Regulation of Innate Immunity in the C. elegans Intestine by Olfactory Neurons

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Regulation of Innate Immunity in the C. elegans Intestine by Olfactory Neurons, Is A Well-Researched Topic, It Is To Be Used As A Guide Or Framework For Your Research.

Abstract

The intestinal epithelium represents one of the first lines of defense against pathogenic bacteria. Immune regulation at this critical barrier is necessary to maintain organismal fitness, and mis-regulation here has been linked to numerous debilitating diseases. Functional relationships between the nervous system and immune system have been found to be critical in the proper coordination of immune defenses at barrier surfaces, however the precise mechanisms underlying theses interactions remains unclear. Through conducting a forward genetic screen utilizing the model organism Caenorhabditis elegans, we uncovered a surprising requirement for the olfactory neuron
gene olrn-1 in the regulation of intestinal epithelial immunity. During nematode development, olrn-1 is required to program the expression of odorant receptors in the AWC olfactory neuron pair. Here, we show that olrn-1 also functions in AWC neurons in the cell non-autonomous suppression of the canonical p38 MAPK PMK-1 immune pathway in the
intestine. Low activity of OLRN-1, which activates the p38 MAPK signaling cassette in AWC neurons during larval development, also de-represses the p38 MAPK PMK-1 pathway in the intestine to promote immune effector transcription, increased clearance of an intestinal pathogen and resistance to bacterial infection. However, derepression of the p38 MAPK PMK-1 pathway also results in severe developmental and reproductive defects, demonstrating the critical function of OLRN-1 to both prime C. elegans intestinal epithelialcells for the induction of anti-pathogen responses, and to limit the deleterious effects of immune hyper-activation. These data reveal an unexpected connection between olfactory receptor development and innate immunity, as well as demonstrate how neuronal regulation of immune responses within the intestinal epithelium is critical for both reproductive and developmental fitness.

Table of Contents

ACKNOWLEDGEMENTS ……………………………………………………………………………………………………….. III
DEDICATIONS …………………………………………………………………………………………………………………………. IV
PUBLISHED WORKS ………………………………………………………………………………………………………………… V
ABSTRACT ……………………………………………………………………………………………………………………………….. VI
TABLE OF TABLES ……………………………………………………………………………………………………………….. XIII
TABLE OF FIGURES ……………………………………………………………………………………………………………… XIV
LIST OF ONLINE FILES ……………………………………………………………………………………………………….. XVI
CHAPTER 1: INTRODUCTION ………………………………………………………………………………………………….. 1
1.1 C. ELEGANS AS A MODEL OF HOST-MICROBE INTERACTIONS ……………………………….. 2
1.2 CHEMOSENSATION AND BEHAVIORAL AVOIDANCE IN C. ELEGANS ……………………….. 5
1.2.1 Chemosensation in C. elegans ………………………………………………………………………………………… 6
1.2.2 Food Preference and Behavioral Avoidance …………………………………………………………………….. 9
1.3 MOLECULAR IMMUNITY IN C. ELEGANS ………………………………………………………….. 13
1.3.1 Introduction to innate immunity in C. elegans …………………………………………………………………. 13
1.3.2 The p38 PMK-1 Innate Immune Pathway ……………………………………………………………………….. 14
1.4 MECHANISMS TO PROMOTE IMMUNE HOMEOSTASIS IN C. ELEGANS ……………………. 18
1.4.1 Aberrant immune activation is toxic to C. elegans …………………………………………………………… 18
1.4.2 Proteostasis: The IRE1-XBP1 Unfolded Protein Response Pathway ………………………………….. 20
1.4.3 FSHR-1: A sensor of reactive oxygen species ………………………………………………………………….. 22
1.4.4 Surveillance Immunity ………………………………………………………………………………………………….. 24

1.4.5 Neuronal regulation of Innate Immunity in C. elegans ……………………………………………………… 26
1.4.5.1 Insulin signaling …………………………………………………………………………………………………………………… 26
1.4.5.2 NPR-1: A Link Between Behavioral Avoidance and Innate Immunity ………………………………………… 28
1.4.5.3 The Octopaminergic Immuno-inhibitory Pathway …………………………………………………………………….. 29
1.4.5.4 NPR-8 and the Regulation of Cuticular Collagens ……………………………………………………………………. 31
1.4.5.5 NPR-9 and the Intersection of Foraging and Immunity ……………………………………………………………… 33
1.4.5.6 Neurotransmitter-dependent Immune Regulation ……………………………………………………………………… 34
1.4.5.7 Acetylcholine and the Wnt Pathway ……………………………………………………………………………………….. 35
1.5 PERSPECTIVES …………………………………………………………………………………………….. 37
CHAPTER 2: INNATE IMMUNITY IN THE C. ELEGANS INTESTINE IS PROGRAMMED BY A
NEURONAL REGULATOR OF AWC OLFACTORY NEURON DEVELOPMENT ………………….. 39
2.1 ABSTRACT …………………………………………………………………………………………………. 40
2.2 INTRODUCTION …………………………………………………………………………………………… 40
2.3 RESULTS ……………………………………………………………………………………………………. 43
2.3.1 Loss-of-function mutations in olrn-1 cause constitutive immune activation …………………………. 43
2.3.2 OLRN-1 suppresses the p38 MAPK PMK-1 innate immune pathway …………………………………. 50
2.3.3 Promotion of intestinal immune homeostasis by olrn-1 is required to ensure reproduction and
development ………………………………………………………………………………………………………………………… 55
2.3.4 Expression of olrn-1 in chemosensory neurons is sufficient to regulate innate immunity in the
intestinal epithelium …………………………………………………………………………………………………………….. 58
2.3.5 Immune effectors regulated by neuronal olrn-1 are dynamically expressed during nematode
development ………………………………………………………………………………………………………………………… 61
2.4 MATERIALS AND METHODS ………………………………………………………………………….. 66
2.4.1 Forward genetic screen ………………………………………………………………………………………………… 66
2.4.2 C. elegans Bacterial Infection and Other Assays ……………………………………………………………… 67
2.4.3 Generation of transgenic C. elegans strains ……………………………………………………………………. 68

2.3.4 Gene expression analyses and bioinformatics …………………………………………………………………. 69
2.3.5 Immunoblot Analyses …………………………………………………………………………………………………… 71
2.3.6 Microscopy …………………………………………………………………………………………………………………. 73
2.3.7 Quantification and Statistical Analysis …………………………………………………………………………… 73
CHAPTER 3: DISCUSSION ………………………………………………………………………………………………………. 74
3.1 INTRODUCTION …………………………………………………………………………………………… 75
3.2 FOOD PREFERENCE AND CHEMOTAXIS MAY BE COUPLED TO INNATE IMMUNE
REGULATION ……………………………………………………………………………………………………. 75
3.3 SUPPRESSION OF THE P38-PMK-1 PATHWAY BY OLRN-1 IS REQUIRED FOR
DEVELOPMENT AND EVOLUTIONARY FITNESS ……………………………………………………….. 79
3.4 NEURONAL REGULATION OF IMMUNE DEFENSES ARE EVOLUTIONARILY ANCIENT …. 81
3.4 NEXT STEPS ……………………………………………………………………………………………….. 84
3.5 POTENTIAL CLINICAL APPLICATIONS OF TOXIC IMMUNE HYPER-ACTIVATION IN
HUMAN PATHOGENIC NEMATODES …………………………………………………………………….. 87
3.6 CONCLUSION ……………………………………………………………………………………………… 90
APPENDIX I: THE NUCLEAR HORMONE RECEPTOR NHR-86 CONTROLS ANTIPATHOGEN
RESPONSES IN C. ELEGANS ………………………………………………………………………………. 91
ATTRIBUTIONS ……………………………………………………………………………………………………………………….. 92
ABSTRACT ………………………………………………………………………………………………………. 93
INTRODUCTION ………………………………………………………………………………………………… 93
RESULTS …………………………………………………………………………………………………………. 95
An RNAi screen identifies a role for the nuclear hormone receptor nhr-86 in the induction of C.
elegans immune effectors ……………………………………………………………………………………………………… 95

nhr-86 activates the transcription of innate immune response genes ………………………………………….. 98
NHR-86 binds to the promoters of innate immune genes to drive their transcription ………………….. 102
The immune response induced by nhr-86 protect a C. elegans from P. aeruginosa infection ………. 105
nhr-86 induces innate immune defenses independent of the p38 MAPK pmk-1 ………………………….. 109
Discussion ………………………………………………………………………………………………………………………… 112
MATERIAL AND METHODS ……………………………………………………………………………….. 115
C. elegans and bacterial strains …………………………………………………………………………………………… 115
C. elegans strain construction ……………………………………………………………………………………………… 116
Feeding RNAi screen ………………………………………………………………………………………………………….. 116
C. elegans bacterial infection and other assays ……………………………………………………………………… 117
mRNA-seq, NanoString ncounter gene expression analyses and qRT-PCR ……………………………….. 117
Immunoblot analyses ………………………………………………………………………………………………………….. 118
ChIP-qPCR, ChIP-seq and bioinformatics ……………………………………………………………………………. 119
Microscopy ……………………………………………………………………………………………………………………….. 121
Statistical analyses …………………………………………………………………………………………………………….. 121
SUPPLEMENTAL FIGURES …………………………………………………………………………………. 122
SUPPLEMENTAL TABLES ………………………………………………………………………………….. 127
APPENDIX II: MEASUREMENTS OF INNATE IMMUNE FUNCTION IN C. ELEGANS ……….. 128
ATTRIBUTIONS ……………………………………………………………………………………………………………………… 129
ABSTRACT …………………………………………………………………………………………………….. 130
INTRODUCTION ………………………………………………………………………………………………. 131
MATERIALS …………………………………………………………………………………………………… 134
C. ELEGANS MAINTENANCE PLATE PREPARATION ……………………………………………….. 136
NGM-OP50 Plate Preparation ……………………………………………………………………………………………. 136

P. aeruginosa PA14 Pathogenesis Assay Plate Preparation ……………………………………………………. 137
C. ELEGANS MAINTENANCE AND SYNCHRONIZATION …………………………………………… 137
PSEUDOMONAS AERUGINOSA PA14 PATHOGENESIS AND C. ELEGANS LIFESPAN ASSAYS
……………………………………………………………………………………………………………………. 138
P. aeruginosa PA14 Pathogenesis Assay Setup ……………………………………………………………………… 139
Assay Scoring and Statistical Analyses …………………………………………………………………………………. 139
C. elegans Lifespan Assay Setup ………………………………………………………………………………………….. 140
C. elegans Lifespan Assay Scoring and Statistical Analyses ……………………………………………………. 140
LAWN OCCUPANCY ASSAY ……………………………………………………………………………… 141
Assay Setup ……………………………………………………………………………………………………………………….. 141
Scoring and Statistical Analysis …………………………………………………………………………………………… 141
INTESTINAL PSEUDOMONAL CFU QUANTIFICATION ……………………………………………. 142
Bacterial Isolation and Plating ……………………………………………………………………………………………. 142
Intestinal CFU Calculations ……………………………………………………………………………………………….. 144
UTILIZING TRANSCRIPTIONAL READOUTS TO EXAMINE IMMUNE PATHWAY FUNCTION
……………………………………………………………………………………………………………………. 144
AN INITIAL EVALUATION OF TISSUE SPECIFICITY IN IMMUNE FUNCTION ………………… 147
BIBLIOGRAPHY …………………………………………………………………………………………………………………….. 152

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Additional information

Author

Kyle J. Foster

No of Chapters

3

No of Pages

191

Reference

YES

Format

PDF

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