Chronic inflammation causes tissue damage and degeneration, thus contributing to aging-associated declines in the functional integrity of tissues and organs. Inflammatory responses to DNA cause polyarthritis and heart failure in mammals, for example when persisting cytoplasmic DNA is not degraded by DNaseII. We identified inflammation-like responses to cytoplasmic DNA in the genetic model system C. elegans. We determined that the systemic functional deterioration is caused by protein folding stress resulting from the production of innate immune peptides. Augmentation of protein folding by inducing the endoplasmic reticulum unfolded stress response (UPRER) or treatment with N-acetylglucosamine alleviated the tissue damage caused by cytoplasmic DNA and limited further functional declines, even in the presence of cytoplasmic DNA. Our results establish C. elegans as an ancestral metazoan model for studying the outcomes of inflammation-like conditions caused by persistent cytoplasmic DNA and provide insight into potential therapies for human conditions involving chronic inflammation.
Chronic inflammation plays a causal role in aging-associated diseases ranging from arteriosclerosis, the development of cancer to neuroinflammation in Alzheimer’s disease. Inflammation results from an aberrant activation of the innate immune response. When transiently activated, the innate immune system is essential for fending off pathogens and maintaining tissue homeostasis. However, when chronically activated, it can cause tissue damage and pathologies. Understanding the mechanisms through which chronic inflammation causes diseases is an important prerequisite to battle age-related pathologies. Given the complexity of the human immune system we sought to establish the simple metazoan Caenorhabditis elegans as model system for gaining new insight into the mechanisms of inflammatory-like conditions. The nematode has been pivotal for understanding a range of conserved biological mechanisms including programmed cell death, development, neuronal connectivity, RNA interference and aging.
In C. elegans, innate immune responses are required as defence mechanisms against bacterial and fungal infections (Ermolaeva & Schumacher, 2014) but also mediate stress resistance when transiently activated upon DNA damage (Ermolaeva et al, 2013). The nematode is particularly suitable for investigating the interaction between distinct tissues, as we recently described in the DNA damage response (Ou et al, 2019). Therefore, the nematode might be highly suitable for investigating the systemic consequences of chronic activation of innate immune responses.
Cytoplasmic DNA, whether originating from invading viruses and bacteria or mislocalized endogenous DNA, is recognized by components of the innate immune system. While the recognition of cytoplasmic DNA comprises an important pathogen defence mechanism, persistence of DNA that can result from a defect in DNase II can result in inflammation. For instance DNase II-deficient mice develop a polyarthritis-like disease. We investigated the consequence of DNase II-deficiency in the response to cytoplasmic DNA in C. elegans.
Upon injection of E. coli DNA into a single intestinal cell, we observed a decline of tissue functionality in nuc-1 mutant animals that carry a deletion mutation in the DNase II ortholog (Williams et al, 2019). Similarto human cells, injection of CpG oligonucleotides was sufficient to trigger this response. In human, uropathogenic E. coli (UPEC) bacteria invade the host cells. We showed that a clinical UPEC isolate when fed to C. elegans invades intestinal cells. In DNase II-deficient animals, the UPEC DNA persisted in the intestinal cells (Figure 1).
This persistence of cytoplasmic DNA resulted in a system functional decline and tissue damage throughout the animals. For example, the disintegration of pharyngeal morphology is readily observable (Figure 2).
In humans, cytoplasmic DNA is sensed by the toll-like receptor TLR9 and through the cGAS-STING pathway. Both are absent in nematodes and thus far it was unknown how pathogen associated molecular patterns (PAMPs) are detected by C. elegans. We probed whether the two major innate immune signalling pathways play a role in the response to cytoplasmic DNA. While the p38 ortholog PMK-1 was dispensable, knockdown of the follicle stimulating hormone receptor-like fshr-1prevented to pathological effect of cytoplasmic DNA. Interestingly, FSHR-1 features a leucine-rich repeat region that is typical for mammalian TLRs. Our data suggest that FSHR-1 functions as sensor of cytoplasmic DNA.
To investigate the molecular basis for the pathological consequences of persistent cytoplasmic DNA, we used electron microscopy imaging of UPEC infected nuc-1 mutant animals. As shown in Figure 3, EM imaging revealed a disruption of the endoplasmic reticulum (ER) morphology with aberrantly interspersed ribosome chains (Figure 3). Indeed, the ER cisternae surface area was enlarged and the ribosome distribution index reduced.
To test whether proteostasis was indeed disrupted, we treated the animals with the proteotoxic agent dithiothreitol (DTT). Indeed, the UPEC-infected nuc-1 mutants were exquisitely sensitive to DTT treatment. We assessed the activation of the unfolded protein response in the ER (UPRER) by quantifying the spliced form of xbp-1 mRNA. Here, we observed a defect in inducing the UPRER. Indeed, the UPRERwas previously established to be required for the animals to sustain the activation of the innate immune response. The UPRERis likely to be necessary for protein folding amid a proteome shift towards the secretion of a large number of immune peptide that constitute the nematode’s innate immune response. We then tested whether activation of the UPRERwas sufficient to avoid or even revert the inflammatory consequences of UPEC infection. We employed independent approaches to activate the UPRER: Knock-down of the sams-1 transcription factor, treatment with tonic concentrations of the protein glycosylation inhibitor tunicamycin, and GlcNAc that was previously shown to elevate UPRERfunction. Strikingly, all three treatments that enhance UPRERactivity prevented the functional decline upon UPEC injection and injection of CpG oligonucleotides in the intestinal cells. Moreover, tonic tunicamycin treatment was sufficient to revert the pathology ensuing form UPEC infection.
Taken together, we established the important genetic system C. elegans as experimental model for investigating inflammation-like conditions. We demonstrated that cytoplasmic DNA leads to the activation of an innate immune response that results in inflammation and systemic tissue disruption when the intestinal cells become chronically infested with cytoplasmic DNA. Moreover, we revealed that protein folding stress amid a chronic innate immune response is the critical mechanism responsible for the inflammation-like phenotype and that activation of the UPRERis sufficient to revert the pathology.
Interestingly, innate immune responses and proteostasis impediments also result from persistent DNA damage that accumulates during the aging process (Edifizi et al, 2017). Our results might therefore impact the understanding of causal mechanisms for age-related inflammation that result from persistent damaged or cytoplasmic DNA.
Our results establish C. elegans as a simple metazoan system for studying fundamental mechanisms of innate immune responses to infectious or endogenous DNA. We have uncovered disrupted proteostasis as a new pathomechanisms of inflammation that could open new perspectives for therapies of inflammation-associated conditions in humans.
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Institute for Genome Stability in Ageing and Disease | CECAD Research Center
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Institute for Genome Stability in Ageing and Disease | CECAD Research Center
Ashley B. Williams (Co-PI)
Matthias Rieckher (Senior Scientist)
Takayuki Miyauchi (Postdoc)
Siyao Wang (Postdoc)
Aakanksha Bansal (doctoral student
João Barata (doctoral student)
Robert Bayersdorf (doctoral student)
Arturo Bujarrabal (doctoral student)
Markus Doll (doctoral student)
David Meyer (doctoral student)
Paulo da Silva (doctoral student)
Najmeh Soltanmohammadi (doctoral student)
Pavana Lakshmi (doctoral student)
Josephine Ecklebe (technician)
Jennifer Engelmeyer (technician)
Robin Lippel (technician)
Devin Mares (technician)