Uncover NETosis. Discover New Treatments

Uncover NETosis. Discover New Treatments

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Nades Palaniyar, PhD
Senior Scientist, Lung Innate Immunity, Program in Translational Medicine, PGCRL, The Hospital for Sick Children
Associate Professor, IMS and LMP, Faculty of Medicine

Discovery of NETs

For centuries, scientists have known that white blood cells such as neutrophils engulf and destroy pathogens. However, until recently, no one knew that neutrophils could cast their DNA as neutrophil extracellular traps (NETs). Despite constituting 60-70% of all white blood cells, innate immune functions of neutrophils had not been studied in depth.

When first discovered, NETs were met with resistance: many members of the scientific community were skeptical of NETosis, and the relevance of NETs to immunity and pathobiology. NETs were considered as an experimental artifact by some, but I was fascinated by the elegance of the biology! Today, however, NETs are so well accepted that they are covered in student textbooks. The discovery of NETs and identifying novel NETosis mechanisms demonstrate that big ideas advance scientific knowledge, in quanta.

How and why I work on DNA and innate immunity

In my PhD, I worked on DNA recombination and replication, and found that many components of the molecular machinery used for these two processes are the same.1 During my postdoctoral studies, I realized that innate immune proteins bind to DNA, and that DNA-protein complexes destroy a host’s own organs and elicit an autoimmune response.2, 3 As a scientist, I have been interested in revealing the mechanisms of NETosis.

It is fulfilling to combine everything that I have learned during my PhD and post-doctoral fellowship studies, to use creative imagination to decipher molecular mechanisms, and to identify drugs to treat diseases. Time flies by, and you are in the “State of Flow” every day. The truth makes you feel humble. Scientist and faculty jobs are great, and allow you to do what you like and make a big difference in the world.

A picture says 1,000 words. I was excited to see the strings of DNA entangled with the innate immune proteins during my postdoctoral fellowship at Oxford University in 2004.3, 4 As a postdoc, I had been contemplating the relevance of my discovery—that innate immune collectins bind to DNA. In the same year, headlines claimed that NETs trap and kill bacteria, and my interest in NETs began.4, 5 While many researchers were focusing on the potential benefits of NETs, I started focusing on the dark side of NETs that is relevant to lung diseases, and other infectious, inflammatory, and autoimmune diseases.6

In the summer of 2004, I moved to Toronto and established my lab at the SickKids Lung Biology Research Program, and became a faculty member in LMP and IMS. Over the last 12 years, my lab has made several major scientific and methodological contributions to two areas—humoral and cell mediated innate immunity.

Collectins and NETs at SickKids

Collectins: I consider collectins as the “antibodies of the innate immune system” (Fig. 1).7 These beautiful proteins look like bouquet of flowers, or asterisks. Collectins have fibrillar collagen-like domains and lectin (carbohydrate-binding) domains. Any pathogens that reach the lower airways or alveoli encounter collectins. Unlike antibodies, collectins bind most of the pathogens because they recognize the molecular patterns present on virtually all bacteria, fungi and virus.7 Importantly, lung collectins do not activate complement, which is a pro-inflammatory mechanism. Too much complement activation is bad for the lungs because if you can’t breathe, nothing else matters!

We identified important roles for collectins—including their specific binding to dying T-cells, altering apoptosis, and promoting apoptotic cell clearance by macrophages.8, 9 We also identified interconnections among collectins, complement, and NETs.10

NETs: In 2004, the field of NETosis was in its infancy, so there weren’t any good in vivo models to study NETs. My lab first established a mouse model to study NETs in the lung, and showed that SP-D simultaneously bind NETs and bacteria.11 Now, for the first time, we have established several in vivo models to study NETs: lipopolysaccharide-mediated acute lung injury, bacterial pneumonia, ventilator induced lung injury, cystic fibrosis (CF), polymicrobial sepsis, and even a recurrent airway obstruction asthma-like horse model.11, 12, 13, 14 I also returned to the University of Guelph, where I did my PhD in Molecular Biology and Genetics, to set up some of these models. Labs from around the world now use these models to study NETs in vivo.

By combining fundamental principles and cutting edge tools available at SickKids’ Peter Gilgan Centre for Research and Learning, we were able to make several NET-related landmark discoveries. Our Proceedings of the National Academy of Sciences, Blood, and Scientific Reports papers showed the importance of mitochondria, and relative importance of specific kinases such as ERK, p38, and Akt in different types of NETosis.15, 16 We showed that Akt is a molecular switch that directs neutrophil death towards NETosis or apoptosis.16

The relevance of the kinase JNK in NETosis was previously unknown. One of our recent studies shows that JNK is a molecular rheostat that senses concentrations of LPS and bacterial load and turns on NETosis.17 These studies help to understand why and how neutrophils turn on NETosis.

Neutrophils are short lived cells. Why would a dying neutrophil transcribe its genome? Hence, the relevance of transcription in these cells was a mystery. We were delighted to assign a new function for neutrophil transcription from our transcriptomics studies. We showed that neutrophils use genome-wide transcription initiation, the first step in gene expression, to help de-condense the entire chromatin for NETosis. Suppressing transcription initiation stops NETosis (Fig. 2).18 This 2017 Scientific Reports is expected to change the field of NETosis, particularly in terms of the understanding of molecular mechanisms and drug discovery.18

Clinical relevance of NETs

Collaborating with clinicians and having the lab in a hospital setting provide me with an amazing opportunity to do clinically relevant projects. We were able to study the relevance of lung collectins and other lung proteins as serum biomarkers in several diseases. About two in ten children develop bronchiolitis obliterans syndrome (BOS) one to two years after receiving a stem cell transplant. BOS is characterized by the permanent narrowing of the airways and reduced lung function, often requiring lung transplantation. We found that a lung protein KL-6 is a great marker in predicting BOS, within one to three months.19 We also found that neutrophils are one of the key culprits in BOS.20 It is now becoming clear that cytotoxic NETs are responsible for damaging airways of the lungs of stem cell transplant patients.21

Females with CF have a higher degree of exacerbations and die mainly of the lung disease four to eight years before male patients.22, 23 We are now determining the roles of T-cells, NETs, and sex differences in immune response in these patients. Nitric oxide metabolism and levels of lipid intermediates such as hepoxilins are also altered in CF airways.24, 25 Lifesaving lung transplantation could fail due to immune damage. Therefore, we are currently studying the relevance of regulating NETosis to address these clinical issues, collaboratively.

The future: Regulating NETosis to treat inflammatory diseases

With the new understanding of NETosis mechanisms, we screened large, focused, FDA-approved cancer drug libraries. These screens identified key classes of drugs that fully suppress NETosis, but do not compromise other neutrophil functions. Intriguingly, inhibitory functions of many of these drugs match with the NETosis mechanisms that we discovered. Therefore, these drugs could be valuable in treating some infectious, inflammatory, and autoimmune diseases. We are in the process of devising personalized medicine strategies using induced pluripotent stem cells to translate this new knowledge.

I am grateful for my mentors, colleagues, students, trainees and staff, and for the funding agencies such as NSERC, CIHR, CF Canada and Mitac, and the referees, who always refer to us as a group with several original ideas. Science is a group effort. When science moves, it moves you. I am confident that my lab and the NETosis research community are on the right track for bringing new knowledge from the lab into clinical care.

We are boldly going where no person has gone before; the motto of the Palaniyar lab is, “Uncover NETosis. Discover New Treatments.” NETs can be good. But, too much of a good thing can be a bad thing. My group and I are determined to fix the NETosis issue in some diseases within the next ten years.

Fig. 1. Collectins are “antibodies of the innate immune systems.” These proteins recognize unique carbohydrate patterns present on most of the bacteria, fungi and virus.7

Fig. 2. Assigning a novel function for neutrophil transcription: Transcriptional firing drives NETosis.18 Neutophils form NETs (Blue, DNA; Red, Citrullinated histone H3; Green, Myeloperoxidase) that can be suppressed by transcription initiation inhibitory drugs. Each intact neutrophil is ~8 mm.

References

  1. Palaniyar N, Gerasimopoulos E, Evans DH. Shope fibroma virus DNA topoisomerase catalyses holliday junction resolution and hairpin formation in vitro. J Mol Biol. 1999 Mar 19;287(1):9-20.
  2. Palaniyar N, Nadesalingam J, Clark H, et al. Nucleic acid is a novel ligand for innate, immune pattern recognition collectins surfactant proteins A and D and mannose-binding lectin. J Biol Chem. 2004 Jul 30;279(31):32728-36.
  3. Palaniyar N, Clar H, Nadesalingam J, et al. Innate immune collectin surfactant protein D enhances the clearance of DNA by macrophages and minimizes anti-DNA antibody generation. J Immunol. 2005 Jun 1;174(11):7352-8.
  4. Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular traps kill bacteria. Science. 2004 Mar 5;303(5663):1532-5.
  5. Takei H, Araki A, Watanabe H, et al. Raid killing of human neutrophils by the potent activator phorbol 12-myristate 13-acetate (PMA) accompanied by changes different from typical apoptosis or necrosis. J Leukoc Biol. 1996 Feb;59(2):229-40.
  6. Cheng OZ, Palaniyar N. NET balancing: a problem in inflammatory lung diseases. Front Immunol. 2013 Jan 24;4:1.
  7. Palaniyar N. Antibody equivalent molecules of the innate immune system: parallels between innate and adaptive immune proteins. Innate Immun. 2010 Jun;16(3):131-7.
  8. Litvack ML, Djiadeu P, Renganathan SD, et al. Natural IgM and innate immune collectin SP-D bind to late apoptotic cells and enhance their clearance by alveolar macrophages in vivo. Mol Immunol. 2010 Nov-Dec;48(1-3):37-47.
  9. Djiadeu P, Kotra LP, Sweezey N, et al. Surfactant protein D delays Fas- and TRAIL-mediated extrinsic pathway of apoptosis in T cells. Apoptosis. 2017 Feb 6.
  10. Yuen J, Pluthero FG, Douda DN, et al. NETosing Neutrophils Activate Complement Both on Their Own NETs and Bacteria via Alternative and Non-alternative Pathways. Front Immunol. 2016 Apr 14;7:137.
  11. Douda DN, Jackson R, Grasemann H, et al. Innate immune collectin surfactant protein D simultaneously binds both neutrophil extracellular traps and carbohydrate ligands and promotes bacterial trapping. J Immunol. 2011 Aug 15;187(4):1856-65.
  12. Yildiz C, Palaniyar N, Otulakowski G, et al. Mechanical ventilation induces neutrophil extracellular trap formation. Anesthesiology. 2015 Apr;122(4):864-75.
  13. Jin L, Batra S, Douda DN, et al. CXCL1 contributes to host defense in polymicrobial sepsis via modulating T cell and neutrophil functions. J Immunol. 2014 Oct 1;193(7):3549-58.
  14. Côté O, Clark ME, Viel L, et al. Secretoglobin 1A1 and 1A1A differentially regulate neutrophil reactive oxygen species production, phagocytosis and extracellular trap formation. PLoS One. 2014 Apr 28;9(4):e96217.
  15. Douda DN, Khan MA, Grasemann H, et al. SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium influx. Proc Natl Acad Sci U S A. 2015 Mar 3;112(9):2817-22.
  16. Douda DN, Yip L, Khan MA, et al. Akt is essential to induce NADPH-dependent NETosis and to switch the neutrophil death to apoptosis. Blood. 2014 Jan 23;123(4):597-600.
  17. Khan MA, Farahvash A, Douda, et al. JNK Activation Turns on LPS- and Gram-Negative Bacteria-Induced NADPH Oxidase-Dependent Suicidal NETosis (submitted)
  18. Khan MA, Palaniyar N. Transcriptional firing helps to drive NETosis. Sci Rep. 2017 Feb 8;7:41749.
  19. Gassas A, Schechter T, Krueger J, et al. Serum Krebs Von Den Lungen-6 as a Biomarker for Early Detection of Bronchiolitis Obliterans Syndrome in Children Undergoing Allogeneic Stem Cell Transplantation. Biol Blood Marrow Transplant. 2015 Aug;21(8):1524-8.
  20. Gassas A, Krueger J, Zaidman I, et al. Infections and neutrophils in the pathogenesis of bronchiolitis obliterans syndrome in chldren after allogeneic stem cell transplantation. Pediatr Transplant. 2016 Mar;20(2):303-6.
  21. Domingo-Gonzalez R, Martínez-Colón GJ, Smith AJ, et al. Inhibition of Neutrophil Extracellular Trap Formation after Stem Cell Transplant by Prostaglandin E2. Am J Respir Crit Care Med. 2016 Jan 15;193(2):186-97.
  22. Sweezey NB, Ratjen F. The cystic fibrosis gender gap: potential roles of estrogen. Pediatr Pulmonol. 2014 Apr;49(4):309-17.
  23. Kushwah R, Gagnon S, Sweezey NB. Intrinsic predisposition of naïve cystic fibrois T cells to differentiate towards a Th17 phenotype. Respir Res. 2013 Dec 17;14:138.
  24. Douda DN, Grasemann H, Pace-Asciak C, et al.. A lipid mediator hepoxilin A3 is a natural inducer of neutrophil extracellular traps in human neutrophils. Mediators Inflamm. 2015;2015:520871.
  25. Ghorbani P, Santhakumar P, Hu Q, et al. Short-chain fatty acids affect cystic fibrosis airway inflammation and bacterial growth. Eur Respir J. 2015 Oct;46(4):1033-45.