A whole-genome clustered regularly interspaced short palindromic repeats (CRISPR) screening study is the first to identify the gene regulating the inflammatory response and immune cell death during sepsis, Australian researchers reported in the October 5, 2020, edition of Nature Immunology.
This research also showed that ablation of the gene, triggering receptor expressed on myeloid cells (Trem) family Trem-like 4 family (Treml4), resulted in almost complete protection from lethal sepsis and secondary pneumonia due to Pseudomonas aeruginosa in mice.
"Calcium is an important second messenger that regulates many cellular functions, including the induction of genes involved in inflammation and apoptosis," study leader Hamsa Puthalakath told BioWorld Science.
"Our study demonstrated that the TREML4 protein encoded by the Treml4 gene regulates calcium flux inside the cell," said Puthalakath, an associate professor in the Department of Biochemistry and Genetics at La Trobe University's Institute for Molecular Science in Melbourne.
A condition of overwhelming systemic microbial infection resulting in widespread organ damage, sepsis is a major global health burden, affecting an estimated 50 million people annually with approximately 8 million deaths.
Currently, there are no effective treatments for sepsis apart from supportive care and antibiotic therapy, but this can result in immunosuppression, leaving patients susceptible to hospital-acquired infections.
Picking the right moment to intervene
Sepsis is a biphasic disease characterized by an initial inflammatory shock phase, which accounts for about 15% of sepsis-related mortality, followed by a prolonged immunosuppression phase accounting for 85% of deaths, mainly due to secondary infections.
Thus far, approximately 100 clinical trials have been conducted of drugs targeting the inflammatory shock phase of sepsis.
Despite many of these studies having been shown to reduce the time that sepsis patients spend in the intensive care unit (ICU), none has shown reductions in overall mortality, presumably because inflammation is necessary for combatting infection.
These failures, collectively, have led to the idea that targeting the immunosuppressive phase of sepsis, which is often caused by the apoptosis of immune cells, may lead to a cure.
However, the challenge has been to identify appropriate targets, as the molecular triggers of these events remain unknown.
There is now increasing evidence that most of the deleterious effects of sepsis are due to host immune responses rather than the microorganisms per se, suggesting that modifying the host response may be a more suitable therapeutic approach.
This provided the rationale for the new Nature Immunology study, which was a collaboration between La Trobe University, the Walter and Eliza Hall Institute of Medical Research and the University of Melbourne.
Using whole-genome CRISPR screening in mice, the researchers identified TREML4 as being a key regulator of inflammation and immune cell death in polymicrobial sepsis.
Genetic ablation of Treml4 in mice using CRISPR gene editing was then shown to result in almost complete protection from lethal sepsis and against secondary pneumonia caused by P. aeruginosa.
Importantly, "Treml4 ablation did not have any observed adverse effects on the development or well-being of mice, which grew normally and reproduced normally," said Puthalakath. Moreover, "gene ablation provided approximately 90% protection against P. aeruginosa-mediated pneumonia."
Genetic Treml4 response, myeloperoxidase activation, endoplasmic reticulum stress response and apoptotic cell death in innate immune cells.
"When subjected to sepsis, Treml4 leads to increased inflammatory cytokine production leading to organ failure and subsequently to the death of innate immune system neutrophils and macrophages," noted Puthalakath.
"Deletion of Treml4 blocked these processes and led to less inflammatory damage and less immune cell death, resulting in better clearance of the pathogen and improved survival," he said.
Indeed, these regulatory effects of Treml4 ablation were shown in turn to lead to an overall increase in the survival rate of the ablated mice to greater than 90%, during both the acute and chronic phases of polymicrobial sepsis.
However, although "there is no guarantee that what works in mice will work in humans, processes such as apoptosis are highly [evolutionarily] conserved phenomena," noted Puthalakath.
"To address this question, we have now identified the human TREML4 receptor homologue and are planning to replace the mouse receptor with the human and attempt to determine whether the human receptor behaves in the same way as the mouse TREML4 receptor."
Regarding sepsis treatment development, said Puthalakath, "blocking TREML4 function via gene deletion, or eventually through a monoclonal antibody (MAb), reduces production of the inflammatory cytokines causing multiple organ failure and immune cell death during polymicrobial sepsis."
To this end, "we have now identified the human TREML4 receptor and are in the process of developing therapeutic MAbs that, if successful, will be treatments for polymicrobial sepsis," said Puthalakath. "The therapeutic potential of these MAbs could be tested in the mouse strain expressing the human receptor."
"We also have evidence that receptor ablation protects against Candidemia, which is a common problem amongst ICU patients with intravenous catheters and on high doses of antibiotics, so we are currently pursuing the mechanisms of this phenomenon." (Nedeva, C. et al. Nat Immun 2020, Advanced publication).