GMP-AMP synthase (cGAS) and a family of DNA binding receptors called the AIM2-like receptors (ALRs). Analysis of cGAS-deficient mice has revealed that cGAS is a key DNA sensor that is required for activation of the ISD pathway; however, whether the ALRs contribute to this pathway remains unclear.

Here, we generated mice lacking all 13 mouse ALR genes as a novel tool to explore the function of the ALRs. We show that all ALRs are dispensable for the type I interferon (IFN) response to transfected DNA ligands, DNA virus infection, and lentivirus infection. We also show that the DNA sensor cGAS, but not the ALRs, is required to drive autoimmune disease in the Trex1-deficient mouse model of Aicardi-Goutieres Syndrome. Finally, we used CRISPR to disrupt the human AIM2-like receptor IFI16 in primary human fibroblasts and show that IFI16 is dispensable for the IFN response to transfected DNA ligands as well as human cytomegalovirus (HCMV) infection. Thus, our data reveal that ALRs are dispensable for activation of the ISD pathway and demonstrate that cGAS is the primary DNA sensor that drives the IFN response to DNA.
TNFAIP8 variant 2 has an anti-inflammatory role in Toll like Receptor 4 signaling
Authors: Lowe Julie Marie , Menendez Daniel , Resnick Michael A , Fessler Michael B
Published: January 2023

The cell response to pathogens involves complex signaling that affects expression of thousands of genes. Characterization of the regulation and function of these genes is critical since failure of this response can foster the development of chronic disorders. In a high-throughput microarray analysis of human macrophages stimulated with the TLR4 ligand LPS, we found high and rapid induction of Tumor Necrosis Factor-a-Inducible Protein-8 (TNFAIP8), a poorly defined gene proposed to regulate cell fate. While very little is known about TNFAIP8 expression and function, recent studies of murine Tnfaip8 suggest a role in antibacterial immunity and inflammation. Our analysis of human TNFAIP8 revealed two previously uncharacterized expression variants (v1 and v2) that possess distinct predicted functional domains. Primary human macrophages predominantly express v1, whereas a panel of human cancer cell lines displayed a marked v2 predominance. Interestingly, we found that v1 and v2 are both induced by LPS in macrophages, but with different kinetics, suggesting that they may have distinct functions in innate immunity. Selective depletion of v2 in A549 cells dramatically enhanced the baseline expression of several pro-inflammatory cytokines including IL-6, IL-8 and IL-1b. Furthermore, in response to LPS, v2-depleted cells had increased induction of IL-8, IL-1b, and TNFa. Together, these data suggest that TNFAIP8 v2 has an anti-inflammatory role in resting and TLR ligand-stimulated cells. Work is currently underway to determine 1) the molecular mechanisms of the anti-inflammatory role of TNFAIP8, for example, whether NF-kB activation is altered by v2 depletion; and 2) whether v1 has a distinct role in immune signaling.