bluuebunny/crossref_metadata_2025 huggingface dataset:
Visceral adipose tissue contributes to the generation of pro-inflammatory B cell subsets in aging mice
Authors: Blomberg Bonnie , Diaz Alain , Romero Maria , Vasquez Thomas , Frasca Daniela
Published: January 2023

Age-related decrease in B cell function is associated with chronic low-grade inflammation and increased pro-inflammatory mediators. This effect is also associated with an increase in visceral adipose tissue (VAT) and metabolic inflammation. Adipocyte-derived chemokines are involved in the chemotaxis of immune cells which infiltrate the VAT and contribute to the inflammatory process. To identify contributors to the phenotypic and functional changes observed in aged B cells, we undertook studies of B cells in the VAT. We recently analyzed immune cells infiltrating the VAT of obese C57BL/6 mice (age 12–24 months). We found macrophages, B cells and T cells in VAT in a ratio of 1:3:5. As to B cells, we measured the major B cell subsets and found higher percentages of pro-inflammatory (age-associated B cells, ABC) and less Follicular (FO) B cells in VAT as compared to spleen. B cells isolated from VAT express higher levels of inflammatory immune activation markers (TNF-α/IL-6/IL-8), significantly higher NF-kB activation and phospho-STAT3, and secrete higher amounts of Ig antibodies specific for fat antigens as compared to splenic B cells. To evaluate the ability of adipocytes from the VAT to promote inflammation and induce pro-inflammatory B cell subsets, we co-cultured adipocytes from the VAT with splenic B cells from the same mice. Results show that co-culture for 72 hrs significantly changed the relative percentages of the B cell subsets, leading to a higher percentage of ABC, similar to what we have observed in the fat. Moreover, we found that the adipocytes produce several pro-inflammatory chemokines. These results are the first to show a direct effect of adipocytes on pro-inflammatory B cells.
Palmitate-induced MMP-9 expression in the human monocytic cells: Significance in metabolic inflammation
Authors: Al Roub areej Abu , Koshy Merin , Sindhu Sardar , Ahmad Rasheed
Published: January 2023

Matrix metalloproteinase (MMP)-9 is predominantly produced by monocytes/macrophages and directs the infiltration of circulatory immune cells into the adipose tissue through damage of vascular endothelial basement membrane. Obese individuals are known to have increased MMP-9 plasma levels and MMP-9 is reported to play an important role in obesity-mediated adipose tissue inflammation. Since in obesity, the levels of circulatory free fatty acid palmitate are increased and modulate the expression of inflammatory mediators, the role of palmitate in the regulation of MMP-9 remains unclear. Therefore, we determined whether palmitate could induce MMP-9 production, and if so, which signaling pathway(s) were involved. Here, we provide the evidence that palmitate induces MMP-9 expression at both mRNA and protein levels in human monocytic cells (P<0.05). Increased NF-κB/AP-1 activity (P<0.05) was also observed in palmitate-treated THP-1 cells. Palmitate-induced MMP-9 secretion was markedly suppressed either by neutralizing anti-TLR-4 antibody or by inhibiting clathrin-mediated endocytosis. Furthermore, MyD88−/− THP-1 cells did not express MMP-9 in response to palmitate treatment. Genetic silencing of TLR4 by siRNA also abrogated the palmitate-induced up-regulation of MMP-9. Altogether, these results show that palmitate induces TLR4-dependent activation of MMP-9 gene expression, which requires the recruitment of MyD88 leading to activation of NF-kB/AP1 transcription factors. Thus, our findings suggest that the palmitate-induced MMP-9 secretion might be an underlying mechanism of its increased levels in obesity and related metabolic inflammation.
Modulatory effect of perforin gene dosage during pathogen associatated blood brain barrier (BBB) disruption
Authors: Willenbring Robin , Jin Fang , Hansen Mike , Johnson Aaron J
Published: January 2023

Neuroinflammation is a putative mechanism of pathogen associated blood brain barrier (BBB) disruption. However, mechanisms of immune mediated vascular permeability remain an active topic of research. Our lab has defined a role for CD8 T cells and perforin in BBB tight junction protein alterations during neuroinflammation. Within the human population, perforin is highly polymorphic, the significance of which is unknown. Therefore, we investigated the capacity of T cells with a single perforin allele, which reduces perforin mRNA and protein expression by half, in modulating BBB disruption in a murine model. We also conducted a genetic analysis to determine the significance of perforin polymorphisms in humans. In vivo, we assessed perforin+/+, perforin+/− and perforin−/− mice using the peptide induced fatal syndrome (PIFS) model for CD8 T cell mediated BBB disruption. Using 3D volumetric analysis of gadolinium enhanced, T1 weighted MRI we determined that perforin alleles are additive in increasing BBB disruption. This finding was not the result of differing immune cell infiltration or virus load. In parallel, we conducted a population genetic analysis of the human perforin allele. Using the Exome Aggregation Consortium and primary literature, we categorized over 450 perforin polymorphisms. Perforin polymorphism rates directly correlated with equatorial distance and environmental pathogen richness. Therefore, our studies demonstrate a single perforin allele alters BBB disruption severity. We hypothesize that diversity at the human perforin allele is a form of inclusive fitness by contributing to differential susceptibility to pathogen associated CNS vascular permeability.
Mechanisms underlying 4-1BB agonist antibody mediated hepatotoxicity
Authors: Bartkowiak Todd , Jaiswal Ashvin R , Ai Midan , Budhani Pratha , Ager Casey , Curran Michael A
Published: January 2023

Agonist antibodies against the T cell costimulatory receptor 4-1BB have proven extraordinarily efficacious in the treatment of a multitude of pre-clinical murine tumors both in mono- and combination therapy settings. Although administration of α4-1BB in Phase I and II clinical trials has led to disease stabilization and partial remission in some cases of melanoma; high grade, dose limiting liver inflammation has slowed the progress of these agents toward FDA approval. We sought to uncover the mechanisms by which 4-1BB agonist antibodies trigger hepatotoxicity in hopes of discovering approaches by which the anti-tumor and hepatotoxic effects could be separated. We show that α4-1BB mediated liver damage initiates through stimulation of myeloid cells, followed by subsequent recruitment and activation of CD8 and CD4 T cells in the liver. Moreover, we show that the inflammatory cytokine IL-27 is essential in myeloid conversion of T cells into mediators of liver damage. Conversely, we show that Foxp3+ regulatory T cells are potent suppressors of 4-1BB agonist mediated hepatotoxicity; as depletion of Tregs exacerbates liver pathology. Our results go on to support the use of combination therapies in the treatment of tumor malignancies, in particular CTLA-4 blockade which may expand Tregs in the liver to ameliorate α4-1BB mediated toxicities while also amplifying anti-tumor immune responses.
Negative regulation of IL-2 signaling by β2 adrenergic receptor
Authors: Ruiz-Medina Blanca E , Esparza Edmundo , Kirken Robert A
Published: January 2023

T, B, and natural killer (NK) cells are required for normal immune response and regulated by cytokines such as interleukin 2 (IL-2). Cell signals are propagated following receptor-ligand engagement to control recruitment and activation of effector proteins. The IL-2 receptor β subunit (IL-2Rβ) serves in this capacity and is known to be phosphorylated on tyrosine, serine, and threonine residues. Regulation of lymphocytes is also influenced by the nervous system through the β2 adrenergic receptor (β2AR), and both pathways ultimately affect cell trafficking, proliferation, differentiation, and cytokine production. Signaling through either IL-2Rβ or the β2AR has been extensively studied, however, their potential cross-talk has yet to be fully characterized. Here we hypothesize that engagement of β2AR regulates IL-2 receptor signaling. Cells were treated with β2AR agonists and then stimulated with or without IL-2. Agonists reduced IL-2 induced proliferation in a dose dependent manner. To identify the mechanism, receptor phosphorylation and activation of downstream targets was assessed using phosphospecific antibodies. Pretreatment of cells with β2AR agonists diminished IL-2-induced phosphorylation of IL-2Rβ as well as the activation of downstream signaling molecules. In contrast, in the absence of IL-2, a biphasic β2AR agonist-induced phosphorylation of specific serine and threonine residues on IL-2Rβ associated to its activation was observed, while tyrosine phosphorylation changes were not detected. Downstream signaling molecules were differentially activated. In conclusion, the cross-talk between IL-2Rβ and β2AR could represent a novel regulatory mechanism of IL-2 signaling important for immunoregulation.
Low molecular weight compounds which inhibit BAFF binding to its receptor, BR3, suppress activation of monocytes.
Authors: Yoshimoto Keiko , Ishioka Eriko , Nishikawa Ayumi , Suzuki Katsuya , Takeuchi Tsutomu
Published: January 2023

Backgrounds and Purpose

We found that the elevated expression of a BAFF receptor, BR3, in monocytes of patients with primary Sjögren’s syndrome (pSS) was involved in overproduction of IL-6 by BAFF-stimulated pSS monocytes. Additionally, the proportion of BR3-positive monocytes to total monocytes was positively and significantly correlated with the serum IgG level of pSS patients. In this study, we investigated the effects of the compounds, which block BAFF binding to BR3, on BAFF-induced IL-6 production in pSS monocytes.

Methods

High throughput screening (HTS) of a chemical library was carried out to search for compounds that block binding of soluble BAFF (sBAFF) to BR3. THP-1 and peripheral monocytes prepared from pSS patients were cultured in the presence of sBAFF and the binding inhibitors. IL-6 production by the cells was measured by ELISA.

Results

A total of 18,562 compounds were examined for inhibitory activities of sBAFF binding to BR3. To eliminate false positives, inhibitory activities of the HTS-hits against IL-6 production by sBAFF-stimulated THP-1 were measured. As a result, we discovered two pyrrolopyrimidine derivatives which showed substantial inhibition of sBAFF-binding. sBAFF-induced IL-6 production by pSS monocytes was significantly suppressed by these compounds in a dose dependent manner.

Conclusion

We have successfully discovered low molecular weight compounds which have inhibitory activities against BAFF signaling in monocytes.
Recombinant variant of Fasciola hepatica fatty acid binding protein impairs the NF-κB activation in response to toll-like receptors agonists
Authors: Ramos-Benítez Marcos Javier , Ruiz-Jimenez Caleb , Espino Ana M
Published: January 2023

In recent years, it has been demonstrated that a strong Th2 immune response is able to suppress the inflammatory type or Th1 response; this Th1 response is linked to septic shock and some autoimmune diseases. Fasciola hepatica promotes a Th2 response during chronic infections, hence the production of anti-inflammatory cytokines. Our research group published that fatty acid binding protein (FABP) purified in native form (Fh12) from F. hepatica adult worm impairs the inflammatory cytokines storm typically induced by exposure to Lipopolysaccharides (LPS). Our work aimed to determine whether a recombinant variant of FABP could have a similar effect. We optimized the expression in E. Coli of an isoform of FABP named Fh15 and performed western blot analysis to confirm an immunological similarity between Fh12 and Fh15. Human monocytes THP1-CD14 cells were treated with Fh15, then stimulated separately with specific activation ligands for TLR 2, 4, 5 and 8 in order to determine the capacity to inhibit the NF-κB activation; we used Quanti-Blue medium to quantify the inhibition. Our results showed that both proteins are immunologically similar. Also, Fh15 was able to block the NF-κB activation induced by specific TLR ligands up to 95%, showing similar capacity as Fh12. The use of bacterial expression lead us to conclude that the glycosylation sites described for Fh12 are not required for the effect. We can anticipate that Fh15 has the potential to exhibit an inflammatory cytokines suppression effect. Fh15 impairs the NF-κB activation induced by different TLR-ligands suggesting a broad spectrum of action. We are working to elucidate the mechanisms for this effect and examine the potential of Fh12 and Fh15 as anti-inflammatory molecules in animal models.
Identification of CD163 as an anti-inflammatory receptor for HMGB1-haptoglobin complexes.
Authors: Yang Huan , Wang Haichao , Wang Yongjun , Chavan Sangeeta S , Al-Abed Yousef , Antoine Daniel J , Andersson Ulf , Tracey Kevin J
Published: January 2023

Secreted by activated cells or passively released by damaged cells, extracellular HMGB1 functions as a pro-inflammatory mediator of infection or injury. It was previously unknown whether HMGB1 can be partner with other endogenous proteins to tilt the balance towards anti-inflammatory activities. Here we demonstrated that an acute phase protein, haptoglobin, effectively binds HMGB1, and triggers anti-inflammatory programs in a CD163-dependent fashion. Specifically, the HMGB1 and haptoglobin complexes elicit the production of anti-inflammatory enzymes (heme oxygenase-1) and cytokines (e.g., IL-10) in wild type, but not in CD163-deficient macrophages. Genetic disruption of haptoglobin or CD163 expression renders animals more susceptible to lethal sepsis, whereas administration of haptoglobin to wild type or to haptoglobin-gene deficient animals confers significant protection. These findings reveal a novel mechanism by which an acute phase protein modulates extracellular HMGB1 activities, and provides a novel strategy for treatment of HMGB1-dependent inflammatory diseases.
T cell-intrinsic ASC critically promotes Th17-mediated experimental autoimmune encephalomyelitis.
Authors: Martin Bradley N , Wang Chenhui , Li Xiaoxia
Published: January 2023

In the present study, we uncovered a novel T cell-intrinsic role of the inflammasome adaptor molecule ASC in Th17-mediated EAE pathogenesis. We found that while T cell-intrinsic ASC is required for the effector stage of active EAE, ASC deficiency in T cells specifically impaired Th17- but not Th1-mediated passive EAE. T cells deficient in IL-1β were similarly impaired in their ability to mediate the effector phase of EAE. Mechanistically, TCR signaling induced pro-IL-1β expression and licensed T cells for inflammasome activation. Polarized Th17 cells expressed IL-1R, and produced mature IL-1β in response to ATP via ASC/NLRP3-dependent caspase-8 activation. Additionally, ATP-treated Th17 cells showed enhanced survival compared to ATP-treated Th1 cells, which was abrogated by IL-1Ra, suggesting an autocrine action of Th17-derived IL-1β. Together, these data suggest a critical role for IL-1β produced by a novel Th17 cell-intrinsic ASC-NLRP3-Caspase-8 inflammasome during CNS inflammation.
The human IL-22 binding protein isoforms are a rheostat for IL-22 signaling.
Authors: Lim Chrissie , Hong MeeAe , Savan Ram
Published: January 2023

The cytokine interleukin-22 (IL-22) activates signal transducer and activator of transcription 3 (STAT3) to drive various processes central to tissue homeostasis and immunosurveillance; its dysregulation causes inflammatory diseases. IL-22 has a soluble receptor, IL-22 binding protein (IL-22BP; IL22RA2), which antagonizes IL-22 activity and has genetic associations with autoimmune diseases. Humans express three IL-22BP isoforms, whereas mice only express an IL-22BPi2 homolog. Given the lack of IL-22BPi1 and IL-22BPi3 expression in mice, there has not been a thorough characterization of their activities to date. In this study, we show that IL-22BPi2 and IL-22BPi3 differentially modulate IL-22 activity: IL-22BPi3 has lower inhibitory activity than IL-22BPi2, but is expressed more abundantly throughout the body during homeostasis. IL-22BPi2 and IL-22BPi3 also differentially alter the cooperative actions between IL-22 and IL-17, which are commonly co-expressed in autoimmune settings. In addition, we demonstrate that IL-22BPi1 is inactive because it is not secreted. Importantly, while IL-22BPi3 is the dominant isoform expressed constitutively, only IL-22BPi2 is induced when myeloid cells are activated by Toll-like receptor 2 (TLR2) or retinoic acid. These data suggest that the IL-22BP isoforms have distinct spatial and temporal roles and coordinately fine-tune IL-22-dependent STAT3 responses in tissues as a rheostat. This work is the first to date to perform a thorough biochemical and molecular characterization of the human IL-22BP isoforms.
Serotonin Receptor Regulation of Eosinophil Transendothelial Migration
Authors: Campbell Jackson Archie , Green Jeremy , Soveg Frank , Abdala-Valencia Hiam , Cook-Mills Joan M
Published: January 2023

Previous research has shown that 5-hydroxytryptophan (5HTP), a metabolic precursor of serotonin, reduces allergic inflammation in the lung by inhibiting eosinophil migration across endothelial monolayers. Furthermore, when the metabolic conversion of 5HTP to serotonin is blocked, 5HTP inhibition of eosinophil migration is reduced. Therefore, 5HTP metabolism to serotonin is required for 5HTP inhibition of eosinophil migration. However, it is currently unknown if serotonin receptors are involved in mediating 5HTP function. We determined serotonin receptor expression on both endothelial cells and eosinophils by western blot and qPCR. Endothelial cells and eosinophils expressed serotonin receptors 1A, 1B, 3A, and 6. We subsequently determined whether inhibition of these serotonin receptors abrogates the effects of 5HTP. Notably, it was shown that blocking serotonin binding to serotonin receptors 1A and 1B by use of selective receptor inhibitors blocks 5HTP inhibition of migration. The function of signaling by serotonin receptors in 5HTP-mediated inhibition of migration is under investigation. Thus, serotonin receptors mediate 5HTP inhibition of transendothelial migration, and may serve as a target for intervention during allergic inflammation. These results will help design ongoing clinical studies addressing whether 5HTP blocks allergic asthmatic responses.
LIGHT, the Tumor Necrosis Superfamily Protein 14 (TNFSF14), Controls Lung and Skin Fibrosis.
Authors: Herro Rana , Sidler Daniel , da Silva Antunes Ricardo , Tamada Koji , Croft Michael
Published: January 2023

Lung and skin fibrosis are characterized by excessive accumulation of collagen and α-smooth muscle actin in these organs. The key molecules that promote these phenotypes are of clinical interest.

Thymic stromal lymphopoietin (TSLP) and Periostin (Pstn) have been found at high levels in patients with Asthma, Idiopathic Pulmonary Fibrosis, and Atopic Dermatitis. TSLP has been proposed as a primary driver of lung fibrotic disease, while Pstn was found to be an important mediator of Asthma and Systemic Sclerosis. We asked whether LIGHT (aka TNFSF14) controls TSLP and Pstn production to initiate fibrosis.

Genetic deletion of LIGHT abolished TSLP and Pstn expression driven by bleomycin used to induce fibrosis, accompanied by near-complete absence of accumulation of lung and skin collagen and α-smooth muscle actin. Furthermore, recombinant LIGHT administered in vivo induced lung and skin expression of both TSLP and Pstn in the absence of other inflammatory stimuli, and strikingly reproduced the primary features of bleomycin-driven disease in a TSLP-dependent manner. Blockade of LIGHT binding to either of its receptors, herpes virus entry mediator and lymphotoxin beta receptor, inhibited clinical symptoms of lung and skin fibrosis, and correspondingly both receptors were found on human epithelial cells and fibroblasts, primary sources of TSLP and Pstn respectively. Moreover, LIGHT induced TSLP directly in human bronchial epithelial cells and keratinocytes and synergized with IL-13 and TGF-β in vivo to promote TSLP expression and drive fibrosis.

These results show that LIGHT is a profibrogenic cytokine that may be a key driver of TSLP and Pstn production during the initiation and development of fibrotic diseases in the lung and skin.
Loss of A Disintegrin And Metalloproteinase 10 (ADAM10) in dendritic cells dampens type 2 immune responses.
Authors: Damle Sheela , Martin Rebecca , Conrad Daniel H
Published: January 2023

Allergy and allergic asthma are significant health burdens in developed countries and are increasing in prevalence. Current therapeutic strategies focus on management of disease symptoms, but are unable to control disease initiation. Previous reports from our lab demonstrated a reduction in type 2 (Th2) immune responses with ADAM10 inhibition. Thus, we sought to understand the role of ADAM10 in immune cells involved in Th2 responses, specifically dendritic cells (DCs). We generated mice which have ADAM10 deleted from DCs using mice that express Cre recombinase driven by the CD11c promoter (A10DCKO mice). We used a house dust mite (HDM) extract model of allergic airway inflammation and demonstrated a drastic reduction in airway resistance in the A10DCKOs as well as a reduction in eosinophils. We also found a reduction in Th2 cytokine expression, HDM specific IgG1 antibody, and lung inflammation in the A10DCKOs. After administration of inhaled antigen we found a significant reduction in antigen specific T cell proliferation in the spleen of the A10DCKOs, but no difference in the mediastinal lymph nodes. To understand the mechanism and given the importance of ADAM10 in Notch signaling, we compared the phenotype of the A10DCKOs to published reports of Notch2 DC-specific KOs. The A10DCKOs had a similar reduction in total conventional, CD172+, and CD172+ ESAM+ DCs in the spleen. However, we saw no decreases in other DC populations in the spleen and mesenteric lymph node demonstrated in the Notch2 KO, indicating a role for ADAM10 separate from Notch2 signaling. Overall, these results point to a mechanism of action of an ADAM10 inhibitor and a novel strategy for modulating Th2 immune responses.
Autocrine and paracrine cytokine feedback augment the production of Th2-associated cytokines.
Authors: Olson Matthew Richard , Kaplan Mark H
Published: January 2023

The Type 2 cytokines (including cytokines from Th2 and Th9 cells) play a critical role in priming of the humoral response, immunity to parasites, and the induction of allergic disease. It has been proposed that unlike Type 1 cytokines (i.e. IFN-γ), the induction of Type 2 cytokines is cell autonomous and requires no additional signals for optimal cytokine production. Contrary to this notion, we found that the addition of golgi transport inhibitors that block cytokine secretion (i.e. monensin (Mon) and brefeldin A (BFA)) during stimulation significantly impaired the induction of Type 2, but not Type 1 cytokine mRNA and protein production. These data suggest that the release of cytokines by activated Th cells is uniquely required for further production of Type 2 cytokines. Mon and BFA strongly inhibited the release of IL-2 after stimulation and blockade of IL-2 during activation in the absence of golgi inhibitors significantly diminished Type 2 cytokine production. Further, transduction of cells with a constitutively active STAT5 negated the need for additional IL-2 signaling in driving Type 2 cytokine transcription, suggesting that STAT5 is the primary mediator of IL-2-driven gene expression. Interestingly, both autocrine and paracrine IL-2 production were capable of driving maximal Type 2 cytokine production. Taken together, these data indicate a novel role for IL-2 feedback in promoting Type 2 cytokine production and likely in promoting allergic inflammation.
Crisaborole Topical Ointment, 2%, a novel nonsteroidal, anti-inflammatory, topical, phosphodiesterase 4 inhibitor, reduced pruritus and signs of atopic dermatitis in 2 phase 3 studies in children and adults with mild-to-moderate atopic dermatitis
Authors: Zane Lee Thomas , Hebert Adelaide A. , Eichenfield Lawrence F. , Lebwohl Mark G. , Paller Amy S. , Simpson Eric L. , Tom Wynnis L. , Hughes Matty H.
Published: January 2023

Atopic dermatitis (AD) is an inflammatory skin disease with intense pruritus and eczematous lesions. Crisaborole Topical Ointment, 2% (Anacor Pharmaceuticals, Palo Alto, CA), is an investigational, nonsteroidal, anti-inflammatory, topical, phosphodiesterase 4 inhibitor for the treatment of AD. Here, we present the impact of crisaborole on pruritus and signs of AD evaluated as supportive efficacy endpoints for 2 Phase 3 studies (301, 302). Patients ≥2 years old with mild-to-moderate AD were enrolled in 2 double-blind, vehicle-controlled, multicenter Phase 3 studies of identical design. Patients were randomized 2:1 to receive crisaborole or vehicle twice daily for 28 days. Supportive efficacy endpoints examined severity of pruritus (measured twice daily) and signs of AD (measured weekly) on a 4-point scale (None [0] to Severe [3]). Success was defined for pruritus or each sign or symptom of AD as achievement of None (0) or Mild (1) with ≥1-grade improvement from baseline. Improvement in pruritus was achieved sooner in crisaborole-treated patients than vehicle-treated patients (pooled data: median, 1.37 vs 1.70 days; P = 0.001). A greater proportion of crisaborole-treated patients achieved success for all clinical signs of AD by Day 29 (301, 302; erythema: 62.8% vs 46.1%; 54.9% vs 33.9%; induration/papulation: 57.7% vs 54.8%; −51.9% vs 40.2%; exudation: 41.0% vs 33.3%; 38.1% vs 27.2%; excoriation: 63.0% vs 51.8%; 57.2% vs 44.2%; lichenification: 51.7% vs 46.5%; 51.4% vs 35.3%). In 2 large Phase 3 studies, crisaborole demonstrated early relief of pruritus and showed improvement in all measured signs of AD. Crisaborole Topical Ointment, 2%, may represent a safe and efficacious treatment for patients ≥2 years with mild-to-moderate AD.
FcγRIII signaling promotes IL-33-dependent monocyte migration to the lung extravascular space.
Authors: Camacho Daniel F , Tjota Melissa Yuwono , Turnquist Heth R , Sperling Anne I
Published: January 2023

IL-33 is implicated in type 2 inflammatory processes, including allergic asthma. Our group previously demonstrated that two Th2 stimuli, allergen-specific IgG immune complexes (ICs) and house dust mite extract (HDM), signal through FcRγ-associated receptors on antigen presenting cells to upregulate IL-33 and induce type 2 allergic airway inflammation. In this study, we find that monocytes from WT, but not IL-33−/− or FcγRIII−/− mice, accumulate in the lung extravascular space within 18 hours of OVA-IC challenge. Exogenous IL-33 treatment in the airways is sufficient to restore monocyte accumulation in the IL-33−/− mice. Further, treatment with pertussis toxin abolishes IL-33-dependent monocyte accumulation, a finding consistent with GPCR-dependent migration of these cells. Our findings suggest that during allergic sensitization, activation of the FcRγ signaling pathway promotes IL-33- and GPCR-dependent monocyte migration to the lung extravascular space, where monocytes may then contribute to type 2 allergic inflammation.
Airway epithelial cells promote allergic sensitization to cockroach allergen through GM-CSF production
Authors: Sheih Alyssa , Ziegler Steven F
Published: January 2023

Allergic asthma is an inflammatory disease of the airways driven by an overactive immune response against allergens. Allergen-specific TH2 cells are critical to the pathogenesis of asthma but factors controlling the initiation of TH2 responses are not well understood. The emerging picture is one that points to a role for airway epithelial cells (AECs) and epithelial-derived cytokines in promoting TH2 immunity to allergens. However, allergens are structurally diverse and can elicit different epithelial-derived cytokines. Thus, there are multiple pathways that lead to TH2 immunity and mechanisms of sensitization that are unique to individual allergens remain to be elucidated. Cockroach allergen (CRA) is an environmental allergen that is strongly associated with the incidence of allergic asthma. Despite its clinical importance, very little is known about the molecular and cellular pathways involved in allergic sensitization to CRA. To determine the mechanisms that regulate allergic sensitization to CRA, we intranasally sensitized and challenged mice with CRA to induce allergic airway inflammation with hallmark features of asthma. Using this model, we demonstrate that allergic sensitization to CRA requires activation of AECs. MyD88 deletion in AECs reduced allergic airway response to CRA as well as production of GM-CSF during sensitization. Using a neutralizing antibody against GM-CSF, we showed that GM-CSF is essential for TH2 priming and allergic sensitization to CRA, likely through activation of dendritic cells. When GM-CSF was intranasally delivered during sensitization, TH2-mediated eosinophilia was restored in the absence of MyD88. Thus, AECs promote allergic sensitization to CRA through production of GM-CSF.
Towards a non-allergenic peptide mix containing the T cell epitopes of the clinically most relevant house dust mite allergens for tolerance induction
Authors: Huang Huey-Jy , Curin Mirela , Banerjee Srinita , Chen Kuan-Wei , Garmatiuk Tetiana , Resch Yvonne , Campana Raffaela , Focke-Tejkl Margarete , Valenta Rudolf , Vrtala Susanne
Published: January 2023

House dust mites are one of the most important allergen sources. Der p 1, Der p 2, Der p 5, Der p 7, Der p 21 and Der p 23 are the clinically most important house dust mite (HDM) allergens. The aim of this study was to define a mix of non-allergenic T cell epitope-containing peptides of these allergens for tolerance induction. According to the amino acid sequences of these allergens, we synthesized and purified 33 overlapping peptides covering the complete sequences of Der p 1, 2, 5, 7, 21 and 23. The peptides were tested for IgE and IgG reactivity with sera from HDM allergic patients in ELISA. PBMCs from 27 HDM allergic and 10 non-HDM allergic individuals were incubated with the synthetic peptides and T cell proliferation was measured using a CFSE dilution-based assay. The peptides could be purified in large amounts. They lacked secondary structure but most of them remained soluble in physiological buffers. ELISA assays indicated that most peptides from Derp 1, 2, 5, 7, 21 and 23 lacked IgE reactivity and thus were non-allergenic. T cell proliferation assays identified 12 predominant epitopes in the Der p allergens. Our data indicates that a reasonable number of non-allergenic peptides including the sequences and thus T cell epitopes of the clinically most relevant house dust mite allergens can be defined for prevention of HDM allergy by tolerance induction.
Blocking CD28 signal in steroid resistant asthma
Authors: Mori Akio , Kouyama Satoshi , Yamaguchi Miyako , Kinoshita Arisa , Kamide Yosuke , Hayashi Hiroaki , Watai Kentaro , Mitsui Chihiro , Oshikata Chiyako , Sekiya Kiyoshi , Tsuburai Takahiro , Fukutomi Yuma , Taniguchi Masami , Ohtomo Takayuki , Kaminuma Osamu
Published: January 2023

To investigate the mechanism of steroid resistance in severe asthma, effect of CD28 signal blockade on T cell activation and asthma model was analyzed.

PBMC obtained from mild (steroid sensitive, SS), steroid dependent (SD), and steroid resistant (SR) asthmatics were stimulated with mitogens. Der f 2-specific Th clones were established and effects of glucocorticoids (GCs) on the proliferation and cytokine synthesis were analyzed. Steroid responsiveness of murine Th clones reactive to ovalbumin were analyzed. Unprimed BALB/c mice were transferred with these clones and then antigen challenged. Effect of GC on BALF eosinophilia was evaluated. CTLA4-Ig was administered through nasal inhalation or venous injection.

IL-5 production by PBMC of SS asthmatics was significantly reduced after ICS administration, but that of SD asthma remained high. IC50 values for the suppression of cytokine synthesis and proliferation responses by dexamethasone was not different among SS, SD, or SR asthmatics. Addition of CD28 signal induced steroid resistance in IL-2 and PI-3 kinase dependent manner. Murine SS and SR Th clones were selected based on the steroid sensitivity of their proliferation responses in vitro. Airway infiltration of eosinophils and lymphocytes of mice transferred with SS clones were effectively inhibited by GC. In contrast, those of mice transferred with SR clones were not significantly inhibited. Administration of CTLA4-Ig significantly suppressed the proliferation of GC-treated SR clones in vitro, and the BALF eosinophilia of mice transferred with SR clones in vivo.

Costimulatory signal mediated through CD28 seems crucial for the induction of steroid resistance and might be a target for therapeutic intervention.
TGFBR1M318R+/− Knock-In mice recapitulate the immune phenotypes of Loeys-Dietz syndrome.
Authors: Laky Karen , Guerrerio Anthony , Weissler Katherine A , Frischmeyer-Guerrerio Pamela
Published: January 2023

Loeys-Dietz syndrome (LDS) is an autosomal dominant disorder caused by mutations in genes encoding proteins in the transforming growth factor beta (TGFβ) signaling pathway. Patients heterozygous for a point mutation in the kinase domain of TGFβR1 (TGFBR1M318R) exhibit classic manifestations of LDS. Mice with this mutation were made with the hope that an animal model of LDS will lead to better understanding of the mechanisms underlying LDS symptoms. TGFBR1M318R+/− mice exhibit the hallmark skeletal and cardiovascular phenotypes of LDS. We sought to determine if TGFBR1M318R+/− mice also recapitulate the immune phenotypes characteristic of LDS. We find that like their human counterparts, TGFBR1M318R+/− mice spontaneously develop elevated serum IgE and eosinophilic esophagitis (EoE), characterized by esophageal dilation, increased collagen, food impaction, and increased infiltration of eosinophils, T cells, mast cells, and myeloid antigen presenting cells. We made bone marrow (BM) chimeras to determine whether altered TGFβR signaling in hematopoietic and/or stromal cells led to EoE. Both irradiated TGFBR1M318R+/− mice reconstituted with WT BM, and WT mice reconstituted with TGFBR1M318R+/− BM had EoE 8 weeks post-reconstitution, indicating that aberrant TGFβ signaling in either BM-derived or non-hematopoietic cells can be associated with EoE, but that neither is absolutely required. To determine which hematopoietic cells are essential to the development of EoE we generated RAG2−/− TGFBR1M318R+/− mice and found that they too developed EoE, demonstrating that neither T or B cells were essential. Using TGFBR1M318R+/− mice to study the immune pathology of LDS should lead to better therapeutic strategies for the treatment of EoE.
Whole Exome Sequencing (WES) of a Small IPEX-like Patient Cohort results in High Hit Rate and Supports Focusing on the Usual Suspects.
Authors: Hagin David , Ochs Hans D , Torgerson Troy R
Published: January 2023

IPEX (Immune dysregulation Polyendocrinopathy Enteropathy X-linked) syndrome is caused by mutations in FOXP3. The term IPEX-Like is used to describe patients with features of IPEX syndrome and normal FOXP3 sequencing. A cohort of 15 IPEX-like patients was submitted for WES and analyzed in search for underlying monogenic defects. Potential causative mutations were identified in 46% (7/15). Major clinical features and mutations identified are presented below. Despite an obvious analysis bias, the high prevalence of previously described causative genes supports a two-step approach for studying a defined population, starting with targeted sequencing of relevant genes followed by trio WES of unsolved cases.

Clinical SymptomsGeneMutationsIEnteropathy, FTT, Autoimmune cytopenias, T1D, LAD, Recurrent infections, Antibody deficiency.CTLA4Heterozygous c.410C>Tp.Pro137LeuIIAutoimmune cytopenias, Enteropathy, FTT, LAD, Recurrent infections.LRBAHomozygousc.2617_2620del CTCTp.Leu873fsIIIEnteropathy, Lymphocytic pneumonitis, Short stature, Eczema, Recurrent infections.STAT3Heterozygous c.1243G>Cp.Glu415GlnIVEnteropathy, FTT, Lymphopenia, LAD and SplenomegalyTTC7ACompound heterozygous:c.1817A>G p.Lys606Argc.2086T>Cp.Ser696ProVEnteropathy, FTT, transient leukopenia. Two siblings died of severe enteropathy.MYO5BHomozygousc.946G>Ap.Gly316ArgVIEnteropathy, Autoimmune cytopenias, Eczema, ArthritisCTLA4Heterozygousc.436G>Ap.Gly146ArgVIIT1D, Thyroid disease, Enteropathy, Glomerulonephritis, Eczema, Fungal osteomyelitisFOXP3Hemizygous splice site mutation:c.712G>CT1D – Type 1 Diabetes, LAD – Lymphadenopathy, FTT – Failure To Thrive, ICH – Intracranial Hemorrhage.
Developing a T cell:Dendritic cell co-culture system to predict idiosyncratic drug induced hypersensitivity reactions
Authors: Oropallo Michael A , Shang Jackie , Ke Zhenlian , Gonzalez Raymond James , Sistare Frank D. , Lebron Jose , Monroe James J.
Published: January 2023

Idiosyncratic drug induced hypersensitivy reactions (IDHR)s pose a significant safety risk to patients. Indeed, IDHRs have been associated with severe, life-threatening conditions such as Stevens Johnson syndrome, Toxic epidermal necrolysis, and hepatotoxicity. Though drug-induced immune system activation is often implicated in IDHRs, the mechanisms by which this occurs are poorly understood and likely involve formation of drug metabolites or haptens. Furthermore, pre-clinical models often fail to detect the potential of drugs to cause these reactions. Thus, there is a need to create models that can 1) examine how drugs or drug byproducts activate the immune system and 2) predict the potential of drugs to induce IDHRs before they are evaluated in clinical trials. To that end, we have examined the in vitro activation of T cells by autologous monocyte derived dendritic cells (DC)s in the presence of drugs that are associated with IDHR. In concert with previous findings, IDHR associated drugs induce T cell proliferation. Additionally, T cells stimulated by IDHR associated drugs upregulate cell surface markers of activation including CD71, CD69, CD45RO, CD25, and MHC-II. Importantly, we have begun to explore how drug metabolites formed either during T cell:DC co-culture or during culture with hepatocytes influence DC presentation and T cell activation.
NK cell phenotype and proliferation in Systemic Lupus Erythematosus
Authors: Hudspeth Kelly L , Shu Wang , Wang Jingya , Rahman Saifur , Smith Michael A , Casey Kerry Anne , Stephens Geoffrey L , Sanjuan Miguel A ,   Autoimmunity Molecular Team, Hasni Sarfaraz A , Ettinger Rachel , Siegel Richard M
Published: January 2023

Systemic Lupus Erythematosus (SLE) is a complex autoimmune disorder whose pathology appears to involve many immune cell types. While it is clear that autoantibody producing B cells as well as CD4+ T cell help are key contributors to disease, little is known regarding the role of innate lymphoid cells such as Natural Killer (NK) cells in the pathogenesis of SLE. We have characterized the phenotype of NK cells by multicolor flow cytometry in a large cohort of SLE patients. While the overall percentage of NK cells was similar or slightly decreased compared to healthy controls, a subset of patients displayed a high frequency of NK cells expressing the proliferation marker, Ki-67, which was not found in healthy donors. Only a moderate increase of Ki-67 was observed on other immune cell types such as total CD4+, CD8+ T cells or CD19+ B cells in the same donors. Increased NK cell proliferation was found to correlate with clinical parameters. Furthermore, proteomics analysis and auto-antibody arrays revealed significant correlations between NK cell expression of Ki-67 and specific serum protein biomarkers, as well as SLE associated auto-antibodies. These results will contribute to the understanding of the mechanistic role of NK cells in immune-mediated pathology of SLE.
Immunoprofiling determines survival and guides therapy in sepsis
Authors: Valentine Catherine , Bergerat Agnes
Published: January 2023

Objectives

The host immune response during sepsis is still not well understood. Our goal was to examine different mediators of immune function to determine mechanistic explanations for the immune dysfunction observed in sepsis, and determine if blockade of PD-L1 would alter survival in sepsis

Methods

Outbred CD-1 mice underwent cecal ligation and puncture (CLP) to induce sepsis. 24h post-CLP, Il-6 levels were measured by ELISA. Splenocytes were analyzed by flow cytometry for expression of MHCII, CSM, and other cell surface markers. Additional mice were treated 6h post CLP with anti PD-L1 and followed for survival.

Results

Mice were stratified into groups predicted to live (P-Live) or die (P-Die) on the basis of plasma IL-6. Although both groups of mice had increased expression of PD-L1 on splenic monocytes relative to healthy mice, P-Die mice expressed higher levels relative to P-Live mice. P-Die mice had higher levels of MHCII compared to the P-Live group. CD28 and CD40L expression was higher on lymphocytes in P-Live mice. CD4 and CD8 cells had higher levels of CD69 in the P-Die mice compared to P-Live, whereas IL-2r was increased in P-Live compared to healthy mice, but unchanged in P-Die mice. Surprisingly, P-Live mice that received anti PD-L1 had a significantly higher mortality than mice treated with isotype.

Conclusions

Studies of CSM, MHCII and activation markers in P-Live vs. P-Die mice demonstrate variability during the host response in sepsis. Cells in P-Die mice are capable of presenting antigen, but induce apoptosis of lymphocytes via PD-L1. Lymphocytes in P-Die mice show evidence of decreased function. Immunoprofiling of septic patients will identify survivors vs. non-survivors and guide appropriate immunotherapy of sepsis
B cells respond via germinal centers to produce anti-lipid IgG antibodies in a murine model of lupus
Authors: Baeza Carlos Wong , Mora Albany Resendiz , Tescucano Alonso , Landa Carla , Zarate Luz Angela , Wong Carlos , Baeza Isabel , Romo Leopoldo Flores
Published: January 2023

Anti-lipid IgG antibodies are produced in some mycobacterial infections and autoimmune diseases (lupus, anti-phospholipid syndrome). However, few studies have addressed the mechanisms that lead to the production of these immunoglobulins. Anti-lipid IgG antibodies are consistently found in a mouse model of lupus induced by chlorpromazine-stabilized non-bilayer phospholipid arrangements (NPA). NPA are transitory lipid associations found in the membrane of most cells; when NPA are stabilized they can become immunogenic and induce specific IgG antibodies, which are involved in the development of the disease. We used this model of lupus to investigate the in vivo mechanisms that lead to the production of anti-lipid IgG antibodies. B cells are activated, in response to most protein antigens, via germinal centers or extrafollicular reactions. In germinal centers, a T cell-dependent response leads to isotype switch, somatic hypermutation, affinity maturation and memory generation, whereas these events do not usually occur in extrafollicular reactions. In this mouse model of lupus induced by chlorpromazine-stabilized NPA, we found plasma cells producing NPA-specific IgGs in the inguinal lymph nodes, the spleen, and bone marrow of mice. We also found a significant number of germinal center B cells specific for NPA in the inguinal lymph nodes and the spleen, and we demonstrated the presence of NPA in these same germinal centers. In contrast, we found very few extrafollicular reaction B cells specific for NPA. Altogether, our data suggest that, in this murine model of lupus, B cells produce anti-NPA IgG antibodies mainly via germinal centers.
Pulmonary anti-α-1,3-glucan IgA-secreting B cells are protective against cockroach-induced allergic disease
Authors: Patel Preeyam S , Kearney John F
Published: January 2023

Currently, an estimated 20% of the population worldwide suffers from an allergic disorder. Children born in industrialized countries have a higher risk of acquiring allergies and asthma than those in developing countries. This difference has been attributed to lower exposure to microbial antigens during early life and the consequent lack of stimulation for the developing immune system. We have previously demonstrated that bacteria and allergens can bear similar epitopes. German cockroach allergen and an enteric Enterobacter species isolated from mice bear similarly-recognized α-1,3-glucan epitopes. Neonatal mice have a large frequency of anti-α-1,3-glucan-IgA secreting cells that significantly decrease by 5 weeks of age. Following challenge with cockroach allergen, neonatal, but not adult, mice immunized with α-1,3-glucan-bearing Enterobacter had a significantly decreased frequency of eosinophils and T cells in the pulmonary parenchyma, antigen-specific memory T cells in the mediastinal lymph node, and IgE in the blood compared to mice immunized with α-1,3-glucan-deficient Enterobacter. A unique population of anti-α-1,3-glucan IgA-secreting cells were identified in the lungs of mice immunized with α-1,3-glucan-bearing Enterobacter as neonates, but not in those immunized as adults. IgA-deficient mice immunized with α-1,3-glucan-bearing Enterobacter as neonates were no longer protected against allergic disease development. We suggest that early immunization with α-1,3-glucan selects neonatal anti-α-1,3-glucan-IgA secreting clones for incorporation in to the adult repertoire such that these B cells have the ability to suppress the development of cockroach-induced allergic disease during adult life.
Small size of membrane-tethered ligands regulates kinase-phosphatase segregation in γδ TCR triggering.
Authors: Choudhuri Kaushik
Published: January 2023

Ligand recognition by the T cell antigen receptor (TCR) is central to effective immunity. Unlike αβ T cells, γδ T cells do not undergo canonical thymic selection, and consequently are not restricted by pMHC antigens or the co-receptors CD4/8. Some of these unconventional T cells recognize invariant membrane-tethered ligands, and their rapid activation and recruitment to sites of inflammation serves as a ‘first line of defense’ against incipient infection. The substantial structural and stoichiometric similarities between αβ and γδ TCRs prompted us to investigate whether γδ TCR ligand-recognition and triggering shared basic physicochemical constraints with conventional αβ TCRs. We chose as a model system the stress-inducible murine surface protein T22 that is recognized by the G8 γδ TCR - for which detailed biophysical and structural information is available. Since T22 is similar in size to pMHC molecules, we tested whether ‘size-based’ kinase-phosphatase segregation, following G8 engagement by T22, controls initiation of TCR signaling. In our ongoing studies, we have established that the small axial size of T22 is critical for G8 triggering. Elongation of the T22 ectodomain leads to failure of TCR phosphorylation and IL-2 responses. Using optical and electron microscopy we show that elongated T22/G8 complexes increases access of inhibitory phosphatases to sites of TCR engagement at cell-cell contacts. We are testing whether truncation of the ectodomain of the large cell surface phosphatases CD45/CD148, which are normally sterically segregated from engaged TCR, affects triggering. Finally, we are adapting these strategies, to investigate mechanisms of triggering by γδ T cells that recognize small-molecule organic compounds.