bluuebunny/crossref_metadata_2025 huggingface dataset:
Somatic DNA mutations in the blood of normal and autoimmune individuals.
Authors: Sylva Tanya R Da , Gordon Carly , Connor Alison , Keystone Edward , Wu Gillian E
Published: June 2021

We have analyzed blood from normal and autoimmune individuals, finding a previously unrecognized level of somatic DNA mutations. Using a PCR‐cloning technique we have found an average mutational frequency of 10−3 mutations/bp in two marker genes (a mitochondrial [ND1] and nuclear gene [DLD]). These somatic mutational data from normal and autoimmune individuals suggests a need for re‐evaluation of self tolerance models to incorporate mutated self. Negative selection requires the presentation of self antigens – but what happens when self mutates? Among the possibilities are: a) there may be no B and T cells capable of launching a reaction against the mutated self because no new epitopes were generated by the mutant antigen, b) an immune cell may recognize the mutant antigen as non‐self and react to it but the number of identical mutant antigens may be too small to promote an auto‐immune reaction, or c) the immune cells may bind and react to the mutant antigen and in doing so form a memory response that can cross‐react to the nonmutant self antigen precipitating or supporting autoimmunity. A model of autoimmunity precipitated and/or supported by mutations in self will be presented. Research funded by CIHR.
The Role of Apoptosis in the Resistance of DRAK2‐Deficient Mice to Autoimmunity
Authors: Hernandez Jeniffer B. , Ramos Stephanie J. , Gatzka Martina , Walsh Craig M.
Published: June 2021

Autoimmune disease often occurs via the escape of autoreactive T cells from apoptosis, implying a breakdown of central or peripheral tolerance mechanisms. We have recently demonstrated that thymocytes and T cells lacking the serine/threonine kinase DRAK2 have a diminished activation threshold, suggesting that this kinase serves as a quiescence factor to maintain tolerance to weak self‐antigens. Drak2−/ − T cells produce higher levels of the cytokine IL‐2 and exhibit enhanced sensitivity to TCR mediated stimulation. Surprisingly, Drak2−/ − mice are resistant to experimental autoimmune encephalomyelitis (EAE). EAE is a disease induced in mice by immunization with MOG35–55 peptide, which elicits multiple sclerosis‐like symptoms due to T cell activation and recruitment into the CNS. We have observed enhanced apoptosis in effector T cells derived from Drak2−/ − mice, suggesting that DRAK2 serves as both a quiescence factor in naïve T cells and a survival factor in effector T cells. T cells from MOG35–55 ‐immunized DRAK2‐deficient mice have decreased levels of interferon gamma, IL‐17, and increased regulatory T cells (Treg) compared to wild‐type mice. We hypothesize that the decrease in these cytokines and increase in Treg is due to the increase in apoptosis of the effector cells. In accord with this, sensitivity to MOG35–55 peptide induced EAE was rescued in DRAK2−/− mice by a T cell‐specific Bcl‐xL transgene, demonstrating that the resistance to this disease is a consequence of defective survival of MOG‐specific T cells. These results highlight the potential for inhibiting autoimmunity without affecting anti‐pathogen responsiveness via DRAK2 blockade. This work was supported by NIH grant R01 AI063419.
Role of Regulatory T Cells in the Antigen Specific Induction of Tolerance in Murine Asthma.
Authors: Boudousquié Caroline , Pellaton Céline , Barbier Nathalie , Spertini François
Published: June 2021

Natural and inducible regulatory T cells (Tregs) are key players in controlling the development of asthmatic inflammation. To examine the respective role of these subsets in the induction of allergen specific tolerance in established asthma, BALB/c mice were sensitized to OVA and tolerized intranasally with OVA. This led to a marked upregulation of CD4+CD25+Foxp3+ T cells in the lungs, which were regulatory as shown by their suppressive and anergic characteristics in vitro. When mice were depleted of CD25+ T cells using the anti‐CD25 mAb PC61, depletion severely hampered tolerance induction as indicated by a strong recruitment of eosinophils into BALF and a vigorous T cell response to OVA upon challenge, in contrast to non depleted mice. However, in vivo transfer of CD4+CD25+ T cells had no effect on lung inflammation whereas transfer of CD4+CD25− T cells led to reduced eosinophils recruitment upon challenge. PKH26 labeled CD4+CD25− T cells did not proliferate in vivo, although they did proliferate in vitro. When stimulated with OVA in vitro, CD4+CD25− T cells produced high amounts of IL10, low IL‐5 and no TGFβ, IL‐17 or IFNγ. They were equally distributed in the spleen, BLN and lungs. In conclusion, both CD4+CD25+ and CD4+CD25− T cells appear to be essential in tolerance induction. The functional relationship between both subsets will have to be further analyzed.
Interferon‐alpha negatively regulates Th2 development
Authors: Huber Jonathan , Farrar J. David
Published: June 2021

Viral infections trigger the production of type I interferon (IFNα/β) as well as Th1 cytokines like IL‐12 and IFNγ. In contrast, IFNα is not associated with Th2 responses, which are controlled by IL‐4. Hence, the effect of IFNα on Th2 development has not been very well characterized despite its importance for understanding how CD4+ T cells respond to virus infections in Th2‐biased conditions like asthma. To address this issue, we studied the effects of IFNα on IL‐4‐dependent Th2 development in human CD4+ T cells. In these studies, we found that IFNα dominantly inhibits the ability of IL‐4 to promote Th2 differentiation. Further, this effect is specific to IFNα, as neither IL‐12 nor IFNγ inhibit IL‐4‐regulated Th2 development. IFNα inhibits Th2 development by blocking the induction of GATA‐3 independently of STAT6 activation. Thus, we have found a novel function of IFNα in the inhibition of Th2 responses that may be useful as a therapeutic approach to the treatment of asthma.

This study is supported by a grant from the NIH/NIAID (AI56222) and by a training grant from the NIH/NIAID (5T32AI005284).
Trichosanthin induces allergic airway inflammation relying on early IL‐4 production derived from innate immune cells
Authors: Sun Bing , Wang Yuan , Mao Kai Rui
Published: June 2021

Trichosanthin (TCS) is a simple protein isolated from the plant Trichosanthes kirilowii. TCS has anti‐tumor and ant‐HIV activity. Also, it can induce IgE production in both humans and mice. In the present study, we show that TCS can induce airway inflammation, a typical Th2‐type response£without the help of alum adjuvant. Furthermore, TCS is capable of replacing alum to promote OVA‐specific airway inflammation. Interestingly, IL‐4 production in the circulation is associated with an increase in Gr‐1+ cells in peripheral blood. Using IL‐4 neutralizing antibody and IL‐4 gene knockout mice, our sudies show that IL‐4 production at an early stage of OVA‐priming is essential in vivo to create an environment favoring Th2 cell differentiation. Taken together, the data indicate that IL‐4 production from innate immune cells triggered by TCS at an early stage plays a critical role in the pathogenesis of OVA‐specific airway inflammation. Our data suggest that TCS is not only a strong allergen, but also a Th2‐type adjuvant modulating immune response switching to a Th2‐type pathway.
Influence on the Pulmonary Ventilation of the Patients with allergic rhinitis by Inhaling Allergen
Authors: Xiaoming Chen MIng
Published: June 2021

Objective: To analysis the relationship between the allergic rhinitis and bronchial asthma, the pulmonary ventilation of the patients with non‐asthma allergic rhinitis were measured after inhaled allergen.

Method: After the patients inhaling the allergen, the clinical score of the rhinitis manifestations and FEV1/FEC1 %£ The first 1 second the rate of exhaled volume£© were recorded. Otherwise£firstly inhaled the hydrocortiscoid for five days, measured again as above.

Result: Obviously increase the score and decrease FEV1/FEC1 % after inhaled allergen immediately. But it was not apparent if inhaling the hydrocortisone firstly.

Conclusion: The allergic rhinitis and bronchial asthma occur in a similar inflammatory mechanism in a common respiratory duct.
Regulatory role of B cells in a Murine Model of Allergic Airway Disease
Authors: Singh Anurag , Guernsey Linda A. , Mcnamara Jeffrey T. , Secor Eric R. , Schramm Craig M. , Thrall Roger S.
Published: June 2021

Mice sensitized to ovalbumin (OVA) develop a biphasic response to OVA aerosols, such that acute exposure results in allergic airway disease (AAD) while chronic exposure results in local inhalational tolerance (LIT), with resolution of local pulmonary responses but persistence of the systemic allergic response. We have previously reported that B cell lymphocytosis persists in hilar lymph nodes (HLN) during LIT and that CD4+CD25+Foxp3+ Treg cells are significantly increased in the HLN of LIT mice. This raised the consideration that B cells were involved in modulating the LIT response.

Methods and Results: CD19+ B cells isolated from HLN of LIT mice were transferred to sensitized mice before inhaled antigen challenge. Compared to vehicle or AAD CD19+ transferred mice, LIT CD19+ transferred mice developed less BAL leukocytosis and eosinophilia (p < 0.05). In vivo, mice with attenuated AAD following LIT HLN B cell transfer showed a significant increase in the percentages of CD4+ Foxp3+ Treg cells in BAL and HLN, but not systemically (p < 0.03). This protection was antigen specific as LIT HLN B cells did not limit inflammation when transferred to bovine serum albumin (BSA) sensitized mice that were subsequently aerosolized with BSA. In vitro, LIT HLN B cells induced conversion of CD4+CD25‐ Teff cells to CD4+CD25+Foxp3+ T regulatory cells (Treg). The ability to induce suppressive Treg cells was regional, since B cells from AAD or systemic sites during LIT failed to do so. CFSE labeling of LIT HLN B cells demonstrated that these B cells home in and are retained at active sites of inflammation.

Conclusions: These observations support a novel mechanism of regional immune regulation, possibly related to the development of a subset of regulatory B cells within the LIT HLN B cell population.

This work was funded by NIH/AI R01 HL‐43573
Leukotriene A4 Hydrolase Inhibitors Decrease Allergic Airway Inflammation and Hyperreactivity by Modulating Leukocyte Recruitment and IL‐13 Production
Authors: Rao Navin L , Banie Homayon , Riley Jason , Sun Binggang , Xue Xiaohua , Fourie Anne , Karlsson Lars , Dunford Paul J
Published: June 2021

Leukotriene A4 hydrolase (LTA44H) produces the pro‐inflammatory mediator LTB4, a potent chemoattractant of allergy‐associated leukocytes such as mast cells, T cells, eosinophils, neutrophils and monocytes. The potential utility of a selective, LTA44H inhibitor was examined in vivo using a murine, mast cell‐dependent model of allergic airway inflammation. Treatment with a LTA44H inhibitor significantly decreased eosinophils, neutrophils and lymphocytes in bronchoalveolar lavage fluid (BALF) compared to vehicle‐treated animals. Cytokines such as IL‐4, IL‐5, IL‐13 and MCP‐1 were also decreased in the BALF. Airways hyperreactivity, measured by the forced oscillation technique, was significantly reduced in LTA44H inhibitor‐treated animals, as were serum levels of ova‐specific IgE and IgG1. Ex vivo antigen stimulation of peribronchiolar lymph node cells from inhibitor‐treated animals resulted in decreased IL‐13 production. Mechanistically, LTA44H inhibitors decreased CD4+ and CD8+ T cell infiltration into the lung and migration of Ova+ dendritic cell into draining lymph nodes. An associated decrease in T cell cytokine production and dendritic cell function in the lung potentially explains the decrease in multiple aspects of airway inflammation and hyperreactivity. These results demonstrate that inhibition of LTA44H has potential therapeutic utility in the treatment of allergic airway disease.
The prevention and treatment of DNA vaccination with encoding the cockroach allergen Bla g 1 on the allergic asthma
Authors: Zhou Bin , Zhou Yixuan , Ensell Mang , Glickstein Jonathan , Kermany Mohammad Habiby , Cai Qing , Cai Chun , Liu Wenxia , Yoo Tai June
Published: June 2021

Cockroaches produce potent allergens that are an important cause of asthma. The hallmark feature of allergic asthma is abnormal expansionof Th2 cells in the lungs. We present a novel immunotherapeutic strategy using a Bla g 1 DNA vaccination that prevents lung inflammation, airwayobstruction, and hyperreactivity to allergen in a mouse modelof allergic disease, even afteranimals were presensitized and a Th2‐polarized immune responsewas established. Animals which received the DNA vaccine were protected against a following allergic sensitization with cockroach extract. The protective effect was characterized by suppression of cockroach allergen‐specific IgE production and enhanced IFN‐γ expression. In a therapeutic situation, treatment of sensitized animals with DNA vaccines decreased IgE production and drastically reduced anaphylactic activity as measured by enhanced IFN‐γ expression. Concerning the cellular immune response, DNA immunization induced a sustaining and dominant shift from a Th2 type response towards a balanced Th1/Th2 type response as indicated by increased IFN‐γ but decreased IL‐4 and IL‐5 levels in lymphoproliferation assays. Moreover, studies using bronchoalveolar lavage fluids of cockroach‐sensitized animals showed marked increase in imflammatory cells and eosinophils in contrast to BALF from Blag 1‐vaccinated animals. The histologic studies showed that the much reduced infiltration of inflammatory cells was observed in lung tissue of the gene vaccinated mice than that of the controls mice. The results demonstrate the allergen‐specific protective and therapeutic efficacy of a DNA vaccine encoding the cockroach allergen Bla g 1.
Altered expression of matrix metalloproteinase‐2 correlates with increased vasoactive intestinal peptide in a murine model of asthma
Authors: Samarasinghe Amali Eashani , Hoselton Scott Alan , Schuh Jane Marie
Published: June 2021

Vasoactive intestinal peptide (VIP) is a pleuripotent neuropeptide and cytokine that we hypothesize may regulate the production of matrix metalloproteinase (MMP)‐2 in asthma. METHODS: As the first step of this investigation, mice were sensitized and challenged with Aspergillus fumigatus to elicit allergic airways disease. Expression of VIP mRNA was determined with real‐time PCR. Total MMP‐2 expression in serum was determined by enzyme‐inked immunosorbent assay (ELISA), and immunohistochemistry was used to identify the spatial expression of VIP and MMP‐2 in the allergic lungs. RESULTS: The mRNA for VIP was markedly increased 14 days post challenge in comparison to naïve controls. Serum MMP‐2 peaked at day 7, and was most prominent in the columnar epithelium. While naïve animals showed constitutive VIP production in the airway columnar epithelium and smooth muscle surrounding arterioles, experimental animals produced more VIP in these regions as well as the alveolar lining. CONCLUSION: VIP production spatially and temporally correlates with MMP‐2 expression, suggesting its role as a regulator of this important matrix proteinase. Research supported by Grant Number 2P20RR015566 from the National Center for Research Resources (NCRR).
Histamine 4 Receptor Antagonism modulates TH2 cell function, IL13 production, and lung remodeling in a model of chronic asthma
Authors: Cowden Jeffery , Riley Jason , Thurmond Robin , Dunford Paul J
Published: June 2021

H4R antagonists are anti‐inflammatory in murine models of asthma. Here, we examined the specific ability of H4R to modulate TH2 cell function and the associated downstream effects of Th2 cytokine modulation such as: inflammation, mucus cell hyperplasia, MMP expression and collagen disposition, using a selective H4R antagonist. Mice were sensitized i.p. to ovalbumin in alum followed by repeated challenge with ovalbumin. Lungs were lavaged and tissues collected. Therapeutic treatment resulted in decreased lung TH2 cytokines, IL13 and IL5, and decreased cell influx into the lung. In addition to the reduction of eosinophils, there was a reduction in CD3+ cells. Cultured T cell re‐stimulated with antigen ex‐vivo had reduced production of TH2 cytokines. IL13 dependent markers of lung remodeling such as goblet cell hyperplasia, MMP‐9 expression, and total lung collagen were reduced. Thus, we demonstrate that the anti‐inflammatory properties of H4R antagonists include modulation of TH2 cell function, resulting in a reduction of TH2 derived IL13. Consequently, H4R antagonists reduce cardinal signs of asthma such as inflammation and lung remodeling, suggesting their potential as a novel human therapeutics.
IRAK‐M selectively modulates the alternative, instead of the classical NFkB pathway in a ligand‐specific fashion
Authors: Li Liwu , Su jianmin , Surace Michael
Published: June 2021

Innate immunity signaling process is controlled by numerous positive as well as negative mediators. The interleukin‐1 receptor associated kinase M (IRAK‐M) is one of the negative regulators that contribute to attenuation of NF‐kappa‐B activation. However, the molecular mechanism involved is poorly defined. We observed that IRAK‐M selectively suppressed the NIK‐IKKalpha‐mediated alternative NF‐kappa‐B pathway. Deletion of IRAK‐M led to NIK stabilization, favored the formation of IKK‐alpha/IKK‐alpha homo‐dimer instead of IKK‐alpha/IKK‐beta heter‐dimer, and enhanced RelB nuclear distribution. In contrast, the p65 nuclear localization and phosphorylation was not affected by IRAK‐M deficiency. We found that IRAK‐M deletion led to accelerated synthesis of I‐kappa‐B‐alpha, as well as increased expression of selected cytokines such as IL‐6. Furthermore, the elevated IL‐6 expression can be ablated when RelB was knocked down with specific siRNA. We also demonstrated that the observed inhibitory effect of IRAK‐M was primarily limited to TLR‐2 ligand, instead of TLR4. Taken together, our findings suggest that IRAK‐M negatively regulates alternative NF‐kappa‐B pathway in a ligand specific manner.
Eosinophil‐derived neurotoxin acts as an alarmin to activate TLR2‐MyD88 signal pathway in dendritic cells and enhance Th2 immune responses
Authors: Yang De , Chen Qian , Su Shao‐Bo , Zhang Ping , Caspi Rachel R. , Micchalek Suzanne M. , Rosenberg Helene R. , Oppenheim Joost J.
Published: June 2021

Eosinophil‐derived neurotoxin (EDN) is an eosinophil granule‐derived secretory protein with ribonuclease and anti‐viral activity. We have previously shown that EDN can induce the migration of dendritic cells (DCs). Here we report that EDN can activate myeloid DCs by triggering the TLR2/Myd88 signaling pathway, thus establishing EDN as an endogenous ligand of TLR2. EDN activates TLR2 independent of TLR1 or TLR6. EDN also enhances antigen‐specific Th2‐biased immune responses as evidenced by predominant production of antigen‐specific IL‐5 and IL‐13 as well as higher levels of IgG1 than IgG2a. Based on its ability to serve as a chemoattractant and activator of DCs, as well as the capacity to enhance antigen‐specific immune responses, we consider EDN to have the properties of an endogenous alarmin that alerts the adaptive immune system for preferential enhancement of antigen‐specific Th2 immune responses. (Funded in whole or in part with federal funds from the NCI, NIH, under contract N01‐CO‐12400).
Induction of adaptive immunity by flagellin does not require TLR5 or robust innate immune activation
Authors: Sanders Catherine , Uematsu Satoshi , Akira Shizuo , Gewirtz Andrew T.
Published: June 2021

The effectiveness of vaccine adjuvants is attributed to their activation of robust and varied innate immune events, which in turn may promote protective adaptive immunity. Flagellin (flg), a TLR5‐ and MyD88‐dependent immunogen, is a potent inducer of both arms of immunity, and thus may be a good vaccine adjuvant candidate. We hypothesized that TLR5 is critical for flagellin to induce both innate and adaptive immunity to co‐administered OVA. Surprisingly, TLR5KO mice boosted with flg+OVA developed strong OVA‐specific IgG titers, suggesting that TLR5 is not needed for flagellin.s adjuvanticity. Responses were not due to common contaminants, as flg‐treated TLR5TLR4DKO and TLR5TLR2DKO mice both made comparable Ab titers to TLR5KO. Adjuvants are thought to function via activation of early immune events. Flg‐treated TLR5KO mice lacked systemic IL‐12, TNFα, IL‐17, and RANTES measured over 24hr, and made very low levels of KC, IL‐6, IL‐1α, MCP‐1, and G‐CSF. No upregulation of CD80, 86, or 40 were detected on splenic DCs removed 6hr after I.P. flg injection of TLR5KO mice. These data indicate flagellin.s adjuvanticity does not require TLR5, detectable DC activation, or the relatively high serum cytokine levels found in WT mice. Our results suggest that vaccine adjuvants may not necessarily require robust innate immune events to generate effective adaptive immune responses.
Characerization of a novel pore‐forming protein in macrophages
Authors: de Armas Lesley R , Ramos Jahir , Lyapichev Kirill , Shiratsuchi Motoaki , Deyev Vadim , Podack Eckhard
Published: June 2021

Perforin 2 (P2) is the predicted protein of Macrophage specific gene 1 (Mpg‐1) expressed in differentiated macrophages. The function of Perforin 2 is not known. When P2 is overexpressed in 293 cells it forms polymeric pore complexes similar to poly C9 and poly Perforin1 in membranes that are observed by negative staining and electron microscopy. The pore structure of poly P2 suggests cytolytic function similar to Perforin1 and C9. P2 has a predicted transmembrane domain followed by a short cytoplasmic domain containing a cluster of lysine residues followed by a conserved tyrosine and serine residue, suggesting functional roles in P2 polymerization and expression of cytolytic activity. In this study, we isolated enriched subcellular fractions containing P2 from 293 cells transfected with eGFP tagged P2. The fractions were digested with TPCK‐treated trypsin for various amounts of time to determine P2 susceptibility to proteolytic cleavage. We found that with increasing exposure to trypsin, P2 was cleaved into 2 pieces whose molecular weights by SDS‐PAGE corresponded to cleavage at the cluster of lysines in the cytoplasmic domain of P2. The electronmicroscopic pore structure of poly P2 however remained intact indicating protease resistance of the pore complex. The proteolytic cleavage of P2 is likely important for P2 function in macrophages.

Supported by NIH CA039201
Neutrophils from burn patients suppress the beta‐defensin production by human epidermal keratinocytes
Authors: Kawasaki Takashi , Jeschke Marc G , Shigematsu Kenji , Kobayashi Makiko , Herndon David N , Suzuki Fujio
Published: June 2021

Recently, we have isolated immunosuppressive neutrophils (PMN‐II) from patients with severe thermal injuries. PMN‐II play a role on the increased susceptibility of burn hosts to various infections. Since antimicrobial peptides have been described as important effector molecules on host antimicrobial innate immunities, in the present study, the influence of PMN‐II on the beta‐defensins (BD) production by human keratinocytes has been studied.

Normal human epidermal keratinocytes (NHEK: 5×105 cells, Lonza) were cultured with neutrophils (1×105 cells, upper chamber), isolated from burn patients or normal volunteers, in dual‐chamber transwells. Culture fluids harvested 24 hrs after cultivation were assayed for BD by ELISA. Also, these culture fluids were analyzed for their antimicrobial activities by a standard colony counting method using Pseudomonas aeruginosa. PMN from burn patients were confirmed as PMN‐II by their cytokine/chemokine producing profiles.

The production of BD2 and BD3 by NHEK was suppressed by PMN from burn patients. The culture fluids of NHEK transwell‐cultured with burn patient PMN showed decreased antimicrobial activities, as compared with controls (culture fluids of NHEK transwell‐cultured with healthy PMN). PMN isolated from peripheral blood of burn patients were confirmed as PMN‐II, because these cells produced IL‐10 and CCL2, but not IL‐12 and CCL3.

These results indicate that PMN‐II appeared in association with burn injury contribute to the decreased production of BD in thermally injured patients. (Supported by SHC NA #8840.)
Regulation of NF‐κB and Inflammasome Function by Pyrin Only Protein‐2 (POP2)
Authors: Atianand Maninjay Kumar , Haller Matthew C. , Duffy Ellen B. , Harton Jonathan A.
Published: June 2021

Inflammation and innate immune responses are mediated through the activation of NF‐κB‐dependent cytokine transcription and the formation of the IL‐1β converting inflammasome complex. Inflammasome assembly requires pyrin domain (PYD)‐dependent interactions between an NLR and ASC with subsequent recruitment of the IL‐1β converting enzyme caspase‐1. Proteins containing only a PYD can act as modulators of NF‐κB activity and inflammasome functions. We have recently characterized a novel protein, pyrin only protein‐2 (POP2) that inhibits p65‐induced NF‐κB activity and blocks inflammasome formation by disrupting the interaction between ASC and various NLRs. While expressed in macrophages, the molecular basis for POP2 mediated inhibition of NFκB and inflammasome assembly are unknown. We present data that the action of POP2 differs from that of POP1. We have also further refined POP2's mode of action and mapped the functional domain of POP2 required for regulation of NF‐κB activity. Further, the residues involved in NF‐κB inhibition appear to be distinct from those involved in ASC binding and inflammasome inhibition, suggesting that NF‐κB and inflammasome inhibition are independent functions. We propose a model for role of these distinct functions of POP2 in regulating innate immunity.
Immunoregulatory role of Toll‐like receptor 9 in septic peritonitis
Authors: Ishii Makoto , Hogaboam Cory M , Standiford Theodore J , Kunkel Steven L
Published: June 2021

Objective: Toll‐like receptor 9 (TLR9) is recognized as a key receptor in the innate immune response. But little is known about the role of TLR9 in sepsis. In this study, experiments were performed to determine the role of TLR9 during septic peritonitis.

Methods: Female wild‐type (WT) and TLR9‐deficient (TLR9−/−) BALB/c mice were subjected to cecal ligation and puncture (CLP) surgery. After 24 hours of CLP surgery, the peritoneal lavage and the serum were collected (n=10). The cytokine expression in peritoneal lavage was measured by sandwich ELISA. The serum chemistry analysis was performed by the Animal Diagnostic Laboratory at the University of Michigan. In a survival study, mice were monitored for 7days after CLP surgery (n=17).

Results: The total number of peritoneal leukocytes was significantly higher in TLR9−/− CLP‐treated mice as compared with WT CLP‐treated mice, which was due to an increase in peritoneal neutrophils. The level of TNF‐α, CCL2, and CCL3 in peritoneal lavage was significantly lower in TLR9−/− mice compared with levels in WT mice. In addition, the serum level of BUN, Creatinine, and LDH were lower in TLR9−/− mice. Finally, we observed that TLR9−/− mice were highly resistant to CLP‐induced peritoneal sepsis.

Conclusion: The absence of TLR9 promotes a protective innate immunity during experimental septic peritonitis.

(Funded by NIH HL31237, NIH HL74024, NIH HL31963)
Functional Analysis of Recombinant Human Mannan‐binding Lectin and its Structural Variants expressed in Insect Cells
Authors: Rawal Nenoo , Salvi Veena P. , Rajagopalan Rema
Published: June 2021

Mannan‐binding lectin alleles (MBL/B, C, and D) arise due to point mutations in the structural gene and are associated with increased susceptibility to recurrent infections in children and adults. Our data show that mutations in the collagen region have no effect on the ability of the recombinant forms of the structural variants (rMBL/B, C, and D) to form higher order oligomers. Purification of structural variants on mannose‐ and mannan‐Sepharose columns indicated that the structural variants bind to sugars. When binding to mannan was determined by ELISA using monoclonal antibody (mAb 131‐01), the structural variants bound poorly compared to wild type MBL purified from plasma (pMBL/A) or rMBL/A. The weak binding is attributed to the 16 to 60‐fold weaker avidity of the mAb for the variants than for pMBL/A or rMBL/A. Preliminary studies using surface plasmon resonance (Biacore) that does not require antibody, show pMBL/A and rMBL/A as well as rMBL/D bind to mannan with high affinity (KD range = 0.47 to 1.2 nM). The variants, rMBL/B and rMBL/C also bind to mannan but with a 35 to 54‐fold weaker affinity than pMBL/A. The effect of point mutations in MBL on complement activation was studied by hemolytic assay using MBL depleted serum and mannan coated sheep erythrocytes. The results show that rMBL/C was ineffective and rMBL/B activated complement weakly where as rMBL/D was fully functional like pMBL/A and rMBL/A.

Research supported by NIH grant HL073804
Soluble gC1qR/p33 accelerates the rate of complement activation in C1 inhibitor (C1‐INH) deficient serum
Authors: Vinayagasundaram Uma , Vinayagasundaram Rama , Glassberg Philip F , Nguyen Phuong , Mathew Sally E , Menzies Catherine A , Peerschke Ellinor I.B. , Ghebrehiwet Berhane
Published: June 2021

C1‐INH is a serine protease inhibitor (serpin) whose deficiency results in a life threatening disease─angioedema. Its primary function is to regulate key enzymes of both the complement and kinin generating systems; but it also plays a key role in controlling the integrity of C1. These studies were undertaken to test the hypothesis that gC1qR, which activates both the complement and kinin systems, can accelerate complement activation in C1‐INH deficient serum. To achieve this, we first prepared C1‐INH depleted serum (NHS−C1inh) by affinity depletion on protein A‐immobilized anti‐C1‐INH, followed by reconstitution with 20 μg/ml C1q and 20 mM CaCl2 and MgCl2. Depletion was verified by ELISA, and its activity by a standard hemolytic assay. Incubation (60 min, 37º C) of NHS alone, followed by measurement of residual hemolytic activity showed no loss in function when compared to untreated NHS. However, NHS−C1inh lost 30–50% of its function due to spontaneous autoactivation. Importantly, whereas incubation of NHS with 5μg/ml gC1qR or aggregated IgG resulted in complete activation after 60 min, it took only 15 min for the same level of activation in NHS−C1inh. This was reversed when the NHS−C1inh was reconstituted with C1‐INH. The data suggest that gC1qR, can trigger and/or exacerbate the inflammatory process associated with angioedema by enhancing activation of complement. [Supported by NIH‐NIAID Grant R01 AI‐060866]
Surfactant Protein D enhances phagocytosis of Cryptococcus neoformans hypocapsular strain cap59Δ by murine macrophages
Authors: Geunes‐Boyer Scarlett Gabriel , Oliver Timothy N , Heitman Joseph , Perfect John R , Wright Jo Rae
Published: June 2021

Cryptococcus neoformans is an opportunistic pathogen and the leading cause of fungal meningitis in humans. Infection occurs following inhalation of C. neoformans cells, which, in the absence of a cellular immune response, results in pulmonary infection. C. neoformans cells produce a polysaccharide capsule composed predominantly of glucuronoxylomannan (GXM), which makes up approximately 90% of the capsular material. In the lungs, surfactant proteins SP‐A and SP‐D contribute to innate defense by facilitating the aggregation, uptake and killing of microorganisms by phagocytic cells. We hypothesized that SP‐D plays a role in C. neoformans pathogenesis by facilitating its clearance. We examined the ability of SP‐D to modulate the innate immune response to C. neoformans serotype A using wild type encapsulated strain H99 and mutant hypocapsular strain cap59Δ. We assessed the ability of SP‐D to bind to and facilitate phagocytosis of both C. neoformans strains. We found that SP‐D binds to cap59Δ approximately six‐fold better than to H99, and enhances phagocytosis of cap59Δ about four‐fold in vitro. To investigate SP‐D binding in vivo, SP‐D−−/− mice were intranasally instilled with AF488‐labeled cap59Δ. We observed a greater number of phagocytosed C. neoformans cells in wild type compared to SP‐D−/− mice, consistent with our in vitro data, using confocal microscopy. These studies suggest that SP‐D plays a role in host defense against C. neoformans by binding to and enhancing its phagocytosis.

This work was supported by NIH grant HL‐30923
Aspergillus fumigatus primes dendritic cells to stimulate IL‐17 production from CD4+ T cells
Authors: Fei Mingjian , Oriss Timothy , Steele Chad , Kolls Jay , Ray Prabir , Ray Anuradha
Published: June 2021

Aspergillus fumigatus (A. fumigatus) is a fungus that induces allergic airways diseases in immunocompromised patients, and in patients with cystic fibrosis or severe asthma. Host defense against this fungus depends on both innate and adaptive immunity. DCs have been shown to transport conidia or hyphae from the airway to the lung draining lymph node and initiate T cell responses. Fungus‐induced Th2 response has been associated with allergic bronchopulmonary aspergillosis while Th1 response favors resistance to the pathogen. We have initiated studies to understand adaptive immune responses against A. fumigatus. We have found that resting conidia are not able to induce activation of bone marrow derived‐DCs, while swollen conidia efficiently promote DC maturation. In order to investigate how Aspergillus‐programmed DCs prime T cell responses, we cocultured fungus‐exposed DCs with CD4+ T cells in the presence or absence of TCR activation by specific antigen or anti‐CD3 antibody. Even without TCR signaling, similar to DCs exposed to the dectin‐1 agonist curdlan, Aspergillus‐conditioned DCs induced IL‐17 from T cells, which was not elicited by control DCs that were not exposed to any stimuli. Collectively, our observations show the ability of Aspergillus to prime DCs for IL‐17 production from CD4+ T cells, which is increasingly being associated with allergic diseases. Funded by: HL84932 and HL 77430 (to A.R.)
Analysis of local innate immune response to Eimeria acervulina using chicken intestinal cDNA microarray
Authors: Lillehoj Hyun Soon , Kim Chul Hong , Keeler Calvin L , Hong Yeong Ho
Published: June 2021

Intestinal intraepithelial lymphocytes (IELs) are the primary immune effector cells in the gut involved in innate immune response to Eimeria, the etiologic agent of avian coccidiosis. In this study, a library of genes expressed by intestinal IELs of Eimeria‐infected chickens was constructed using the expressed sequence tag (EST) strategy and was used to analyze gene expression profiles during primary or secondary Eimeria acervulina infections. The avian intestinal IEL cDNA microarray (AVIELA) contained duplicates of 9,668 individual ESTs, of which 5,872 contained at least one replicate spot that provided high quality, analyzable data. Following primary infection with E. acervulina, the expression levels of 74 genes were significantly altered compared with the uninfected controls. Gene function analysis indicated that many of the modulated genes were related to innate and adaptive immune responses (interleukin signaling, TLR signaling, antigen presentation, apoptosis). Duodenum RNA samples obtained at various times after infection was obtained and Gene Ontology analysis revealed that the genes related to apoptosis, immune response, host‐pathogen interaction, cell growth, and cell communication were significantly altered in their expression following both primary and secondary infections. The results of our high‐throughput gene expression analysis of intestinal cDNA microarray provide for the first time comprehensive understanding of host‐pathogen interaction in the duodenum following E. acervulina.
The cannabinoid 2 receptor mitigates survival and tissue damage during sepsis
Authors: Caldwell Charles C. , Tschoep Johannes , Goetzman Holly S. , Choi Lisa G. , Adediran Samuel
Published: June 2021

Cannabinoids have both psychotropic and immunomodulatory effects. It has been suggested that immune effects are mediated by the cannabinoid 2 receptor (CB2R), which is expressed upon leukocytes. In this study, we obtained CB2R knockout mice (KO) and subjected them to cecal ligation and puncture (CLP) to define the role of this receptor in sepsis. We observed that CB2R KO mice showed a significantly higher mortality as compared to the wild type (WT) control group. CB2 KO mice also showed increased serum IL‐6 levels 6 hours after CLP as compared to the WT group. At 24 hours following CLP: 1) We found a significant increase in bacteremia in CB2R KO mice. 2) The chemokines KC and MIP‐2 were significantly elevated in CB2R KO mice as compared to the WT control. 3) We determined that numbers of neutrophils in septic CB2R KO mice showed a significant increase at the site of infection. 4) Lung bacterial load and the tissue damage were significantly higher in CB2 KO mice as compared to the WT control. 5) Furthermore, CB2R KO mice showed significantly higher blood urea nitrogen as well as increased histological evidence of kidney damage. These data suggest that the CB2R beneficially mediates the immune response during sepsis. This study is funded by NIH R01 GM72760.
Evaluating gene expression patterns induced by SEB and LPS in human PBMCs
Authors: Mendis Chanaka , Jett Marti
Published: June 2021

Both Staphylococcal enterotoxin B (SEB), an exotoxin produced by staphylococcal aureus and lipopolysaccharide (LPS) released from various bacterial cell walls are commonly associated with food poisoning and capable of triggering flu‐like symptoms. Here we compared the host response to these distinct toxins at a genetic level by investigating the time dependent expression pattern of a set of genes that have been known to explain some of the symptoms induced by the toxins through their functions. After obtaining a specific gene expression pattern for each toxin through microarray analysis a set of genes were selected for further analysis based on their function, such as apoptosis, cytokine induction, DNA binding, phosphorylation or transporter activity. After designing primers for the selected genes, real time PCR and RT‐PCR were carried out to confirm the genetic expression pattern initially established by microarray analysis. The combination of these gene analysis techniques such as microarrays, real‐time PCR and RT‐PCR will provide us the opportunity to effectively and accurately identify/confirm a set of toxin‐specific genes as well as to gain some useful information of the mechanism of action.
TLR9 activation is a key event for the maintenance of a mycobacterial antigen‐elicited pulmonary granuloma
Authors: Ito Toshihiro , Schaller Matthew , Hogaboam Cory M. , Stolberg Valerie , Chensue Stephen W. , Kunkel Steven L.
Published: June 2021

We previously described a model of polarized Th1 cell‐mediated anamnestic pulmonary granulomatous inflammation in sensitized mice induced by pulmonary embolization of agarose beads coated with covalently bound antigens of Mycobacteria bovis purified protein derivative (PPD). We have also demonstrated TLR9 plays an important role in maintaining the appropriate phenotype in a Th1 granulomatous response. We demonstrate in this study that TLR9−/− mice showed greatly increased granuloma formation as assessed by impaired myeloid dendritic cells and increased regulatory T cells, and Th‐2 skewed cytokine profile compared with wild‐type. Moreover, the expression of Th17 cytokine family members (IL‐17 and IL‐21) was abrogated in TLR9−/− mice. These data also support the concept that dendritic cells, but not macrophages, from bone marrow can promote the differentiation of Th‐17 cells, while both macrophages and dendritic cells promote Th‐1 cell differentiation after BCG stimulation. These findings support the concept that the impaired recruitment of granuloma dendritic cells alters the Th17 cytokine profile in TLR9‐deficient mice.

This work was supported by NIH grants HL031963, HL074024, and HL031237.
The effects of two glycosphingolipid inhibitors on Pneumocystis‐induced lung inflammation
Authors: Vuk‐Pavlovic Zvezdana , Standing Joseph E , Limper Andrew H
Published: June 2021

Pneumocystis, an opportunistic fungus, causes pneumonia in immunocompromized hosts. Inflammation is induced, at least partly, by β‐eta‐glucan, the major component of Pneumocystis cell wall. Airspace‐lining epithelial cells recognize glucan by membrane glycosphingolipid (GSL) lactosyl ceramide (LacCer). We treated Pneumocystis‐infected mice with two GSL inhibitors: PDMP and NB‐DNJ (Miglustat‐Actelion Pharmaceuticals). Starting at 8 weeks post infection, animals received either 150 mg PDMP or 1200 mg Miglustat/kg of body weight ‐ dissolved in saline, or equal volume of saline alone (daily i.p. injections for 10 days). Results show that PDMP‐treated mice had fewer leukocytes in bronchoalveolar lavage fluid (BALF) than saline‐treated controls, with the most marked difference in the number of neutrophils. Interestingly, BALF concentrations of MIP‐2 and TNF‐alpha (determined by ELISA) did not differ between these two groups. The analysis of Pneumocystis burden in the lung, as determined by Q‐PCR, showed decrease in PDMP‐treated animals. The anti‐inflammatory effects of Miglustat were less pronounced than PDMP effects. Neither inhibitor induced lung injury as measured by albumin leak into airspaces. Results suggest that GSL inhibitors might have an impact on inflammatory response to Pneumocystis, leading to new options in treatment of Pneumocystis pneumonia.

Supported by NIH‐RO1‐HL62150 and Mayo Clinic
Korean‐American merchants’ perceptions, main barriers, and motivators related to implementing a nutrition program in Baltimore City
Authors: Song Hee‐Jung , Gittelsohn Joel , Kim Miyong , Anliker Jean , Suratkar Sonali , Sharma Sangita
Published: June 2021

There are over 25,000 Korean American food stores in the U.S, most in low‐income urban communities, making them a potential venue for nutrition interventions. However, such programs should be perceived to be feasible by storeowners. As part of the data collection for a store‐based intervention (Baltimore Healthy Stores), we examined motivators and barriers to the program participation and implementation, and perceived effectiveness of the intervention. In‐depth interviews with seven corner storeowners were conducted during the intervention. Detailed field notes by interventionists during the 10 month trial were also used to explore how the program was implemented at each store. A follow‐up survey with store owners was conducted to evaluate sustainability of the program. Stores were differentiated by ‘active involvement into activities’, ‘compliance with intervention protocol’ and ‘overall supportive atmosphere’. Stores which showed strong supports for the program by providing high traffic locations for interventionists, encouraging customers to participate in activities, and having good relationships with interventionists were more likely to perceive the program to be feasible leading to more sustainable promoted food stocks compared to the stores which showed weak support for the program. These findings could be used to develop successful corner store based nutrition interventions in urban communities.

Funded by USDA/FANRP and the Center for a Livable Future.
Consumption of three supplements with identical micronutrient content in Mexican children
Authors: Neufeld Lynnette M , Dominguez Clara P , Cabrera Amira Hernandez , Guerra Armando Garcia
Published: June 2021

The objective of this analysis was to compare consumption of three nutritional supplements with identical micronutrient content for children. Communities (18 per supplement) were randomly assigned to receive a fortified food (FF, 44 g of milk‐based powder dissolved in water to form a pap), syrup (SY, 5 ml) or Sprinkles (SK, 1 g dissolved in food). Children 6 to 12 mo of age, beneficiaries of the Oportunidades program were recruited. Supplements were delivered daily (6 mo) then weekly from recruitment to 24 mo of age. Compliance and consumed amount (0, ¼, ½, ¾, 1 doses) were recorded for the dose (SY) or the portion served (FF and SK). 927 children were assigned to treatment (FF n=265; SY n=323; SK n=339). There were no observed differences between groups at baseline. There was no difference (p>0.10) between groups in the mean proportion of days offered that the supplements were consumed (FF 85.8±13.4; SY 87.6±13.6; SK 88.9±10.5%). The proportion of days that the entire dose was consumed was significantly (p<0.05) higher in the SK (80.9±17.1%) than the FF (56.5±29.3%) and SY (67.8±24.6%) groups. Over the first 12 mo of the study, children in the SK group consumed significantly (p<0.01) more doses (225.6±54.2) than the FF (187.8±64.3) or SY (184.3±53.4) groups. Despite the controlled nature of this study, consumption of the entire dose varied considerably by group. Further analyses are needed to determine how this might relate to consumption of each in the context of a large scale program such as Oportunidades.

The Oportunidades program financed this study and holds the rites to the data presented.
Effect of three supplements with identical micronutrient content on language acquisition in Mexican children
Authors: Feregrino Raquel Garcia , Neufeld Lynnette M , Guerra Armando Garcia , Dominguez Clara P , Mejia Fabiola
Published: June 2021

The objective of this analysis was to assess the impact of three nutritional supplements with identical micronutrient content on language acquisition in children. Communities (18 per supplement) were randomly assigned to receive a fortified food (FF), syrup (SY) or Sprinkles (SK). Children 6 to 12 m of age, beneficiaries of the Oportunidades program were eligible to participate. Supplements were delivered daily (6 mo) then weekly and compliance recorded. Language acquisition was assessed with the McAuthur test which assessed both comprehension and expression and was applied to all children 9 mo of age and older and reported as number of words. Appropriate statistical analyses for cluster randomized trials were used. 727 children were recruited (FF n=214; SY n=246; SK n=267) of whom 358 were ≥9 m of age. After 4 m supplementation, 695 children were tested and we present results for all (cross‐sectional sample) and for change in language comprehension and expression on a longitudinal sub‐sample (n=288). There was no significant difference in language comprehension in either sample. In the sub‐sample, change in language expression was higher in the FF (p<0.05) and SK (p<0.10) groups than the SY group (FF: 7.4±12.1 SK: 6.7±7.8 SY: 5.0±5.1 words used). These results suggest that FF and SK may have provided greater benefit for language development than SY. However, there results should be considered preliminary until analysis are conducted after longer supplementation periods. The Oportunidades program financed this study and holds the rites to the data presented.