20241003Lecture9.pdf

Investigating the role of MRGPRX2/b2 in S. aureus induced atopic dermatitis Hannah Dychtenberg MSc Student Pundir Lab 1 What is Atopic Dermatitis (AD)? Chronic inflammatory skin disease Commonly called eczema Complex etiology: microbial...

Investigating the role of MRGPRX2/b2 in S. aureus induced atopic dermatitis Hannah Dychtenberg MSc Student Pundir Lab 1 What is Atopic Dermatitis (AD)? Chronic inflammatory skin disease Commonly called eczema Complex etiology: microbial dysbiosis, genetic mutations, familial history, skin barrier disruption, immune dysregulation, allergen exposure 2 https://nationaleczema.org/eczema/types-of-eczema/atopic-dermatitis/ https://www.westminsterclinic.ae/blog/atopic-dermatitis/ Prevalence & Epidemiology AD impacts approximately 20% of children and up to 10% of adults AD places the highest burden on children, females, and those living in high-income countries 3 Urban et al., JAAD International, 2021 The Burden of Atopic Dermatitis 4 Silverberg, Annals of Allergy, Asthma & Immunology, 2019 Skin Microbial Dysbiosis in Atopic Dermatitis Skin barrier disruption 5 Eun Kim & Sung Kim, Journal of Clinical Medicine, 2019 Mast Cells Mast cells are innate immune cells derived from multipotent hematopoietic stem cells (HSC) in the bone marrow Belong to the myeloid lineage of immune cells Mast cells are granulocytes! Taken by Dr. Mariana Castells 6 IgE Mediated Mast Cell Activation 7 Created with BioRender.com & https://www.dreamstime.com/vasoconstriction-vasodilatation-image271553036 Mast Cells in Pathogen Recognition In addition to their role in inflammation and allergies, mast cells play a critical role in pathogen detection & surveillance Localize in barrier tissues (respiratory tract, tongue, skin, gastrointestinal tract, meninges, etc.) Spatial and temporal advantage over other leukocytes 8 Marshall, Nature Reviews Immunology, 2004 MRGPRX2/b2 Mediated Mast Cell Activation Human connective tissue mast cells express MRGPRX2 Mice express a homolog, Mrgprb2 9 Pundir et al., Cell Host & Microbe, 2019 MRGPRX2/b2 Detect Bacterial Ligands MRGPRX2/b2 are receptors for cationic (positively charged) ligands Non-immunologic mast cell activation (IgE-independent) 10 Created with BioRender.com MRGPRX2 Activation 11 Pundir et al., Cell Host & Microbe, 2019 The Interplay between Mast Cells and S. aureus in Atopic Dermatitis S. aureus produces δ-toxin which activates mast cells and induces atopic dermatitis-like skin inflammation in mice 12 Created with BioRender.com My Project… We know that mast cells play a role in S. aureus induced atopic dermatitis-like skin inflammation Mast cells express the receptors MRGPRX2/b2 which Gram-positive bacteria can activate to induce a proinflammatory response We hypothesize that S. aureus skin colonization in AD leads to excessive inflammation mediated by MRGPRX2/b2 13 Skin Anatomy 14 Image from https://www.britannica.com/science/human-skin Methods: Epicutaneous Sensitization 15 Created with BioRender.com Methods: Intradermal Injection 16 Created with BioRender.com Mouse Trial Results Preliminary results are conflicting: in my previous trial, Mrgprb2- wildtype mice exhibited a more severe phenotype than Mrgprb2- mutant mice In my most recent trial, Mrgprb2-mutant mice exhibited a more severe phenotype The main issue: the mice are taking the Tegaderm off prior to day 7 The mice need to be infected with bacteria for the same duration to accurately compare them! 17 Infected Mouse 18 Significance Painful, itchy skin Leveraging the interaction between Impaired Depression MRGPRX2/b2 and S. aureus will pave the sleep way for the design of more targeted AD therapeutics Atopic The ultimate goal is to reduce the side Dermatitis effects of current therapeutics and improve overall quality of life Social Missed isolation work/school Financial burden 19 Checkpoint Questions 1. What are the two primary modes of mast cell degranulation? How are they similar/different? 2. Explain the role of the host-microbiota cross-talk in atopic dermatitis pathogenesis 3. How does atopic dermatitis impact overall quality of life? 20 The role of MRGPRs in infected wound healing through the neuroimmune axis Colin Guth Pundir Lab October 3rd, 2024 21 Antibiotic Resistance: A Global Crisis Antibiotic resistance is rapidly increasing Deaths due to antibiotic resistant organisms could reach 10 million by 2050 Novel therapies to fight this threat are desperately needed O’Neil 2016 22 Wound healing significance Surgical site infections in the US cost $3.3 billion annually Diabetic chronic wounds and foot ulcers affect up to 26.1 million worldwide Impact of infection can only be reduced with antibiotics Sourced from Phoenician Foot and Ankle Specialists 23 Mast Cells (MCs): Sentinels of the Body Tissue-resident cells found at host-environment junctions Possess a broad repertoire Gaudenzio et al., 2022. Unpublished of receptors to detect a variety of molecules Activation occurs in seconds to minutes Well known for roles in allergy but also play a crucial role in infection Generated with BioRender 24 MRGPRX2/ Mrgprb2 Expression and Activation Found exclusively in connective tissue mast cells Mast Cells Mrgprb2 (CTMCs) Adapted from McNeil et al., 2015 Activated by a variety of cationic compounds Mediates susceptibility to bacterial infection Generated with BioRender 25  Mast Cells Inhibit Biofilm Formation Through MRGPRX2 Bacteria Bacteria Bacteria Alone + + Mast Cells Mast Cells Without MRGPRX2 Adapted from Pundir et al.,262019  Uninjured Injured Catestatin: A Multi-Faceted Neuroendocrine AMP Cationic cleavage product of chromogranin A (ChgA) Adapted from Radek et al., 2008 Induced in the skin upon injury Antimicrobial and immunomodulatory properties MRGPRX2 activator? Wildtype Catestatin-KO Adapted from Radek et al., 2010 27 28 29 Hypothesis: MRGPRX2-dependent activation of mast cells plays a pivotal role in the healing process of wounds infected with bacteria and healing can be improved by activation with catestatin. 30  Determine if catestatin activates MCs through the MRGPRX2/Mrgprb2 receptor Objectives Determine the effect of topical application of catestatin to bacterially infected wounds 31  Generated with BioRender Objective 1 Cell Types 32  Generated with BioRender Objective 1 Workflow 33  LAD2 WT LAD2 MRGPRX2-KO ✱ ✱ 500 ✱ Catestatin 400 Histamine (ng/ml) induces MC 300 histamine release through 200 MRGPRX2 100 0 0 1.25 2.5 5 10 48/80 CST (uM)  LAD2 WT LAD2 MRGPRX2-KO 400 ✱ ✱ Prostaglandin D2 (pg/ml) Catestatin 300 induces MC PGD2 release 200 through MRGPRX2 100 0 Buffer CST 48/80 Catestatin induces MC cytokine/chemokine gene expression through MRGPRX2 36 So, CST induces robust activation of human mast cells via MRGPRX2 in vitro. How does this apply in vivo? 37  Generated with BioRender Objective 1 Mouse Lines 38  Generated with BioRender Evan’s Blue Assay 39  Catestatin Induces Inflammation in WT But Not Mrgprb2-Deficient Mice Dye Leakage 40  Generated with BioRender Catestatin-Treated Wound Infections 41 Catestatin Reduces Bacterial Load in MRSA Infected Wounds in an Mrgprb2-Dependent Manner Bacterial Load 42 43 Exploring Therapeutics and Filling a Gap in Our Knowledge The threat of AMR needs to be addressed immediately Potential to significantly worsen wound healing impact Catestatin is a potential antibiotic alternative Activation of MCs through MRGPRX2 could be a promising immunomodulatory therapy O’Neil 2016 44 Checkpoint Questions 1. Mast cells are often referred to as ‘sentinels’. List three characteristics which makes this a fitting description. 2. What bacterial molecules do MRGPRX2/Mrgprb2 detect to induce mast cell activation? 3. What are some advantages of using therapeutics which enhance the body’s immune response against infections to treat infections over conventional antibiotics with direct antimicrobial effects? 45 References McNeil BD, Pundir P, Meeker S, Han L, Undem BJ, Kulka M, Dong X. 2015. Identification of a mast-cell-specific receptor crucial for pseudo-allergic drug reactions. Nature 519:237–241. Pundir P, Liu R, Vasavda C, Serhan N, Limjunyawong N, Yee R, Zhan Y, Dong X, Wu X, Zhang Y, Snyder SH, Gaudenzio N, Vidal JE, Dong X. 2019. A Connective Tissue Mast-Cell-Specific Receptor Detects Bacterial Quorum-Sensing Molecules and Mediates Antibacterial Immunity. Cell Host Microbe 26:114-122.e8. Radek KA, Lopez-Garcia B, Hupe M, Niesman IR, Elias PM, Taupenot L, Mahata SK, O’Connor DT, Gallo RL. 2008. The neuroendocrine peptide catestatin is a cutaneous antimicrobial and induced in the skin after injury. Journal of Investigative Dermatology 128:1525–1534. Radek KA, Elias PM, Taupenot L, Mahata SK, O’Connor DT, Gallo RL. 2010. Neuroendocrine nicotinic receptor activation increases susceptibility to bacterial infections by suppressing antimicrobial peptide production. Cell Host Microbe 7:277–289. 46 Staphylococcus aureus: Virulence and immune evasion Ruina Bao MICR* 3230 Immunology Oct 3, 2024 Key concepts for today Ø S. aureus pathogenesis Ø Host response to infection Ø Virulence factors: Cell wall anchored proteins and toxins Ø Strategies to evade the host immune response 48  S. aureus: Facultative pathogen 49 Naghavi et al. The Lancet 2024  S. aureus: Facultative pathogen Pathobiont Community-acquired and nosocomial pathogen 50 Piewngam et al.. Lancet Microbe 2024  S. aureus: Facultative pathogen Wide range of diseases 51 Berry et al. Ann N Y Acad Sci 2022  Host defences against S. aureus DeLeo et al. Infect Dis Clin North Am 2010 52 Lee et al. Nat Rev Dis Primers 2018 Adhesion to the host mediated by CWA proteins nasal vestibule Berry et al. Ann N Y Acad Sci 2022 53 Lee et al. Nat Rev Dis Primers 2018  Other functions of CWA proteins: Spa 54 Howden et al. Nat Rev Microbiol 2023 Using an ELISA based method to detect S. aureus adhesion Quench YYY YY Y Y YYYY YYY YY YYY Y YY YYYY Y Y Y Y Y YY Y YY Leonard et al., Manuscript in preparation Petrie and Leonard et al., 2019 55  Virulence factors: Toxins Protein/Molecule Function Toxic shock syndrome Activates T- toxin-1, TSST1 cells Staphylococcal Activate T- enterotoxins cells Leukocidins Causes leukocyte lysis Alpha-hemolysin (α- Causes host hemolysin) cell lysis, compromise Fig. 6.25 physical barriers e.g. 56 epithelial cells  Evade the immune system Interference with complement activation Fig 13. 22 57  Evade the immune system Staphylococcal complement inhibitor (SCIN) stabilizes inactive C3 convertase Fig 13. 22 58  Evade the immune system: Fibrin shield ClfA proteins bind to host structural proteins to promote adhesion Shield MAMPs by coating the bacterium with host proteins to block detection of peptidoglycan 59 Berry et al. Ann N Y Acad Sci 2022  Putting it all together: Bacteremia How S. aureus invades host cells and evades the host immune system 60 Berry et al. Ann N Y Acad Sci 2022 Checkpoint questions 1. Why might a patient be given mupirocin, ahead of surgery? 2. What distinguishes a superantigen and what does it lead to the production of? 3. Describe two examples of how S. aureus may evade the host immune system. 61