Center for Molecular Medicine Cologne

Björn Schumacher / Ashley Williams - B 5

Chronic inflammatory responses to persistent cytoplasmic DNA

Introduction 

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.

A new model for inflammatory responses

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.

Pharmacological restoration of ER proteostasis reverses the inflammation-like pathology

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.

Conclusions

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.

Perspectives

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. 

 

Edifizi D, Nolte H, Babu V, Castells-Roca L, Mueller MM, Brodesser S, Krüger M & Schumacher B (2017) Multilayered Reprogramming in Response to Persistent DNA Damage in C. elegans. CellReports20:2026–2043

Ermolaeva MA & Schumacher B (2014) Insights from the worm: the C. elegans model for innate immunity. Seminars in Immunology26:303–309

Ermolaeva MA, Segref A, Dakhovnik A, Ou H-L, Schneider JI, Utermöhlen O, Hoppe T & Schumacher B (2013) DNA damage in germ cells induces an innate immune response that triggers systemic stress resistance. Nature501:416–420

Ou H-L, Kim CS, Uszkoreit S, Wickström SA & Schumacher B (2019) Somatic Niche Cells Regulate the CEP-1/p53- Mediated DNA Damage Response in Primordial Germ Cells. Developmental Cell50:167–183.e8

Williams AB, Heider F, Messling J-E, Rieckher M, Bloch W & Schumacher B (2019) Restoration of Proteostasis in the Endoplasmic Reticulum Reverses an Inflammation-Like Response to Cytoplasmic DNA in Caenorhabditis elegans. Genetics:genetics.302422.2019–67

 

Bayersdorf, R., and Schumacher, B. (2019). Recent advances in understanding the mechanisms determining longevity. F1000Res 8.

Jachimowicz, R.D., Beleggia, F., Isensee, J., Velpula, B.B., Goergens, J., Bustos, M.A., Doll, M.A., Shenoy, A., Checa-Rodriguez, C., Wiederstein, J.L., Baranes-Bachar, K., Bartenhagen, C., Hertwig, F., Teper, N., Nishi, T., Schmitt, A., Distelmaier, F., Ludecke, H.J., Albrecht, B., Kruger, M., Schumacher, B., Geiger, T., Hoon, D.S.B., Huertas, P., Fischer, M., Hucho, T., Peifer, M., Ziv, Y., Reinhardt, H.C., Wieczorek, D., and Shiloh, Y. (2019). UBQLN4 Represses Homologous Recombination and Is Overexpressed in Aggressive Tumors. Cell 176, 505-19 e22.

Ou, H.L., Kim, C.S., Uszkoreit, S., Wickstrom, S.A., and Schumacher, B. (2019). Somatic Niche Cells Regulate the CEP-1/p53-Mediated DNA Damage Response in Primordial Germ Cells. Dev Cell 50, 167-83 e8.

Williams, A.B., Heider, F., Messling, J.E., Rieckher, M., Bloch, W., and Schumacher, B. (2019). Restoration of Proteostasis in the Endoplasmic Reticulum Reverses an Inflammation-Like Response to Cytoplasmic DNA in Caenorhabditis elegans. Genetics 212, 1259-78.

Bianco JN, and Schumacher B (2018). MPK-1/ERK pathway regulates DNA damage response during development through DAF-16/FOXO. Nucleic Acids Res 10.1093/nar/gky404.

Jachimowicz RD, Beleggia F, Isensee J, Velpula BB, Goergens J, Bustos MA, Doll MA, Shenoy A, Checa-Rodriguez C, Wiederstein JL, Baranes-Bachar K, Bartenhagen C, Hertwig F, Teper N, Nishi T, Schmitt A, Distelmaier F, Ludecke HJ, Albrecht B, Kruger M, Schumacher B, Geiger T, Hoon DSB, Huertas P, Fischer M, Hucho T, Peifer M, Ziv Y, Reinhardt HC, Wieczorek D, and Shiloh Y (2018). UBQLN4 Represses Homologous Recombination and Is Overexpressed in Aggressive Tumors. Cell 10.1016/j.cell.2018.11.024.

Notara M, Behboudifard S, Kluth MA, Masslo C, Ganss C, Frank MH, Schumacher B, and Cursiefen C (2018). UV light-blocking contact lenses protect against short-term UVB-induced limbal stem cell niche damage and inflammation. Sci Rep 8, 12564.

Rieckher M, Bujarrabal A, Doll MA, Soltanmohammadi N, and Schumacher B (2018). A simple answer to complex questions: Caenorhabditis elegans as an experimental model for examining the DNA damage response and disease genes. J Cell Physiol 233, 2781-2790.

Torgovnick A, Heger JM, Liaki V, Isensee J, Schmitt A, Knittel G, Riabinska A, Beleggia F, Laurien L, Leeser U, Jungst C, Siedek F, Vogel W, Klumper N, Nolte H, Wittersheim M, Tharun L, Castiglione R, Kruger M, Schauss A, Perner S, Pasparakis M, Buttner R, Persigehl T, Hucho T, Herter-Sprie GS, Schumacher B, and Reinhardt HC (2018). The Cdkn1a(SUPER) Mouse as a Tool to Study p53-Mediated Tumor Suppression. Cell Rep 25, 1027-1039 e1026.

Torgovnick A, Schiavi A, Shaik A, Kassahun H, Maglioni S, Rea SL, Johnson TE, Reinhardt HC, Honnen S, Schumacher B, Nilsen H, and Ventura N (2018). BRCA1 and BARD1 mediate apoptotic resistance but not longevity upon mitochondrial stress in Caenorhabditis elegans. EMBO Rep 10.15252/embr.201845856.

Ou HL, and Schumacher B (2018). DNA damage responses and p53 in the aging process. Blood 131, 488-495.

Bujarrabal A, and Schumacher B (2017). Tracking senescent cells: A new biomarker assay opens new avenues in senescence research. Mech Ageing Dev 162, 106-7.

Edifizi D, Nolte H, Babu V, Castells-Roca L, Mueller MM, Brodesser S, Kruger M, and Schumacher B (2017). Multilayered Reprogramming in Response to Persistent DNA Damage in C. elegans. Cell Rep 20, 2026-43.

Garinis GA, Schwer B, and Schumacher B (2017). Editorial: DNA damage & immunity. Mech Ageing Dev10.1016/j.mad.2017.04.006.

Rieckher M, Bujarrabal A, Doll MA, Soltanmohammadi N, and Schumacher B (2017). A simple answer to complex questions: Caenorhabditis elegans as an experimental model for examining the DNA damage response and disease genes. J Cell Physiol10.1002/jcp.25979.

Williams AB, and Schumacher B (2017). DNA damage responses and stress resistance: Concepts from bacterial SOS to metazoan immunity. Mech Ageing Dev 165, 27-32.

 

 

Former Funding Period 01/2017 - 12/2019

Information from this funding period will not be updated anymore. New research related information is available here.

Prof. Dr. Björn Schumacher CMMC Cologne
Prof. Dr. Björn Schumacher

Institute for Genome Stability in Ageing and Disease, CECAD Cologne

Principal Investigator - A 11

+49 221 478 84202

+49 221 478 86510

Institute for Genome Stability in Ageing and Disease, CECAD Cologne

Joseph-Stelzmann-Str. 26

50931 Cologne

http://schumacher.cecad-labs.uni-koeln.de/Home.66.0.html

CMMC Profile Page

Curriculum Vitae (CV)

Publications on PubMed

Publications - Björn Schumacher

Link to PubMed

Dr. Ashley Williams CMMC Cologne
Dr. Ashley Williams

Inst. for Genome Stability in Aging and Disease and CECAD Cologne

+49 221 478 84205

+49 221 478 32400

Inst. for Genome Stability in Aging and Disease and CECAD Cologne

Joseph-Stelzmann-Str. 26

50931 Cologne

CMMC Profile Page

Curriculum Vitae (CV)

Publications - Ashley Williams

Link to PubMed

Group Members

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)

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