PaperPlayer biorxiv immunology

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.14.246132v1?rss=1

Authors: Hollingsworth, L. R., Sharif, H., Griswold, A. R., Fontana, P., Mintseris, J., Dagbay, K. B., Paulo, J. A., Gygi, S. P., Bachovchin, D. A., Wu, H.

Abstract:

NLRP1 is a cytosolic inflammasome sensor that mediates activation of caspase-1, which in turn induces cytokine maturation and pyroptotic cell death. Gain-of-function NLPR1 mutations cause skin inflammatory diseases including carcinoma, keratosis, and papillomatosis. NLRP1 contains a unique function-to-find domain (FIIND) that autoproteolyzes into noncovalently associated subdomains. Proteasomal degradation of the autoinhibitory N-terminal fragment (NT) activates NLRP1 by releasing the inflammatory C-terminal fragment (CT). Cytosolic dipeptidyl peptidases 8 and 9 (DPP8/9) interact with NLRP1, and small-molecule DPP8/9 inhibitors activate NLRP1 by poorly characterized mechanisms. Here, we report cryo-EM structures of the human NLRP1-DPP9 complex, alone and in complex with the DPP8/9 inhibitor Val-boroPro (VbP). Surprisingly, the NLRP1-DPP9 complex is a ternary complex comprised of DPP9, one intact FIIND of a non-degraded full-length NLRP1 (NLRP1-FL) and one NLRP1-CT freed by NT degradation. The N-terminus of the NLRP1-CT unfolds and inserts into the DPP9 active site but is not cleaved by DPP9, and this binding is disrupted by VbP. Structure-based mutagenesis reveals that the binding of NLRP1-CT to DPP9 requires NLRP1-FL and vice versa, and inflammasome activation by ectopic NLRP1-CT expression is rescued by co-expressing autoproteolysis-deficient NLRP1-FL. Collectively, these data indicate that DPP9 functions as a "bomb-diffuser" to prevent NLRP1-CTs from inducing inflammation during homeostatic protein turnover.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.13.249441v1?rss=1

Authors: Ayala, M. V., Bonaud, A., Bender, S., Lambert, J.-M., Lechouane, F., Carrion, C., Cogne, M., Pascal, V., Sirac, C.

Abstract:

Plasma cells (PC) represent the last stage of B cell development and are mainly characterized by their capacity of secreting large quantities of antibodies. They can be implicated in a broad-spectrum of neoplastic disorders, including Multiple Myeloma, Waldenstrom macroglobulinemia or Monoclonal Gammopathy of Clinical Significance, all characterized by the abnormal proliferation of a PC clone. Up to date, there are only few reporter models to specifically follow PC development, migration and homing in mouse and none allowing the genetic manipulation of these cells. We created a transgenic mouse model in which a green fluorescent protein gene was placed under the control of the well-characterized regulatory elements of the murine immunoglobulin J (IgJ) chain locus. Thanks to this model, we demostrated that IgJ is an early and specific marker of antibody secreting cells (ASCs) and appears before the expression of CD138, making it a good candidate to targeted genetic modifications of plasma cells. Therefore, a conditional deletion model using a Tamoxifen-dependent Cre recombinase inserted into the IgJ locus was characterized. Using a reporter model, we showed that, in contrast with existing models of B cell lineage genetic modification, the activity of the CRE recombinase only affects ASCs after tamoxifen treatment. Additionally, we used this model in a functional in vitro assay, to show that Ig modifications directly affect plasma cell survival. These two new mouse models, IgJGFP and IgJCreERT2 represent exquisite tools to study PCs. In pathology, the IgJCreERT2model opens new frontiers for in vivo genetic modifications of PCs to better reflect the pathophysiology of PC-related diseases.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.12.248450v1?rss=1

Authors: Braudeau, C., Salabert-Le Guen, N., Chevreuil, J., Rimbert, M., Martin, J. C., Josien, R.

Abstract:

Background: Immune profiling by flow cytometry is not always possible on fresh blood samples due to time and/or transport constraints. Besides, the cryopreservation of peripheral blood mononuclear cells (PBMC) requires on-site specialized lab facilities, thus severely restricting the extent by which blood immune monitoring can be applied to multicenter clinical studies. These major limitations can be addressed through the development of simplified whole blood freezing methods. Methods: In this report, we describe an optimized easy protocol for rapid whole blood freezing with the CryoStor CS10 solution. Using flow cytometry, we compared cellular viability and composition on cryopreserved whole blood samples to matched fresh blood, as well as fresh and frozen PBMC. Results: Though partial loss of neutrophils was observed, leucocyte viability was routinely >75% and we verified the preservation of viable T cells, NK cells, monocytes, dendritic cells and eosinophils in frequencies similar to those observed in fresh samples. A moderate decrease in B cell frequencies was observed. Importantly, we validated the possibility to analyze major intracellular markers, such as FOXP3 and Helios in regulatory T cells. Finally, we demonstrated good functional preservation of CS10-cryopreserved cells through the analysis of intracellular cytokine production in ex vivo stimulated T cells (IFNg, IL-4, IL-17A,) and monocytes (IL-1b, IL-6, TNFa). Conclusions: In conclusion, our protocol provides a robust method to apply reliable immune monitoring studies to cryopreserved whole blood samples, hence offering new important opportunities for the design of future multicenter clinical trials.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.13.250241v1?rss=1

Authors: Chai, J., Zhong, F. L., Huang, M., zhang, X., Wang, J., Wu, B., Han, Z., Ann, T. G., Gong, Q.

Abstract:

The nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)-containing receptors (NLRs) mediate innate immunity by forming inflammasomes. Activation of the NLR protein NLRP1 requires auto-cleavage within its FIIND domain1-7. In resting cells, the dipeptidyl peptidase DPP9 interacts with NLRP1-FIIND and together with a related enzyme DPP8, suppresses spontaneous NLRP1 activation8,9. The mechanisms of DPP8/9-mediated NLRP1 inhibition, however, remain elusive. Here we provide structural and biochemical evidence demonstrating that rat NLRP1 (rNLRP1) interacts with rDPP9 in a stepwise manner to form a 2:1 complex. An auto-inhibited rNLRP1 molecule first interacts with rDPP9 via its ZU5 domain. This 1:1 rNLRP1-rDPP9 complex then captures the UPA domain of a second rNLRP1 molecule via a UPA-interacting site on DPP9 and dimeric UPA-UPA interactions with the first rNLRP1. The 2:1 rNLRP1-rDPP9 complex prevents NLRP1 UPA-mediated higher order oligomerization and maintains NLRP1 in the auto-inhibited state. Structure-guided biochemical and functional assays show that both NLRP1-binding and its enzymatic activity are required for DPP9 to suppress NLRP1, supporting guard-type activation of the NLR. Together, our data reveal the mechanism of DPP9-mediated inhibition of NLRP1 and shed light on activation of the NLRP1 inflammasome.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.13.249243v1?rss=1

Authors: Gugsa, G., Bifo, H., Kifleyohannes, T., Abebe, E., Ahmed, M.

Abstract:

Bovine brucellosis is an infectious bacterial disease caused by members of genus Brucella, affecting both animals and humans, and resulting in a serious economic loss in animal production sector and deterioration of public health. This cross-sectional study was conducted from November 2014 to April 2015 to determine the sero-prevalence and associated risk factors of bovine brucellosis in Sendafa, Oromia special Zone, Ethiopia. A total of 503 blood samples were collected using simple random sampling technique from dairy cattle of above 6 months of age with no history of previous vaccination against brucellosis. All sera samples were demonstrated using both Rose Bengal Plate Test for screening and Compliment Fixation Test for confirmation. Accordingly, the overall sero-prevalence of bovine brucellosis in this area was 0.40%. The result showed that the sero-prevalence of bovine brucellosis in the study area was not statistically significant with all proposed risk factors. Thus, the study revealed the absence of significant statistical variation in the sero-prevalence of bovine brucellosis in different age, sex groups, breeding method and history of previous abortions (P>0.05). No reactors were observed in male animals. Sero-prevalence of 0.40% was observed in animals without previous history of abortion. Moreover, information was gathered on individual animal and farm-level risk factors and other farm characteristics using a questionnaire. The awareness among the society was poor, so the positive animals can be a potential hazard to animals and humans in the study area. Therefore, public education should be done in order to improve the awareness of people on bovine brucellosis and its public health impact with due consideration on the safely consumption of food of animal origin.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.12.248021v1?rss=1

Authors: Noon, J. B., Sharma, A., Platten, J., Quinton, L. J., Reinhardt, C., Bosmann, M.

Abstract:

Interleukin-27 (IL-27) is a heterodimeric cytokine of the IL-12 family, formed by non-covalent association of the promiscuous EBI3 subunit and selective p28 subunit. IL-27 is produced by mononuclear phagocytes and unfolds pleiotropic immune-modulatory functions through high affinity ligation to IL-27 receptor alpha (IL-27RA). While IL-27 is known to contribute to immunity and to end inflammation following numerous types of infections, its relevance for host defense against multicellular parasites is still poorly defined. Here, we investigated the role of IL-27 during infection with the soil-transmitted hookworm, Nippostrongylus brasiliensis, in its early intrapulmonary life cycle. IL-27(p28) was detectable in broncho-alveolar lavage fluids of C57BL/6J wild type mice on day 1 after subcutaneous N. brasiliensis inoculation. The expression of IL-27RA was most abundant on lung invading {gamma}{delta} T cells followed by CD8+ T cells, CD4+ T cells and NK cells. IL-27RA was weakly present on CD19+ B cells and absent on neutrophils, alveolar macrophages and eosinophils. Il27ra-/- mice showed increased parasite burden together with aggravated pulmonary hemorrhage and higher alveolar albumin leakage as a surrogate for disruption of the epithelial/vascular barrier. Conversely, recombinant mouse IL-27 injections of wild type mice reduced parasite burdens and lung injury. In multiplex screens, we identified higher airway accumulations of IL-6, TNF and MCP-3 (CCL7) in Il27ra-/- mice, while rmIL-27 treatment showed a reciprocal effect. Finally, {gamma}{delta} T cell infiltration of the airways required endogenous IL-27 expression. In summary, this report demonstrates protective functions of IL-27 to control the early larval stage of hookworm infection in the lungs.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.12.248252v1?rss=1

Authors: Kiritsy, M. C., Ankley, L. M., Trombley, J. D., Huizinga, G. P., Lord, A. E., Orning, P., Elling, R., Fitzgerald, K. A., Olive, A.

Abstract:

Cytokine-mediated activation of host immunity is central to the control of pathogens. A key cytokine in protective immunity is interferon-gamma (IFN{gamma}), which is a potent activator of antimicrobial and immunomodulatory effectors within the host. A major role of IFN{gamma} is to induce major histocompatibility complex class II molecules (MHCII) on the surface of cells, which is required for CD4+ T cell activation. Despite its central role in host immunity, the complex and dynamic regulation of IFN{gamma}-induced MHCII is not well understood. Here, we integrated functional genomics and transcriptomics to comprehensively define the genetic control of IFN{gamma}-mediated MHCII surface expression in macrophages. Using a genome-wide CRISPR-Cas9 library we identified genes that control MHCII surface expression, many of which have yet to be associated with MHCII. Mechanistic studies uncovered two parallel pathways of IFN{gamma}-mediated MHCII control that require the multifunctional glycogen synthase kinase 3 beta (GSK3{beta}) or the mediator complex subunit MED16. Both pathways are necessary for IFN{gamma}-mediated induction of the MHCII transactivator CIITA, MHCII expression, and CD4+ T cell activation. Using transcriptomic analysis, we defined the regulons controlled by GSK3{beta} and MED16 in the presence and absence of IFN{gamma} and identified unique networks of the IFN{gamma}-mediated transcriptional landscape that are controlled by each gene. Our analysis suggests GSK3{beta} and MED16 control distinct aspects of the IFN{gamma}-response and are critical for macrophages to respond appropriately to IFN{gamma}. Our results define previously unappreciated regulation of MHCII expression that is required to control CD4+ T cell responses by macrophages. These discoveries will aid in our basic understanding of macrophage-mediated immunity and will shed light on mechanisms of failed adaptive responses pervasive in infectious disease, autoimmunity, and cancer.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.12.247510v1?rss=1

Authors: Agerholm, R., Rizk, J., Kadekar, D., Borch, A., Hadrup, S. R., Bekiaris, V.

Abstract:

The vitamin A metabolite retinoic acid (RA) and its receptor (RAR) are one of the key interactions regulating cellular immunity and neural signaling. Whether endogenous RA-RAR interactions contribute to the development of neuroinflammation and diseases like multiple sclerosis, remains to be elucidated. Herein, we used the murine experimental autoimmune encephalomyelitis (EAE) model and an established genetic RAR silencing approach to decipher its role in pathogenic T cell responses. We show that RAR is necessary for the development of interleukin(IL)-17-driven, cell-mediated immunopathology in the brain and that it fine tunes the homeostasis of IL-17-producing gamma delta ({gamma}{delta}T17) and CD4+ T cells (TH17). At steady-state, RAR was required in the {gamma}{delta}T17 compartment to sustain optimal cell numbers and maintain expression of genes involved in cell cycle progression. In contrast, RAR negatively regulated T helper-17 (TH17) cell homeostasis. Our data show that RAR is required during the early phases of EAE in order to induce a {gamma}{delta}T17 response and that its activity is necessary throughout the course of the disease to allow TH17 and {gamma}{delta}T17 cells to infiltrate the brain. This is correlated with failure of RAR deficient cells to express surface integrin-alpha4, a major brain homing molecule. Collectively, our work demonstrates that endogenous RA-RAR interactions are important for the homeostasis of IL-17-producing T cells and necessary for their pathogenicity during neuroinflammation.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.12.247965v1?rss=1

Authors: Vincent, C. M., Dionne, M. S.

Abstract:

Male and female animals exhibit differences in infection outcomes. One possible source of sexually dimorphic immunity is sex-specific costs of immune activity or pathology, but little is known about the independent effects of immune-induced versus microbe-induced pathology, and whether these may differ for the sexes. Here, through measuring metabolic and physiological outputs in wild-type and immune-compromised Drosophila melanogaster, we test whether the sexes are differentially impacted by these various sources of pathology. We find that in the fruit fly, the sexes exhibit differential immune activity but similar bacteria-derived metabolic pathology. We show that female-specific immune-inducible expression of PGRP-LB, a negative regulator of the Imd pathway, enables females to reduce immune activity in response to reductions in bacterial numbers. In the absence of this regulator, females are more resistant of infection, indicating that female-biased immune restriction comes at a cost. These data suggest that male and female flies exhibit broadly similar abilities to resist infection, but females exhibit greater tolerance of infection as a result of increased immune regulation.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.11.247395v1?rss=1

Authors: Walls, A. C., Fiala, B., Schaefer, A., Wrenn, S., Pham, M. N., Murphy, M., Tse, L. V., Shehata, L., O'Connor, M. A., Chen, C., Navarro, M. J., Miranda, M. C., Pettie, D., Ravichandran, R., Kraft, J. C., Ogohara, C., Palser, A., Chalk, S., Lee, E.-C., Kepl, E., Chow, C. M., Sydeman, C., Hodge, E. A., Brown, B., Fuller, J. T., Dinnon, K., Gralinski, L., Leist, S. R., Gully, K. L., Lewis, T. B., Guttman, M., Chu, H. Y., Lee, K. K., Fuller, D. H., Baric, R. S., Kellam, P., Carter, L., Pepper, M., Sheahan, T. P., Veesler, D., King, N. P.

Abstract:

A safe, effective, and scalable vaccine is urgently needed to halt the ongoing SARS-CoV-2 pandemic. Here, we describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 copies of the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) in a highly immunogenic array and induce neutralizing antibody titers roughly ten-fold higher than the prefusion-stabilized S ectodomain trimer despite a more than five-fold lower dose. Antibodies elicited by the nanoparticle immunogens target multiple distinct epitopes on the RBD, suggesting that they may not be easily susceptible to escape mutations, and exhibit a significantly lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the protein components and assembled nanoparticles, especially compared to the SARS-CoV-2 prefusion-stabilized S trimer, suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms for inducing potent neutralizing antibody responses and have launched cGMP manufacturing efforts to advance the lead RBD nanoparticle vaccine into the clinic.

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