East Tennessee State University Innate Immunity Reprogramming in Sepsis Mohamed Elgazzar, PhD Assistant Professor Internal Medicine Background 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 Sepsis 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 Pathophysiology 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 Sepsis phenotypes SIRS MARS CARS acute phase chronic phase (immunoactivation) (immunosuppression) Inflammation index - anti-inflammatory cytokines -T-cell apoptosi -reduced antigen presentation -expansion of MDSCs - proinflammatory cytokines - decreased bacterial clearance 1 to 5 6 to Time course changes in sepsis (days) Phenotypes of severe inflammation & sepsis baseline (infection/injury) outcomes mortality or survival mild threat activated (poised promoters) resolving hyperinflammatory phase (reversal of gene reprogramming) (cytokine storm) silenced hypoinflammatory phase severe threat Clinical Significance 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 Mechanism 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: 1. an epigenetic component that silences transcription of inflammatory genes, 2. a microRNA (miRNA) component that represses translation of these genes, and 3. a cellular component manifested by disruption of myeloid cell development and expansion of MDSCs, and Induction of endotoxin tolerance in THP-1 human monocyte cell model 1st LPS 2nd LPS relative expression (fold) Responsive 140 120 100 80 60 40 20 0 Tolerant RNA Protein 0 2 4 6 8 10 12 0 time in LPS (h) 1 2 4 TNFa Modules of proinflammatory gene transcription silencing (the epigenetic component) K9 me S10 p activated basal H3 p50:p50 p65:p50 K9 me RelB ? silenced p50:p50 p65:RelB El Gazzar et al (2007); J Biol Chem Chromatin remodeling is a dynamic process in sepsis McCall & El Gazzar (2010); J Innate Immun 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 MicroRNA-dependent translation repression in sepsis 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 biogenesis Model of translation repression of TNFa in sepsis (The microRNA component) LPS TLR4 NF-kB -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 mi-RISC Ago2 2 AUF1 TTP miR-221 3 miR-125b 1 miR-579 AAAAAA 3` 5`Gppp ORF 5’UTR repression ARE CDE 3’UTR El Gazzar et al (2010); J Biol Chem Model of translation repression by microRNAs Tolerant p Responsive RBM4 RBM4 cytoplasm MKP-1 cytoplasm nucleus p Ago2 Ago2 RBM4 RBM4 RBM4 AAAAA cap and/or Ago2 RBM4 AAAAA translation arrest eIF4A/4G mRNA degradation AAAAA RBM4 p translation AAAAA Conclusions … 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 Hypothesis 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. 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 % survival 80 60 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 Circulating levels of proinflammatory cytokines days 2-4 (acute phase) * * 600 IL-6 (pg/ml) TNFa (pg/ml) 800 400 200 0 Sham CLP 1400 1200 * * 1000 800 600 400 200 0 CLP + Sham CLP CD34 CLP + CD34 days 14-16 (chronic phase) IL-6 (pg/ml) TNFa (pg/ml) 800 600 400 200 0 Sham CLP 1400 1200 1000 800 600 400 200 0 CLP + CD34 Resolved inflammation Sham CLP CLP + CD34 Peritoneal macrophages from chronically septic mice reconstituted with CD34+ cells have normal immune rsponse days 2-4 4000 IL-6 (pg/ml) TNFa (pg/ml) 2000 1500 1000 500 3000 2000 1000 0 0 Sham CLP Sham CLP + CLP CLP + CD34 CD34 days 14-16 4000 * 1500 1000 500 IL-6 (pg/ml) TNFa (pg/ml) 2000 * 3000 2000 1000 0 0 Sham CLP CLP + CD34 Ex vivo stimulation Sham CLP CLP + CD34 CD34+ cells enhance bacterial clearance in chronically septic mice Bacterial load days 2-4 4.0 108 3.0 108 2.0 1.0 108 Blood 600 CFU/1 ml CFU/mouse Peritoneum 108 400 200 0 0 4.0 108 3.0 108 2.0 108 1.0 108 0 600 *p=0.001 CFU/1 ml CFU/mouse days 14-16 400 200 0 CD34+ cells improve bacterial phagocytic activity of innate cells in chronically septic mice B Phagocytic activity Macrophages Neutrophils 100 80 60 40 20 CLP + CD34 CLP 80 60 40 20 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 CLP 100 (585 nm) CLP + CD34 120 0 CLP CLP + CD34 CLP + CLP CD34 Fluorescein-conjugated E. coli emission (585 nm) CLP + CD34 mean fluorescence (585 nm) days 14-16 120 * 100 80 60 40 20 0 120 100 (585 nm) CLP mean fluoresence CLP + CD34 mean fluoresence CLP 120 (585 nm) 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 mean fluorescence days 2-4 counts Days 14-16 Days 2-4 A * 80 60 40 20 0 CLP CLP + CD34 CLP CLP + CD34 CD34+ cell-derivatives home to sites of inflammation bone marrow Day 2 Day 5 Peritoneum Spleen Conclusions 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 Myeloid-derived suppressor cells (MDSCs) underlie chronic sepsis pathogenesis 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 100 101 102 103 104 day 3 100 101 102 103 104 day 6 100 101 102 103 104 100 101 102 103 104 day 0 100 101 102 103 104 Gr1-FITC 100 101 102 103 104 A 100 101 102 103 104 Dramatic expansion of Gr1+ CD11b+ MDSCs cells in late sepsis day 12 100 101 102 103 104 CD11b-PE Gr1+ CD11b+ cells (%) B C * 100 * 80 60 40 20 0 0 3 6 12 Days post CLP day 3 day 6 MDSCs can enhance or attenuate the systemic inflammatory response saline (n=20) MDSCs from D3 (n=30) MDSCs from D12 (n=35) 100 % survival 80 60 40 20 0 0 2 4 6 8 10 12 14 Days post CLP 16 18 20 22 MDSCs from chronically septic mice lose differentiation potential CD11b-PE 100 101 102 103 104 MHC II-FITC CD11c-PE 100 101 102 103 104 MHC II-FITC CD11c-PE F4/80-APC 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 CD11b-PE MHC II-FITC CD11c+-PE F4/80-APC Day 12 100 101 102 103 104 100 101 102 103 104 CD11b-PE 100 101 102 103 104 CD11b-PE CD11b-PE F4/80-APC 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 CD11c+-PE F4/80-APC 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 Grr1-FITC Day3 CD31+-enriched MDSCs Total MDSCs B 100 101 102 103 104 A 100 101 102 103 104 MHC II-FITC Pathway of hematopoietic stem cell differentiation and development of innate cell repertoire normal LT-HSC ST-HSC MPP CMP ST-HSC MPP CMP Monocyte GP Granulocyte GMP septic LT-HSC MP MP Monocyte GP Granulocyte MP Immature monocyte GP Immature granulocyte GMP GMP MiRNAs disrupt myeloid cell repertoire during sepsis Gfi1+ Gfi1+ miR-21+ miR-181+ miR-21+ miR-181+ monocyte normal miR-21+ miR-181+ granulocyte MPP CMP GMP dendritic cell monocyte miR-21+++ sepsis miR-181+++ miR-21+++ miR-181+++ miR-21b+++ miR-181+++ granulocyte MPP CMP Immature MDSC dendritic cell Fig. 9. Depicts disruption of myeloid cell repertoire by miR-21 and miR-181b Conclusions and directions… 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 Acknowledgement LAB Laura Brudecki Research Assistant Jessica Jordan (PhD student) Keeley Haggard (undergrad.) COLLABORATORS Charles McCall, MD Wake Forest University Benjamin Garcia, PhD Princeton University Donald Feruson, PhD ETSU