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Innate Immunity Reprogramming in Sepsis

East Tennessee State University. Innate Immunity Reprogramming in Sepsis. Mohamed Elgazzar, PhD Assistant Professor Internal Medicine. When Toll-like receptors (TLRs) sense a threat they signal innate cells such as neutrophils and macrophages to initiate the acute phase of inflammation

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Innate Immunity Reprogramming in Sepsis

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  1. East Tennessee State University Innate Immunity Reprogramming in Sepsis Mohamed Elgazzar, PhD Assistant Professor Internal Medicine

  2. When Toll-like receptors (TLRs) sense a threat they signal innate cells such as neutrophils and macrophages to initiate the acute phase of inflammation If the threat is limited, the inflammatory response resolves within hours If the threat is severe, the acute phase is replaced by cell reprogramming that sustains a chronic inflammatory phase Background

  3. Sepsis represents an uncontrolled immune response to exposure to microbes and microbial products, such as during a traumatic injury Reflects dysregulation of temporal sequence that normally protects against threats Develops into two contrasting phenotypes: SIRS & CARS SIRS is induced by bacterial infection or non-infectious causes such as trauma or major surgery Sepsis

  4. During SIRS- hyperinflammation characterized by excessive production of inflammatory mediators “cytokine storm,” damage to the vasculature, and hypotension, and if not reated early can result in vascular shock, organ dysfunction and death During CARS- hypoinflammation and immunosuppression characterized by down-regulation of inflammatory mediators due to tolerance of neutrophils and macrophages to bacterial toxins, significant apoptosis of lymphocytes and dendritic cells, and persistent primary and secondary infection Pathophysiology

  5. Sepsis phenotypes SIRS MARS CARS chronic phase acute phase (immunoactivation) (immunosuppression) • - anti-inflammatory cytokines • T-cell apoptosi • reduced antigen presentation • expansion of MDSCs Inflammation index - proinflammatory cytokines - decreased bacterial clearance 6 to 1 to 5 Time course changes in sepsis (days)

  6. baseline (infection/injury) Phenotypes of severe inflammation & sepsis outcomes mortality or survival activated (poised promoters) mild threat resolving (reversal of gene reprogramming) hyperinflammatory phase (cytokine storm) severe threat silenced hypoinflammatory phase

  7. Mortality rates are higher in humans and animals with chronic sepsis Treatment modalities targeting the hyperinflammatory phase (SIRS) were often effective in animal models but failed in human clinical trials; Reason: a delay between the onset of sepsis and the delivery of anti-inflammatory therapy when most patients enter the immunosuppressive (chronic) phase Clinical Significance

  8. Tolerance or hyporesponsiveness of innate cells to stimulation by bacterial toxins sustains immunosuppression and chronic infections We detected this phenotype in in vitro cell model of sepsis and in septic patients There is: an epigenetic component that silences transcription of inflammatory genes, a microRNA (miRNA) component that represses translation of these genes, and a cellular component manifested by disruption of myeloid cell development and expansion of MDSCs, and Mechanism

  9. Induction of endotoxin tolerance in THP-1 human monocyte cell model 140 120 100 80 relative expression (fold) 60 40 20 0 0 4 6 8 10 12 2 1st LPS 2nd LPS Responsive Tolerant RNA TNFa Protein 0 1 2 4 time in LPS (h)

  10. Modules of proinflammatory gene transcription silencing (the epigenetic component) K9 S10 p activated basal p50:p50 p65:p50 H3 me me K9 RelB ? silenced p50:p50 p65:RelB El Gazzar et al (2007); J Biol Chem

  11. Chromatin remodeling is a dynamic process in sepsis McCall & El Gazzar (2010); J Innate Immun

  12. Conclusions • NF-kB transcription factors, and DNA and histone based epigenetic processes cooperatively interact to silence proinflammatory gene expression during the systemic hyperinflammatory phase • Do interactions between epigenetic signals and transcription factors contribute to chromatin remodeling? • Although we can reverse the epigenetic-mediated transcription silencing of inflammatory genes, we cannot recover protein levels This Suggests an additional layer of (translational) repression

  13. MicroRNAs (miRNAs) are small (~22 nucleotide-long) non-coding RNAs that have emerged as key posttranscriptional regulators of gene expression In mammals, miRNAs are predicted to control ~30% of all protein-coding genes By base pairing to complementary AU-rich sequences in the 3`UTR region of the target mRNA, miRNAs mediate mRNA degradation or translational repression miRNA sequences and their predicted target genes can be analyzed using a number of prediction algorithms such as miRBase (http://microrna.sanger.ac.uk) and micoRNA targets (http://www.microrna.org) MicroRNA-dependent translation repression in sepsis

  14. microRNA biogenesis

  15. Model of translation repression of TNF in sepsis (The microRNA component) miR-221 miR-579 miR-125b LPS • miR-125b at 94-115 • miR-579 at 489-502 • miR-221 at 591-613 • miR-181a at 487-507 • ARE= 34-nt at 462-495 • CDE= 15-nt at 570-585 TLR4 NF-kB mi-RISC Ago2 2 AUF1 1 3 TTP TIAR repression AAAAAA 3` 5`Gppp ORF ARE CDE 5’UTR 3’UTR El Gazzar et al (2010); J Biol Chem

  16. Model of translation repression by microRNAs Tolerant p Responsive RBM4 RBM4 cytoplasm MKP-1 cytoplasm nucleus p Ago2 p-body Ago2 RBM4 RBM4 RBM4 cap AAAAA and/or eIF4A/4G AAAAA RBM4  AAAAA Ago2 RBM4 p translation AAAAA mRNA degradation translation arrest

  17. We discovered the epigenetic and microRNA codes that sustain chronic sepsis, by repressing proinflammatory gene expression We can reverse the epigenetic and miRNA-based gene repression program This is clinically significant because reversing gene repression correlates with resolution of sepsis and survival: patients who survive late sepsis exhibit innate cell competency and inflammatory gene activation Conclusions …

  18. Inflammation-induced reprogramming (i.e., during SIRS) of innate cells may underlie the development of the hyporesponsive/immunosuppessive state Evidence supports expansion of bone marrow progenitor cell populations during inflammation We hypothesize that the initial hyperinflammatory (acute) phase of sepsis induces reprogramming of innate cell differentiation and/or maturation which may sustain immunosuppression and the chronic sepsis phenotype. Hypothesis

  19. Adoptive transfer of CD34+ hematopoietic progenitors improves late sepsis survival Sham (n=20) CLP (n=20) CLP + vehicle control (n=25) CLP + CD34+ cells (n=30) 100 80 60 % survival 40 20 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Days post CLP Brudecki et al (2011); Infect Immunity

  20. 1400 1200 800 1000 * 800 * 600 IL-6 (pg/ml) 600 400 400 TNFa (pg/ml) 200 200 0 Sham CLP CLP + 0 CD34 Sham CLP CLP + CD34 800 600 400 200 1400 1200 0 1000 Sham CLP CLP + CD34 800 IL-6 (pg/ml) 600 400 200 0 Sham CLP CLP + CD34 Circulating levels of proinflammatory cytokines days 2-4 (acute phase) * * days 14-16 (chronic phase) TNFa (pg/ml) Resolved inflammation

  21. 2000 1500 1000 500 0 Sham CLP CLP + CD34 4000 3000 IL-6 (pg/ml) 2000 * 1000 2000 * 4000 0 1500 3000 Sham CLP CLP + 1000 IL-6 (pg/ml) CD34 2000 500 1000 0 0 Sham CLP CLP + Sham CLP CLP + CD34 CD34 Peritoneal macrophages from chronically septic mice reconstituted with CD34+ cells have normal immune rsponse days 2-4 TNFa (pg/ml) days 14-16 TNFa (pg/ml) Ex vivo stimulation

  22. 4.0 4.0 108 108 ´ ´ 600 3.0 3.0 108 108 ´ ´ CFU/mouse CFU/mouse 400 2.0 2.0 108 108 ´ ´ CFU/1 ml 1.0 1.0 108 108 ´ ´ 200 0 0 0 CLP CLP CLP + CD34 CLP + CD34 600 *p=0.001 400 CFU/1 ml 200 0 CLP CLP CLP + CD34 CLP + CD34 CD34+ cells enhance bacterial clearance in chronically septic mice Bacterial load days 2-4 Peritoneum Blood days 14-16

  23. CD34+ cells improve bacterial phagocytic activity of innate cells in chronically septic mice A 120 120 100 100 80 mean fluorescence 80 (585 nm) counts mean fluoresence 60 (585 nm) 60 40 0 1 2 3 4 10 10 10 10 10 40 20 20 0 0 CLP CLP + CLP CLP + CD34 CD34 Days 2-4 Fluorescein-conjugatedE. coli emission (585 nm) 120 120 100 0 1 2 3 4 10 10 10 10 10 100 80 * mean fluorescence 80 (585 nm) 60 mean fluoresence 60 (585 nm) 40 40 20 20 0 1 2 3 4 Days 14-16 10 10 10 10 10 0 0 CLP CLP + CLP CLP + CD34 CD34 0 1 2 3 4 10 10 10 10 10 0 1 2 3 4 10 10 10 10 10 0 1 2 3 4 10 10 10 10 10 CLP CLP + CD34 CLP CLP + CD34 0 1 2 3 4 10 10 10 10 10 0 1 2 3 4 10 10 10 10 10 CLP CLP + CD34 CLP CLP + CD34 B Phagocytic activity Macrophages Neutrophils days 2-4 days 14-16 *

  24. bone marrow Peritoneum Spleen Day 2 Day 5 CD34+ cell-derivatives home to sites of inflammation

  25. The initial hyperinflammatory (acute) phase of sepsis reprograms innate cell differentiation and/or maturation to initiate and sustain immunosuppression and chronic inflammation These processes may be linked to inflammation-driven myelopoiesis Conclusions

  26. MDSCs expand in BM, spleen, lymph nodes in nearly all inflammatory conditions They are a mixed population that includes progenitors of macrophages, plymorphonuclear and dendritic cells In mouse, they are phenotyped as GR1+ CD11b+ myeloid cells. In human, they are CD33+ CD11b+ cells They are potently immunosuppressive, affecting innate and adaptive immunity In tumor-bearing animals and human, their elimination improve anti-tumor immunity Myeloid-derived suppressor cells (MDSCs) underlie chronic sepsis pathogenesis

  27. 100 80 Gr1+ CD11b+ cells (%) 60 40 20 0 0 3 6 12 Days post CLP 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 Gr1-FITC 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 CD11b-PE Dramatic expansion of Gr1+ CD11b+ MDSCs cells in late sepsis A day 0 day 3 day 6 day 12 B B C day 3 day 6 * *

  28. saline (n=20) MDSCs from D3 (n=30) 100 MDSCs from D12 (n=35) 80 60 % survival 40 20 0 0 2 4 6 8 10 12 14 16 18 20 22 Days post CLP MDSCs can enhance or attenuate the systemic inflammatory response

  29. Total MDSCs CD31+-enriched MDSCs Grr1-FITC F4/80-APC CD11c-PE CD11c-PE CD11c+-PE CD11c+-PE F4/80-APC F4/80-APC F4/80-APC MHC II-FITC MHC II-FITC CD11b-PE CD11b-PE CD11b-PE MHC II-FITC MHC II-FITC CD11b-PE CD11b-PE 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 MDSCs from chronically septic mice lose differentiation potential A B Day3 Day 12

  30. Pathway of hematopoietic stem cell differentiation and development of innate cell repertoire Monocyte MP normal normal normal LT-HSC ST-HSC MPP CMP GMP Granulocyte GP Monocyte MP septic septic GMP LT-HSC ST-HSC MPP CMP Granulocyte GP Immature monocyte MP GMP Immature granulocyte GP

  31. Gfi1+ Gfi1+ monocyte miR-21+ miR-181+ miR-21+ miR-181+ miR-21+ miR-181+ normal granulocyte MPP CMP GMP dendritic cell monocyte miR-21+++ miR-181+++ miR-21+++ miR-181+++ miR-21b+++ miR-181+++ sepsis granulocyte Immature MDSC MPP CMP dendritic cell MiRNAs disrupt myeloid cell repertoire during sepsis Fig. 9.Depicts disruption of myeloid cell repertoire by miR-21 and miR-181b

  32. The initial hyperinflammatory (acute) phase of sepsis reprograms innate cell differentiation and/or maturation to initiate and sustain immunosuppression and chronic inflammation Expansion of Gr1+ CD11b+ myeloid-derived suppressor cells (MDSCs) may underlie the immunosuppression in chronic sepsis MDSC expansion in sepsis is a programmed response to inflammation, regardless of its sources microRNAs are likely to play a role in this sepsis-induced innate immunity cell reprogramming and MDSC expansion Conclusions and directions…

  33. LABCOLLABORATORS Laura Brudecki Charles McCall, MD Research AssistantWake Forest University Jessica Jordan (PhD student) Benjamin Garcia, PhD Keeley Haggard (undergrad.) Princeton University Donald Feruson, PhD ETSU Acknowledgement

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