bluuebunny/crossref_metadata_2025 huggingface dataset:
Macrophage depletion results in anemia, neutrophilia, and is not an effective therapy for rescuing obesity-linked metabolic impairments
Authors: Bader Jackie E. , Enos Reilly T. , Velazquez Kandy T. , Carson Meredith S. , Sougiannis Alexander T. , McGuinness Owen P. , Nagarkatti Mitzi , Nagarkatti Prakash S. , Robinson Cory M. , Fan Daping , Murphy E. Angela
Published: January 2023

Low-grade, chronic inflammation is thought to play a role in obesity-associated metabolic dysfunction. In obesity, macrophages account for approximately 50% of adipose tissue cells and can secrete a variety of proinflammatory cytokines. In this study, we sought to deplete macrophages in order to decrease macrophage-mediated inflammation and rescue metabolic dysfunction. Following 16 weeks of high-fat diet (HFD) (40% of total kcal from fat) or AIN-76A control diet consumption (n=30), male C57BL/6J mice within each diet cohort were assigned to either a clodronate (CLD)-liposome or PBS-liposome-treatment (n=15/group). Mice received 200 μl (1mg) i.p. injections of CLD or PBS-encapsulated liposomes (control group) twice weekly for 4 weeks to deplete macrophages. Metabolic function was assessed via the HOMA-IR, glucose and insulin tolerance tests, and serum free fatty acids. Adipose tissue, liver, and blood were analyzed for macrophage infiltration, inflammatory mediators and circulating cell populations. HFD-fed mice exhibited an obese phenotype compared to the control diet; however, macrophage depletion was unable to rescue metabolic dysfunction. Interestingly, macrophage-depleted mice had >35% increase in circulating neutrophils and adipose tissue Ly6G content. The increase in neutrophils, likely a compensation for the depletion of macrophages, was linked to an increase in the proinflammatory cytokines, IL-6 and IL1β, in the adipose tissue. Additionally, the decrease in macrophages resulted in iron-deficient anemia. Our study suggests that depleting macrophages in an obese setting is not an effective therapy for rescuing metabolic dysfunction and may increase the risk for anemia and adipose tissue inflammation.
Increased salivary levels of Urokinase Plasminogen Activator Receptor (uPAR) are induced by acute stress and correlate with levels of pro-inflammatory cytokines
Authors: Fernandez-Botran Rafael , Szabo Yvette Z. , Lyle Keith B. , Newton Tamara L.
Published: January 2023

Acute and chronic psychological stress are known to trigger components of the inflammatory system resulting in increased levels of pro-inflammatory cytokines (i.e., IL-1β, IL-6 and TNFα). Stress can also result in the activation of the coagulation and fibrinolytic systems via the sympathoadrenal medullary system, resulting in net hypercoagulability. The aim of this study was to investigate the effects of acute stress on the salivary levels of a key component of the fibrinolytic system, urokinase plasminogen activated receptor (uPAR) and determine the relationship with changes in pro- and anti-inflammatory cytokines. Healthy subjects, ages 18–40 (74% female), were randomly assigned to complete a modified Trier Social Stress Test (n = 44) or to recall an angry autobiographical memory (n = 46). Saliva samples, taken at baseline and 60 min post-stress, were used to measure levels of cytokines and uPAR. Acute stress resulted in a significant increase in the salivary levels of uPAR (p<0.0001) when comparing baseline (median [IQR]: 1.83 [2.31] ng/mL) to post-stress samples (2.57 [2.87] ng/mL). The magnitude of the increase in uPAR levels was similar to those for pro- and anti-inflammatory mediators. There were no significant differences between the two types of stressors. There was moderate to moderate-high correlations with the salivary levels of pro-inflammatory, but not anti-inflammatory cytokines, particularly in the post-stressor samples. These results suggest that components of the fibrinolytic system may also influenced by acute stress, prompting further research into understanding their physiological implications. In addition, uPAR levels in saliva may have potential application as stress biomarkers in saliva.
Changes in peritoneal macrophage composition during sepsis.
Authors: Walker Wendy , Rubio Andres , Kumar Devender
Published: January 2023

Sepsis occurs when an infection activates a systemic inflammatory response. The mortality rate for severe sepsis remains high (30–50%). Additionally, many patients who survive sepsis develop a persistent inflammation, immunosuppression and catabolism syndrome (PICS).

Macrophages are early sentinels of infection and exert both beneficial and detrimental functions during sepsis. As phagocytes, they clear pathogens. Macrophages also express pattern recognition receptors, which trigger inflammatory cytokine production. This process plays a key role in host defense; however during sepsis excessive inflammation leads to immune dysregulation and organ dysfunction.

Tissue-resident macrophages include diverse populations with heterogeneous cell surface markers and functions. Two populations exist in the peritoneum of mice: large peritoneal macrophages (LPM; CD11b+, F4/80-hi, MHC-II-lo) and small peritoneal macrophages (SPM; CD11b+, F4/80-lo, MHC-II-hi). These subsets exhibit different levels of phagocytosis and cytokine production.

Importantly, no prior study has comprehensively examined the migration of monocytes, SPM and LPM during sepsis. We used the mouse cecal ligation and puncture model (CLP) to study this process. Our data demonstrate that LPM exit the peritoneum by 18h post-CLP or sham surgery, accompanied by substantial infiltration of monocytes. Within 14 days after sham surgery, LPM have returned to the peritoneum. In contrast, 14 days after CLP there are still few LPM and a large number of monocytes in the peritoneum. These data indicate that, despite an apparent return to homeostasis, an inflammatory process persists in the peritoneum of these mice. This persisting inflammation may contribute to PICS.
Exacerbation of intestinal IL-17 activity by mycoplasma superantigen leads to increased HMGB1 and chemokines in the joints of autoimmune arthritis mice
Authors: Mu Hong-Hua , Tran Justin
Published: January 2023

An infection can amplify an autoimmune disease by either exacerbating an ongoing disorder, including a relapse, or by leading to chronic progressive disease. We have been studying a mouse autoimmune arthritis model by infecting mice with the Mycoplasma arthriditis. M. arthritidis produces a potent superantigen (SAg) MAM, which is known to be responsible for the autoimmune arthritis induced in susceptible mouse strains. Because the mechanisms leading from systemic autoimmunity to joint-specific autoimmune disease are still unclear, we questioned if autoimmunity initiates at mucosal sites, i.e., gut and lung. In the present study, mice were systemically immunized with collagen II and MAM superantigen, a week later, IL-17 and HMGB1 (the high mobility group box 1, a stress protein) were highly represented within intestinal lamina propria. Levels of IL-17 and HMGB1 in the gut were closely related to the severity of arthritis. The infiltration of inflammatory leukocytes in the joints were found 3 to 5 weeks after IL-17/HMGB1 were seen in the gut. We also showed that IL-17/HMGB1 synergetically stimulate synoviocytes to release a spectrum of chemokines, CXCL1, CXCL2, CXCL12, CCL2 and CCL5, and attracted inflammatory leukocytes. In vivo blocking of IL-17 and/or HMGB1 greatly reduced arthritis severity and joint destruction. Our results suggests that, the synergetic effect of IL-17 and HMGB1 which initiate autoimmunity in the gut is critical for bridging the inflammatory signals between the gut and the joints, and promoting the infiltration of inflammatory cells in the joints. Thus we might develop a therapeutic strategy to reduce inflammatory response and joint destruction by targeting IL-17 and HMGB1 at mucosal sites in autoimmune arthritis.
Pre-existing atopy protects mice from paramyxoviral respiratory infection weight loss
Authors: Hussain Syed-Rehan A. , Rohlfing Michelle , Santoro Jennifer , Grayson Mitchell H.
Published: January 2023

Severe paramyxoviral infections early in life have been associated with development of asthma and allergies; respiratory viral infections are known to exacerbate asthma. We have a mouse model of a severe paramyxviral respiratory infection using murine parainfluenza virus 1, Sendai virus (SeV). C57BL6 mice inoculated with SeV develop a severe acute infection demonstrated by significant weight loss over 8–10 days. The mice recover and are left with long-lasting airway hyper-reactivity and mucous cell metaplasia. Exposure to a non-viral antigen during the acute infection leads to IgE production against the non-viral antigen (allergic sensitization). While this model provides an explanation for development of allergies and asthma, it is not known whether being atopic before viral infection alters the disease state. To address this, we intranasally sensitized and challenged C57BL6 mice with house dust mite extract (HDM) or PBS, and one week later inoculated the mice with 2×105 pfu SeV or ultraviolet light inactivated SeV (UV-SeV). All UV-SeV groups lost no weight. SeV infection in PBS sensitized/challenged (PBS/PBS/SeV) mice led to a 10.8+/−2.5% weight loss from baseline (mean+/−SEM, n=4); surprisingly, HDM sensitized and challenged mice lost no weight with SeV infection (−1.7+/−0.6% weight loss, n=4, p=0.0022 versus PBS/PBS/SeV). Mice HDM sensitized but not challenged loss 10.8+/−1.6% weight with SeV infection (n=4, p>0.99 versus PBS/PBS/SeV). Whether the weight loss protection from HDM sensitization/challenge alters the antiviral immune response and/or protects against post-viral atopic disease is being investigated. These data suggest atopy may protect against symptoms of a severe paramyxoviral respiratory infection.
Principal Component Analysis to Evaluate Relationships in Reactivity to Commercial Extracts and Uncharacterized Fungi among Asthmatic and Allergic Rhinitis Subjects
Authors: Rivera-Mariani Felix Emanuel , Bolaños-Rosero Benjamín
Published: January 2023

Rationale

Identifying patterns in reactivity to allergens can uncover shared respiratory health risks among subjects suffering from chronic respiratory diseases. Therefore, there is a need to identify statistical approaches that can expose inter-allergen relationships.

Methods

: We implemented spearman, exponential, and logistic principal component analysis on specific immunoglobulin-E (sIgE) levels, reactivity to commercial extracts, basidiomycetes (Ganoderma sp, Chlorophyllum sp, Pleurotus sp) crude extracts, and to previously uncharacterized fungal airborne particulate among asthmatics, allergic and non-allergic rhinitis subjects.

Results

: Positive correlations (p = 0.03 to p < 0.001) were identified between reactivity of two commercial fungal extracts (Aspergillus sp, Alternaria sp), and among all three basidiomycete crude extracts (Ganoderma sp., Chlorophyllum, sp, Pleurotus sp.; p = 0.01 to p < 0.001). sIgE levels correlated with all five categories of airborne fungal particulates (un-identified particulates, ascospores, basidiospores, fungal fragments, and mitosporic fungi), but only with three extracts (Aspergillus sp., Alternaria sp, and Chlorophyllum sp). Based on reactivity to fungal airborne particulate, we identified three groups: subjects reactive to ascospores, reactive to basidiospores and unidentified particulate, and reactive to mitosporic fungi and fungal fragments.

Conclusions

: Our finding suggests that PCA of sIgE, together with reactivity to commercial and previously uncharacterized allergens present a suitable strategy to identify respiratory health risks among individuals suffering from chronic respiratory diseases, such as asthma and allergic rhinitis.
A potential role for adipose tissue eosinophils in allergy and asthma.
Authors: Bolus William Reid , Hasty Alyssa
Published: January 2023

Allergy can take many forms, but a mutual trait is the hypersensitive immune response to what the body detects as foreign. A common form of airway allergy is asthma, which affects >235 million people worldwide and is the most common chronic disease among children. Interestingly, obese individuals have a staggering 92% increased risk of asthma, displaying enhanced asthmatic symptoms, airway hyper-responsiveness, & atopy. Due to the clinical correlation between obesity & asthma, scientific endeavors have sought to explain the causality. Studies have shown adipokines secreted from adipose tissue (AT) can have either pro- or anti-inflammatory effects on the lungs. Furthermore, diet-induced obesity increases migration of eosinophils from bone marrow to the lung, as well as delays exit from the airway epithelium to the lumen. Curiously, investigators have tried to understand the correlative link between asthma & obesity without closely examining the AT itself. Our data shows for the first time that AT mounts a hypereosinophilic state upon repeated exposure to a foreign substance (i.e. a hallmark of allergy & asthma in lung); increasing eosinophils from ~5% to 60% of AT leukocytes in obese mice. A single exposure does not elicit an eosinophilic response, potentially suggesting a role for the adaptive immune system. We found that obesity increased sensitivity of this immune response. Furthermore, the surge in AT eosinophils was dependent on IL33, a required cytokine for eosinophilic accumulation in lung during allergy. We are determining if other classic indicators of allergy are present in AT of our model. Ultimately, we hope to elucidate whether AT & lung act within a feedback loop that contributes to the incidence/severity of allergy & asthma.
Adeno-associated viral vector-mediated ATPase gene transfer alleviates asthmatic airway inflammation in OVA-sensitized mice model
Authors: Kuo Ming-Ling
Published: January 2023

Bronchial asthma is a chronic inflammatory disorder of the airway characterized by airway hyperresponsiveness (AHR), pulmonary eosinophilia, elevated serum IgE, and dysregulated T helper (Th) 2 cytokines. It is becoming urgent to identify target molecules for the preventive and therapeutic purpose. ATP accumulation in bronchoalveolar lavage fluid (BALF) of asthmatic subjects indicates that extracellular ATP and the downstream responses are involved. ATP is tightly regulated by CD39, a nucleoside triphosphate diphosphohydrolase (ATPase) encoded by entpd1. Upregulated BALF ATP levels and decreased CD39 RNA were accompanied with asthmatic cardinal features in ovalbumin (OVA)-sensitized mice. Recombinant adeno-associated virus (rAAV) has been developed to carry exogenous genes with safe and low toxic therapeutic potential. The different serotypes and tropisms of AAVs benefit tissue specific gene targeting. This study aims to determine whether lung-targeting rAAV carrying CD39 (AAV/CD39) is able to reduce extracellular ATP in BALF and inhibit the OVA-induced allergic inflammation in an asthmatic murine model. rAAV vectors were administrated into the lungs of mice 3 days before the first OVA exposure. The data indicate that ATP levels in BALF were downregulated accompanied with rescued CD39 expression. The administration of AAV/CD39 was able to suppress AHR, pulmonary eosinophilia, and the production of IL-5 and IL-13 from OVA-activated mediastinal lymph nodes (MLNs) cells. IL-33 in BALF was also reduced. These data indicate that the attenuation of asthmatic airway inflammation may affect innate lymphoid cells (ILC) 2 activation. Thus, ATP level could be a potential target for asthma gene therapy.
Targeting MARCKS phosphorylation site domain shows therapeutic potential for allergic asthma
Authors: Lee Chen-Chen , Chen Ching-Hsien , Lee Ko-Wei , Shu Wen-Chi , Kenyon NIcholas , Wang Chien-Neng , Wu Reen
Published: January 2023

Prevalence of asthma is increased all over the world, especially in Taiwan, study analyzed data of 997,729 enrolees from the National Health Insurance register from 2000 to 2007, the prevalence of asthma increase from 2.9% to 11.9%. Various signaling pathways and mechanisms have been identified and targeted for therapeutic application. Among them, one treatment, based on the use of MANS peptide, which is corresponding to the myristoylated N-terminal 24-amino acid portion of the MARCKS (myristoylated alanine-rich C kinase substrates) protein, to compete MARCKS’ binding to membrane has produced useful results. There is a lack of information regarding to the level/activity of phosphorylated MARCKS (p-MARCKS) in allergic asthma. It is also unclear if this activity can be directly suppressed for the allergic asthma treatment application. In this study, we have found elevated p-MARCKS in most of asthmatic tissues from human as well as from the animal model. This elevation can be directly suppressed by a peptide, MPS, targeting MARCKS phosphorylation site domain, prior to allergen sensitization. In addition, MPS pre-treatment also suppressed all of the symptoms associated with allergic airway asthma, which includes a reduction of inflammatory cells influx and TH2 cytokine presence in the lumen, and also a reduction of airway mucous cell metaplasia and airway hyperreactivity. In addition to inhibition of TH2 responses, lung dendritic cells migrate to mediastinal lymph nodes also decreased by MPS treatment. These results suggest that MARCKS phosphorylation site domain is a potential therapeutic target for the treatment of allergic asthma.
Natural killer cells regulate allergic lung inflammation by acting on group 2 innate lymphoid cells.
Authors: Jaffar Zeina , Ferrini Maria , Roberts Kevan
Published: January 2023

In allergic asthma, inhalation of airborne allergens such as the house dust mite (HDM) effectively activates both innate and adaptive immunity in the lung. Endogenously generated cannabinoids and eicosanoids (acting via CB2 or PGI2-IP receptors, respectively) play important roles in both homeostatic and inflammatory processes including allergic airway inflammation. In this study, HDM responses in CB2 receptor–deficient (CB2−/−) or IP−/− mice were compared with wild type (WT) counterparts. Surprisingly, both CB2−/− and IP−/− mice had increased numbers of pulmonary natural killer cells (NK cells) producing IFN-γ that was inversely associated with the number of group 2 innate lymphoid cells (ILC2s) expressing IL-33Rα and IL-13 compared with WT animals. Remarkably, CB2−/− or IP−/− mice were less responsive to HDM challenge than WT counterparts since intranasal instillation of the allergen induced markedly reduced levels of airway eosinophils, CD4+ lymphocyte infiltration, and mucus production, as well as depressed levels of type 2 cytokines. NK cells and their production of IFN-γ were responsible for such attenuated inflammatory responses because depletion of NK cells or inhibition of IFN-γ production in vivo in CB2−/− or IP−/− mice restored both the HDM-induced allergic inflammation and ILC2 numbers in the lungs. Collectively, these data demonstrate a role for these eicosanoids in regulating the number and properties of NK cells resident in lung tissue and reveal a crucial role for NK cells in the regulation of lung tissue ILC2s and the development of allergic lung inflammatory responses.
The Role of Myristoylated, Alanine-rich C-kinase Substrate (MARCKS) in a Murine Model of Chronic Airway Inflammation
Authors: Wang Chien-Neng
Published: January 2023

Severe asthma, a complicate chronic lung inflammation. Several chronic inflammatory cells that involved in the inflammatory responses, such as macrophages, neutrophils, and eosinophils, produce large amounts of ROS, the excess level of ROS would increase lung inflammation and lung injury. In fact, this inflammation is well controlled by steroid, but some subset of patients these glucocorticoids are less effective. Neutrophil recruitment is a prominent feature of acute exacerbations of chronic asthma. In patients with difficult-to-control asthma, neutrophilic inflammation rather than eosinophils often predominate.

Myristoylated alanine-rich C kinase substrate (MARCKS), is a protein kinase C (PKC) substrate. MARCKS also plays a crucial role that phosphorylation of MARCKS by PKC leads its release from plasma membrane into the cytosol, participating in regulating cell migration such as macrophage and neutrophil. We found that increase of airway MARCKS phosphorylation in asthmatic patients and murine model of chronic steroid-resistance allergic asthma. In this report, our observations indicate that 1) targeting of MARCKS phosphorylation inhibited chronic asthmatic symptoms, such as eosinophil and neutrophil infiltrate into lung. MPS peptide inhibits mucus production, collagen deposition and airway hyperresponsiveness rather than steroid-resistance group; 2) targeting of MARCKS phosphorylation decreased IFN-r, IL-4, IL-17A, IL-33, eotaxin and KC production. MPS peptide also inhibit NADPH oxidase DUOX-1, 2 protein expression and end product 3-nitrotyrosine production; We found that MPS peptide shows a therapeutic potential for steroid-resistance allergic asthma.
Antibodies to a peptide derived from EBNA-1 may be a potential biomarker for SLE
Authors: Spatz Linda A. , Hajtovic Sabastian , Singh Divya
Published: January 2023

The Epstein Barr virus (EBV) has been associated with the autoimmune disease Systemic lupus erythematosus (SLE). We previously demonstrated that mice injected with EBNA-1, a major nuclear protein of EBV, develop anti-dsDNA antibodies that cross-react with EBNA-1. We generated two monoclonal antibodies, 3D4 and 16D2, from EBNA-1 injected mice, and showed that they bind to both EBNA-1 and dsDNA. We also identified a peptide, PFM-15, in EBNA-1 (PQPGPLRESIVCYFM) that is recognized by 3D4 and 16D2. We hypothesize that this peptide can act as a molecular mimic of dsDNA and elicit antibodies to EBNA-1 that cross-react with dsDNA in some individuals who develop lupus. In the present study, we screened the sera of 17 lupus patients and 19 healthy individuals to determine if they have antibodies to PFM-15. We observed a highly significant increase (p<.05) in the number of SLE patients that have antibodies to PFM-15 compared to the number of healthy individuals with these antibodies. Furthermore, statistical analysis revealed that the anti-PFM-15 test has an 80% positive predictive value for SLE suggesting potential to be used diagnostically to detect a subset of patients with lupus. We also observed that 75% of the lupus patients tested, who had antibodies to PFM-15, also had antibodies to dsDNA and a moderately strong correlation was observed between the level of antibodies to PFM and dsDNA in these patients. This is consistent with our observation that antibodies to PFM-15 cross-react with dsDNA. These studies suggest that antibodies to PFM-15 may be a useful biomarker for a subset of patients exposed to EBV who develop lupus. Future studies with a much larger cohort of patients and controls will be needed to confirm this.
Profiling Autoantibodies in Idiopathic Inflammatory Myopathies
Authors: Li Quanzhen , Wang Li , Zhu Chengsong , Yan Mei , Zuo Xiaoxia , Zhang Huali
Published: January 2023

Objects

To screen autoantibodies (autoAbs) in sera of Idiopathic Inflammatory Myositis (IIM) patients and to assess their clinical significance for diagnosis, classification and disease activity evaluation.

Methods

IgG and IgM autoAbs were comprehensively profiled from 138 IIM patients, 70 SLE, 50 SSc and 100 normal controls (NC) using a customized autoantigen array bearing 125 myositis-specific antigens (MSA) and other autoantigens. Western blot and Immunoprecipitation were used for autoAb verification. The profile of autoAbs and correlation with clinical criteria were evaluated.

Results

78% IIM patients showed positive reactivity to at least 1 MSA. The autoAbs which exhibited high prevalence in IIM are anti- Jo-1 (24.6%), MDA5 (23.2%), SAE2 (13%), SRP54 (11.6%) and DNA polymerase II (8.7%). We identified the autoAb clusters that best distinguish the 3 autoimmune disease: anti-Jo-1 and anti-MDA5 for IIM, and anti-CENP-A&B and Scl-70 for SSc, and anti-chromatin/Nucleosome, anti-dsDNA, and anti-Sm/SmD for SLE. The coexistence of MSA autoAbs was observed in IIM with about 35% IIM patients carried more than 2 MSA autoAbs. The IIM patients who developed interstitial lung disease (ILD) exhibited significantly higher autoAbs against Jo-1, MDA5, Mi-2, Ro-SSA and Trptophanyl compared with the IIM without ILD (<0.05). The IIM patients who carried positive autoAbs against JS, TIF1γ, Mitochondrial antigen and La/SSB showed a significantly higher risk of malignancy (p<0.05). Based on autoAbs, we established an autoAb score for IIM prediction and evaluation.

Conclusion

Autoantibody profiling revealed distinct and heterogenic autoAbs in IIM which are potential biomarker for diagnosis, prognosis and classification of the disease.
The respective contributions of P2X7 receptor variants to the development of psoriasiform dermatitis
Authors: Yang Yin , Killeen Meaghan E. , Diaz-Perez Julio A. , Mathers Alicia R.
Published: January 2023

Psoriasis is a chronic inflammatory cutaneous disease caused by environmental triggers in genetically predisposed individuals which account for 2–3% of the world population. However, the mechanisms contributing to the initialization of psoriasis are largely unknown. ATP is a particularly interesting candidate that, via P2X7 receptor (P2X7R) signaling, can activate NF-κB and the IL-23/IL-17 axis, both of which have been shown to be critical in psoriasis pathogenesis. Moreover, P2X7R expression is increased in lesional and non-lesional skin of psoriatic patients compared to healthy donors. Specifically, we have determined that the canonical P2X7R variant A (P2X7RA) is expressed comparably in both lesional psoriatic tissues and normal human skin. However, it is the alternatively spliced P2X7R variant B (P2X7RB) that is highly expressed in human psoriatic lesions. P2X7RB is widely distributed in human tissues and P2X7RB activation enhances cellular proliferation, decreases ATP-induced apoptosis, and in heterotrimers potentiates the inflammatory responses induced by P2X7RA. Importantly, our studies have established that signaling through the P2X7R induces an acute psoriasiform dermatitis in mice. Thus, our ongoing work is focusing on the specific role of P2X7R variants in inflammatory diseases. We confirmed in human blood derived DCs that signaling through the P2X7RB can induce proinflammatory cytokines, such as IL-1β, IL-6 and TNF-α, to same degree as P2X7RA. In conclusion, this work provides a basic mechanistic insight into local inflammation induced following purinergic signaling through the P2X7R that is likely involved in the pathogenesis and potential treatment of many cutaneous inflammatory diseases.
Characterization and isolation of rhesus macaque eosinophils
Authors: Legrand Fanny , Stokes Kindra , Hong So Gun , Tocheny Claire , Mayer-Barber Katrin , Dunbar Cynthia E. , Klion Amy D.
Published: January 2023

With the recent increase in targeted therapies for the treatment of eosinophil-associated disorders, the availability of animal models that mimic human eosinophil responses is a priority. Although murine models have been useful in this regard, differences in eosinophil biology and pathogenesis between humans and mice have limited their utility in some settings. Rhesus macaques provide a potential solution to this problem. Multi-parameter flow cytometry was used to compare the surface phenotype of human and rhesus macaque eosinophils in whole blood. Based on the findings, a strategy for magnetic bead purification of rhesus eosinophils was devised. Activation of purified eosinophils was assessed by surface staining with anti-CD69 antibody, and function was evaluated in a standard chemotaxis assay. Similar to human eosinophils, rhesus macaque eosinophils were found to express EPX and functional CCR3. Siglec-8 expression was not detected. Rhesus neutrophils do not express CD16, the surface marker most commonly used for eosinophil purification in humans. Purity was established by counting 300 cells on cytospin slides stained with Diff-Quik and by flow cytometry. Rhesus eosinophils were isolated from six Rhesus monkeys by Ficoll sedimentation followed by immunomagnetic negative selection using anti-CD64 antibody. Purity of >90% was achieved in 2 animals. Purified eosinophils were not activated, as assessed by CD69 expression, and chemotaxis to eotaxin was preserved. These data provide a comprehensive comparison of the surface expression patterns of human and rhesus granulocytes and a method for purification of viable and functional rhesus eosinophils for in vitro studies.
Autoreactive-prone CD21lo B cells increased in scleroderma patients with interstitial lung disease
Authors: Vowell Katherine Nicholas , Wilfong Erin M. , Crofford Leslie J. , Kendall Peggy L.
Published: January 2023

Systemic sclerosis (SSc) is a devastating autoimmune disease characterized by fibrosis of the skin and vital organs. The pathogenesis of SSc is unknown, but detection of autoantibodies in >90% of SSc patients suggests that B lymphocytes play a role. Therefore, we explored B cell phenotypes in SSc patients using fluorescence cytometry and the multi-dimensional unsupervised analysis tool viSNE. We found that compared to healthy controls, SSc patients had significantly higher proportions of an autoreactive-prone B cell subset identified by low expression of CD21 (CD21lo). Previously, circulating CD21lo B cells were shown to express autoantibodies and were increased in other autoimmune diseases. When considering the clinical phenotypes of the SSc patients, we found that the frequency of CD21lo B cells was only increased in SSc patients with interstitial lung disease (ILD). ILD is a complication of SSc that is associated with increased morbidity and mortality. To our knowledge, this is the first time that a peripheral blood B cell subset has been identified in association with ILD in patients with a systemic autoimmune disease. We investigated the signaling properties of these anergic cells and found lower expression of Bruton’s tyrosine kinase (BTK) in CD21lo B cells compared to normal B cells. This was surprising, as BTK is thought to be overexpressed in autoimmunity, but may indicate that the anergic state is due in part to suppression of BTK-mediated signaling. Our data suggest that CD21lo B cells should be investigated for their role in SSc pathogenesis, and for their utility as a biomarker of ILD. In addition, these findings demonstrate a potential mechanism by which B cell depleting therapy may specifically benefit SSc patients with ILD.
Direct correlation between plasma microbial translocation and autoantibodies in first-degree relatives of patients with systemic lupus erythematosus
Authors: Jiang Wei , Ogunrinde Elizabeth , Zhou Zejun , Kamen Dianne , Oates Jim , Li Quanzhen , Alexander Alekseyenko , Gilkeson Gary S.
Published: January 2023

Background

The process of events underlying autoantibody production in systemic lupus erythematosus (SLE) is unknown.

Methods

Plasma from 36 female African-Americans (18 unrelated healthy controls (UHCs) and 18 first-degree relatives of SLE patients (FDRs)) were assessed for autoantibodies by autoantibody array, lipopolysaccharide (LPS) levels by the limulus amebocyte assay and microbiome by 16S rDNA analysis.

Results

Compared to UHCs, FDRs exhibited elevated plasma autoantibody levels but not similar total IgGs. The median plasma IgGs were 346 (interquartile range, 236–465) and 480 (286–891) for anti-double strand DNA (anti-dsDNA, P = 0.04, Mann-Whitney U-test), 3422 (2863–4076) and 4815 (3447–8464) for anti-single strand DNA (anti-ssDNA, P = 0.03), and 4593 (4050–5521) and 4714 (4004–5278) for total IgGs (P = 0.95), in UHCs and FDRs respectively. Furthermore, parent/child of patients exhibited elevated plasma LPS compared to UHCs. Plasma LPS levels were 59.1 (53.3–68.6) and 71.2 (62.2–80.4) for UHCs and parent/child of patients respectively (P = 0.03). Notably, plasma LPS was positively correlated with IgG anti-dsDNA (r = 0.53, P = 0.02, Spearman correlation) and anti-ssDNA (r = 0.53, P = 0.02) in FDRs but not in UHCs (P > 0.70). Plasma microbiome revealed a reduced diversity (P < 0.02) and lower abundance of Streptococcaceae (unadjusted P = 0.01, adjusted for multiple comparisons P = 0.10) in FDRs compared to UHCs. The ratios of Firmicutes to Bacteroidetes were 6.75 (0.66–42.5) in UHCs and 3.37 (0.63–22.5) in FDRs but did not achieve significance.

Conclusion

Taken together, these results indicate a possible role for plasma microbial translocation and microbiome composition in autoantibody development in SLE.
Role of Vps33B in regulation of inflammatory responses
Authors: McDaniel Margaret , Tracy Charles , Kramer Helmut , Pasare Chandrashekhar
Published: January 2023

Macrophages and dendritic cells use pattern recognition receptors (PRRs) to sense their surroundings and respond to pathogenic stimuli. Engagement of PRRs, such as Toll-like receptors (TLRs), results in accelerated phagocytosis of cargo, activation of anti-microbial responses, and induction of phagosomal and endosomal maturation. Endo/phagosomal maturation and endo/phagolysosome fusion likely plays a critical role in regulation of TLR signaling and outcome of inflammatory responses following microbial recognition. Previous work in the lab has shown that Vps (Vacuolar protein sorting) 33B specifically regulates endosomal maturation following TLR activation, thus influencing the outcome of downstream signaling. In the absence of Vps33B, drosophila hemocytes and mouse macrophages elicit an enormous inflammatory response to live or dead bacterial challenge, yet are unable to degrade the bacterial cargo. This exaggerated immune response is a direct result of accumulated TLRs and ligands that continue to signal while stuck in late endosomal compartments. We have now discovered that this inability to degrade pathogenic cargo has critical implications for adaptive immunity. Absence of Vps33B leads to enhanced maturation of DCs, yet these DCs are unable to effectively prime CD4 T cells, presumably due to lack of processing endocytosed cargo. Mice that specifically lack Vps33B in DCs were unable to activate antigen-specific CD4 T cells in vivo, leading to defective Th1 and Th17 priming. Collectively, our results suggest an important role for Vps33B in both innate and adaptive immune responses. Further work will elucidate the molecular mechanisms of regulation of Vps33B downstream of TLRs and its impact on host immunity.
ILC2-derived IL-13 likely promotes development of peanut allergy
Authors: Krempski James W. , Iijima Koji , Kobayashi Takao , Kita Hirohito
Published: January 2023

Background

Peanut (PN) allergies are life-long diseases that cause severe anaphylactic responses to affected individuals. Allergic immune responses are typically mediated by CD4+ T cells producing type 2 cytokines. However, there is little information available regarding the immunological mechanisms that initiate the development of PN allergen-specific T cells and production of PN specific-IgE (PN-IgE).

Goal

The goal of this study was to elucidate the mechanisms involved in initiation of peanut allergy.

Results

Naïve BALB/c mice (wt) chronically exposed intranasally (i.n.) to PNf developed PN allergy as demonstrated by increased serum levels of PN-IgE as well as acute anaphylactic response when challenged intraperitoneally with PN extract. Acute i.n. exposure of naïve mice to PNf increased the lung levels of IL-13. By using IL-13eGFP reporter mice, we found that group 2 innate lymphoid cells (ILC2s) produce IL-13 when mice are exposed to PNf. Further, IL-13−/− mice showed significant decrease in the numbers of follicular helper T cells in dLNs and serum levels of PN-IgE relative to wild type (wt) mice. When naïve mice were exposed daily to PNf for 2 days, wt mice showed a significant increase in the numbers of dendritic cells (DCs) in the lung dLN; in contrast, overall dLN cellularity as well as number of DCs was decreased in IL-13−/− mice. Furthermore, there were fewer CD11c+ MHC2+ DCs in dLN of IL-13−/− mice, indicating a role for IL-13 in maturation of DCs.

Conclusions

Our findings show that airway exposure to PN induces allergy sensitization by mechanisms involving ILC2-derived IL-13 that mediate the migration and maturation of lung DCs to the dLN. The interplay between ILC2s and DCs are likely critical for development of peanut allergy.
Investigating the role of CX3CR1 in a GITRL- dependent signal 4 checkpoint during LCMV clone 13 infection
Authors: Chang Yu-Han , Wang Kuan-Chung , Watts Tania H.
Published: January 2023

T cell-APC interactions early during chronic viral infection are critical for determining viral set point and disease outcome. Our recent work (Chang et al. Immunity. 2017) revealed a division of labour whereby monocyte-derived APCs (infAPCs), with low MHC II and CD80/86 expression compared to classical DCs (cDCs), preferentially up-regulate TNF receptor superfamily ligands, GITRL, 4-1BBL, OX40L and CD70, in response to type I interferon (signal 3) to provide a post-priming checkpoint (signal 4) for CD4 T cell responses. While cDCs contribute to signal 1 & 2 during priming in response to LCMV clone 13, GITRL on infAPCs binding to GITR on CD4 T cells results in increased surface expression of pro-survival receptors, including CD25, CD127 and OX40 to sustain Th1 helper cell accumulation, help for virus-specific CD8 T cell responses and viral control. Interestingly, CX3CR1, the receptor for fractalkine, was also upregulated on CD4 T cells by GITR signalling during clone 13 infection. CX3CR1 was recently shown to contribute to tumor-infiltration of CD4-helped CD8 CTLs (Ahrends et al. Immunity. 2017). In the chronic LCMV model, we observed that CX3CR1high virus-specific CD4 T cells in the spleen exhibit a markedly increased effector profile when compared to their CX3CR1int-low counterparts, with both higher frequency and per cell expression of Tbet and IFNg production. To investigate how CX3CR1 on CD4 T cells contributes to CD4 T cell help for the CD8 T cell response, we have crossed CX3CR1 deficient mice with TCR transgenic SMARTA mice, expressing an MHC II-restricted TCR specific for LCMV gp61–80. Overall, our data so far indicate that upregulation of CX3CR1 marks the most activated splenic CD4 Th1 effectors during chronic LCMV infection.
Aryl hydrocarbon Receptor Activation in DCs Regulates thymic Function and Immune Tolerance Induction.
Authors: Cole Shelby L. , Shepherd Celine Ann Beamer , Kreitinger Joanna , Shepherd David M.
Published: January 2023

The thymus is a primary immune organ that mediates central tolerance through proper education of developing thymocytes and contributes to peripheral tolerance via the production of thymic regulatory T cells (tTregs). Differentiation of thymocytes involves interactions with both thymic epithelial cells and antigen presenting cells including thymic dendritic cells (tDCs). The thymus is highly sensitive to dioxin, an environmental pollutant that is the prototypical ligand for the Aryl hydrocarbon receptor (AhR) and can induce severe thymic atrophy; however, the underlying mechanisms and ultimate consequences of AhR-mediated thymic atrophy are not currently defined. Moreover, determining how AhR activation in thymic DCs affects thymopoiesis and tTreg induction will further enhance our understanding of immune tolerance. We hypothesized that AhR activation in DCs blocks thymopoiesis and induces tTregs via increased CTLA-4 expression on tDCs. Using CD11ccre x AhRfx(AHRCD11c) conditional knockout mice, we observed that AhR deficiency in CD11c+ tDCs blocked dioxin-induced thymic atrophy and the generation of tTregs, an effect that occurred with other AhR ligands including ITE. Moreover, elevated CTLA-4 mRNA and protein expression was detected in CD11c+ tDCs suggesting a possible mechanism underlying these effects. Together, these findings support the premise that AhR activation in CD11c+ thymic DCs is a critical regulator of thymopoiesis, and the generation of central and peripheral immune tolerance. This research was supported by R01ES013784 (DMS) and NIH Diversity Supplement (SLC).
Complement 5a galvanizes immune inflammation in Hunter syndrome
Authors: Pandey Manoj K. , Prada Carlos Enrique , Magnusen Albert Frank , Nyamajenjere Tsitsi Carol , Mckay Mary Ashley , Rapien Jillian , Köhl Jörg
Published: January 2023

Hunter syndrome, is a lysosomal storage disease (LSD) caused by mutation in IDS that elicits deficiency of iduronate-2-sulfatase. IDS defect initiates excess storage of glycosaminoglycans (GAGs), i.e., heparan sulfate (HS) and dermatan sulfate (DS) in almost every cell of different organs, (e.g., liver, spleen, lung, heart, bones, skin, brain, and nervous system). Affected individuals exhibit abnormal appearance, mental development, organ function, physical skills and death at the age of 10–20 years. Mechanism by which such GAGs propagate disease in Hunter syndrome is wanted. We directly compared inflammatory responses to DS in different groups of BALB/c mice by injecting (i.p./day for 4 weeks) them separately with altered doses of DS, (0, 1, 2, 4, and 8 mg). Complement 5a (C5a) and their receptor (C5aR1) were measured in mice sera and monocytes. As compared to vehicle (PBS), DS treated mice sera and monocytes showed dose dependent increases of C5a and C5aR1. Human patients with Hunter syndrome also showed elevated serum level of C5a when compared to healthy controls. C5a-C5aR1 axis has been valued for increased staffing of immune cells and their venomous influence on tissue impairment in LSD and non LSDs. To explore this risk in Hunter syndrome, mice were injected in presence or absence of indicated amounts of DS and circulatory subsets of immune cells were measured. As compared to vehicle, DS treated mice showed dose dependent rises of monocytes, eosinophils, and neutrophils and their link to excess generation of pro-inflammatory cytokines. Our data showed that DS-induced C5a-C5aR1 axis wave the immune inflammation, which is fatal in Hunter syndrome. Targeting C5a/C5aR1 axis could be potential alternative therapy of Hunter syndrome.
Calcineurin positively regulates TCR-induced LFA-1-dependent cell adhesion via dephosphorylation of Lck Ser-59.
Authors: Otsuka Shizuka , Dutta Debjani , Mittelstadt Paul R. , Ashwell Jonathan D.
Published: January 2023

T cell adhesion via the interaction of the integrin leukocyte function-associated antigen-1 (LFA-1) with its ligand ICAM-1 is essential for the initiation of immune response. Upon ligation of T cell receptor (TCR), activation-induced LFA-1 conformational change occurs via phosphorylation of Thr-758 resulting in the enhancement of binding to ICAM-1. Recently, we reported that dephosphorylation of Lck Ser-59 (LckS59) by calcineurin results in enhanced TCR-proximal signaling, indicating that phosphorylated LckS59 negatively regulates T cell activation. Here we find that inhibition of calcineurin activity by immunosuppressive drugs such as cyclosporin A (CsA) rapidly reverses existing TCR-induced LFA-1-dependent cell adhesion as well as the phosphorylation of LFA-1 Thr-758 in Jurkat and primary human T cells. In addition, TCR-induced cell adhesion of T cells from LckS59A knock-in mice was stronger than T cells from LckWT mice. Consistent with enhanced cell adhesion, some of the signaling events downstream of TCR proximal signaling were also enhanced in LckS59A mouse T cells. Notably, LckS59A T cell adhesion was resistant to CsA inhibition. These observations provide new insights into a previously unrecognized role for calcineurin inhibitors in reversing ligand-dependent adhesion independently of its effects on NFAT activation.
AID phosphorylation regulates MMR during class switch recombination
Authors: Vuong Bao Q. , Choi Jee Eun , Matthews Allysia , Michel Genesis
Published: January 2023

Processing of AID (activation-induced cytidine deaminase)-dependent dU:dG mismatches by the base excision repair (BER) and mismatch repair (MMR) pathways generates untemplated mutations during somatic hypermuation (SHM) or DNA breaks during class switch recombination (CSR). Genetic deletion of UNG or MSH2, which are essential for BER or MMR, respectively, impairs CSR and alters the spectrum of mutations in SHM. Ablation of both UNG and MSH2 completely blocks CSR and limits SHM to GC transition mutations. Phosphorylation of AID at Ser38 (pS38-AID) is required for wild-type levels of CSR and SHM. B cells with a homozygous knock-in mutation of the AID phosphorylation site (AIDS38A/S38A) have intermediate levels of CSR and SHM as compared to wild-type and AID−/− B cells. AID phosphorylation is hypothesized to regulate BER through the interaction of pS38-AID with APE1, which functions downstream of UNG. Consistent with this hypothesis, AIDS38A/S38AMSH2−/− B cells have a complete block in CSR and significantly impaired SHM of Sμ and the JH4 intron. Surprisingly, CSR is also completely blocked in AIDS38A/S38AUNG−/− B cells; however, these cells generate significant mutations in Sμ and JH4 intron. These data demonstrate a critical role for pS38-AID in regulating BER and MMR and uncover a novel pS38-AID-dependent mechanism of mediating MMR-dependent DNA break formation and/or ligation during CSR.
The role of ERK2 in germinal center B cell selection
Authors: Negron Austin Joseph , Jeffreys Sean Alec , Forsthuber Thomas
Published: January 2023

The regulation of programmed cell death is a crucial control point for the maintenance of B cell tolerance in the germinal center (GC) and involves the coordination of several intracellular signaling pathways. Dysregulation of these pathways can lead to the survival of autoreactive B cells, resulting in autoimmune disease. The mitogen-activated protein kinase (MAPK) pathway is involved in the transduction of both survival and apoptotic signals received through the B cell receptor (BCR) and the Fas receptor (CD95), respectively. Extracellular signal regulated kinases 1 and 2 (ERK1/2) are important effectors of this pathway and play a major role in the B cell selection process. Though it is known that ERK2 specifically regulates the pro-apoptotic protein, Bmf, its function in GC B cells remains poorly understood. Our lab has developed a novel mouse model in which the deletion of Erk2 is accompanied by the expression of the fluorescent reporter protein, eYFP, allowing us to identify and isolate viable Erk2 Δ B cells using flow cytometry. Preliminary data using this model has shown that Erk2Δ B cells exhibit impaired survival ex vivo and express several activation-associated surface markers. This suggests that ERK2 plays a major role in facilitating GC B cell selection. Thus we hypothesize that Erk2 Δ B cells are impaired in their ability to participate in the GC reaction. We are currently investigating the ability of Erk2 Δ B cells to respond to activating and pro-apoptotic stimuli in vitro as well as to differentiate in to antibody-secreting plasma cells. Our results will contribute to a better understanding of the molecular programs involved in B cell selection, potentially offering new approaches to treating B cell-driven autoimmune diseases.