Defining the role of Antibiotic Treatments in Shaping Host Innate Inflammatory Responses to Bacterial Infection Restricted; Files Only

Abstract

Despite extensive clinical use, the innate immune consequences of using bactericidal versus bacteriostatic antibiotic treatments to treat susceptible infections are not clear. Using a murine peritonitis model, we observed that a bacteriostatic treatment was more protective than a bactericidal treatment. To understand the mechanisms underlying this unexpected difference, we compared macrophage responses to bactericidal-killed bacteria or those growth-arrested by bacteriostatic antibiotics. We found that clinical Gram-negative bacterial isolates exposed to bactericidal drugs induced more proinflammatory cytokines than those treated with bacteriostatic agents. 

Data from Tlr4-/- macrophages and reporter cells showed that released LPS levels were comparable across antibiotic treatment types and thus not responsible for the bactericidal/bacteriostatic difference we observe. By contrast, bacterial DNA – released only by bactericidal treatments – exacerbated inflammatory signaling through TLR9. In the absence of TLR9 signaling, the in vivo efficacy of bactericidal drug treatment was completely rescued. This demonstrates that antibiotics can act in important, indirect ways distinct from bacterial inhibition: such as causing treatment failure by releasing DNA that induces overwhelming, TLR9-dependent inflammation. These data establish a novel link between how an antibiotic affects bacterial physiology and subsequent pattern recognition receptor (PRR) engagement, which may have relevance for tailoring antibiotic treatments to optimize both bacterial clearance and the resulting innate immune inflammatory responses.

Table of Contents

TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION………………………………………………………..........….…… 1

Antibiotics and Antibiotic Resistance…………………………………………......….….……. 2

Importance of Antibiotics………………………………………………………................... 2

Challenges Associated with Commercial Development of New Antibiotics……...... 3

Growing Threat of Antibiotic Resistance……………………………………............…... 4

Necessity of New Approaches……………………………………………................……… 5

Impact of Antibiotic Treatments on Bacteria……………………………………………....... 7

Bactericidal and Bacteriostatic Drugs: Agents and Mechanisms…………….......…... 7

Bacterial Pathogen Associated Molecular Patterns (PAMPs)……………….........…… 8

Environment Shapes Macrophage Behavior……………………………………….........…… 11

Macrophages Function as Sentinel Sensors of Infectious Stimuli…………….........… 11

Macrophages Function as Phagocytic Killers of Bacteria……………………............... 12

Macrophages Interrogate Endocytosed Cargo to Determine if it is Dangerous......... 15

Multi-Ligand Signaling Determines Macrophage Inflammatory Outcomes……..…… 17

Defining Proportional Response…………………………………………….................…... 17

PAMP Location Matters…………………………………………………....................……... 18

Impact of Antibiotic-Treated Bacteria on Host Inflammatory Outcomes……………..… 20

Beneficial Direct Effects…………………………………………………….....................….. 20

Deleterious Direct Effects…………………………………………....................…………… 21

Beneficial Indirect Effects…………………………………………....................…………… 22

Deleterious Indirect Effects………………………………………..................…………….. 23

Other Effects……………………………………………………………......................………. 25

Summary……………………………………………………………...................…………………. 27

CHAPTER 2: DEFINING THE INFLAMMATORY PHENOTYPES THAT ANTIBIOTIC TREATED BACTERIA INDUCE FROM MACROPHAGES ...... 31

Abstract………………………………………………………………………………..................…. 32

Introduction……………………………………………………………………................………... 33

Methods……………………………………………………………………………..................……. 35

Results……………………………………………………………………………..................……... 45

Discussion……………………………………………………………………...................………… 51

Acknowledgements…………………………………………………………...............…………... 52

Author Contributions……………………………………………………...............……………… 52

Table 1. Drug information and strain specific MIC values……………...………................................................................……... 53

Figure 1. Antibiotic concentrations and exposure intervals that cause killing (cidal drugs) and growth arrest (static drugs)……55

Figure 2. Cidal treated bacteria induce more TNF from macrophages than static treated bacteria………..........................…….. 57

Figure 3. Cidal antibiotic treatments induce more proinflammatory cytokines pattern preserved across clinical strains……….. 59

Figure 4. Class of antibiotic influences host survival and cytokine responses in an in vivo peritonitis model……………….......... 61

Supplemental Figure 1. Extended K12 antibiotic screening data…………………...............................................................……. 64

Supplemental Figure 2. Macrophage viability is maintained during bacterial infections…………………………………….…………... 66

Supplemental Figure 3. Full Luminex Screening……………………………………....................................................................….. 68

CHAPTER 3: MECHANISTIC AND TRANSLATIONAL CHARACTERIZATION OF THE INFLAMMATORY IMPACTS OF ANTIBIOTIC TREATMENTS ON INFECTION OUTCOMES…… 70

Abstract…………………………………………………………………………....................……… 71

Introduction………………………………………………………………..................…………….. 72

Methods………………………………………………………………....................………………… 74

Results……………………………………………………………………....................…………….. 77

Discussion…………………………………………………………………...................……………. 86

Acknowledgements…………………………………………………………………................…... 89

Author Contributions…………………………………………………………................………… 89

Figure 1. Cidal treated bacteria differ morphologically from static treated bacteria….….................................................... 90

Figure 2. Visualization of macrophages infected with cidal and static treated bacteria....................................................... 92

Figure 3. Differential LPS release is not responsible for cidal induced increases in inflammatory cytokines…...................... 94

Figure 4. TLR9 is required for cidal induced increases in inflammatory cytokines…........................................................… 96

Figure 5. Cidal antibiotics liberate more DNA from bacteria than static drugs….......................................................……... 98

Figure 6. Antibiotic class mediated survival difference depends on host TLR9 signaling …………………….................…….... 100

Supplemental Figure 1. Cell comparison internal controls………………………...........................................…..................…. 103

Supplemental Figure 2. STING is not responsible for cidal drug treatment mediated increases in inflammatory markers…… 105

Supplemental Figure 3. Live imaging of antibiotic treated bacteria……………….............................................................…. 107

Supplemental Figure 4. Overall model………………………………....................................................................……………….. 109

Supplemental Movies 1-3. Untreated bacteria, Meropenem treated bacteria, and Chloramphenicol treated bacteria……………. 111

CHAPTER 4: DISCUSSION…………………………………………………….....................................................................…………... 112

Bringing Inflammation into the Antibiotic Prescribing Decision Framework………..........................................................…113

Antibiotic Treated Bacteria Change Macrophage Responses in Distinct Ways …............................................................. 113

New Questions for Antibiotics Prescribers……………………………….........................................................................…… 113

Translational Phenotypes and Outstanding Questions…………………….......……..........................................................…… 115

High Acuity Cidal-treated infections are more inflammatory (and lethal) than Static-treated infections……………………... 115

Additional Translational Questions…………………………………………..........................................................................… 116

Mechanistic Phenotypes and Outstanding Questions………………………………................................................................…. 118

Imaging of Antibiotic Treated Bacteria………………………………..........................................................................……….. 118

TLR9 Sensing is Required for cidal drug specific enhancement of inflammation…...................................................…….. 119

Therapeutic Possibilities of TLR9 targeting and other immunomodulatory strategies for improving patient outcomes……. 119

REFERENCES…………………………………………………………………………........................................................................…….. 121

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Immunology and Molecular Pathogenesis
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Health Sciences, Immunology Biology, Cell Biology, Microbiology
关键词	innate immunity sepsis immunology cidal static macrophage antibiotics microbiology
Committee Chair / Thesis Advisor	David S. Weiss, Emory University
Committee Members	Iain Fraser, NIAID Christopher LaRock, Emory University Luisa Cervantes-Barragan, Emory University Joshy Jacob, Emory University

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