Taming the Immaturity of Neutrophils Driving Injury in Lung Transplantation

Amongst all recipients of solid organ transplants, those receiving a lung transplant (LTx) currently have the worst long-term survival. Although the etiology of chronic rejection is multifactorial, and the timing of the contributing insults is unpredictable, a known occurrence in the immediate postoperative period is primary graft dysfunction (PGD). PGD is a form of acute lung injury (ALI), manifesting as acute respiratory distress syndrome (ARDS) within 72 hours post-LTx that is associated with worse short- and long- term outcomes¹. A major driver of PGD pathophysiology is ischemia-reperfusion injury (IRI)². Although improved surgical techniques have resulted in less ischemic times, the incidence of PGD continues to increase, especially due to sicker recipients¹. IRI is primarily driven by exuberant innate immune responses². PGD is driven by tissue-infiltrating classical monocytes and tissue-resident nonclassical monocytes activating alveolar macrophages to release chemokines, which recruit neutrophils². Moreover, damage associated-molecular patterns (DAMPs) released from cells dying during IRI also activate neutrophils, which perpetuate the injury². Thus, neutrophils have been an attractive target for mitigating lung IRI and improving both short and long-term outcomes after LTx.

Activated neutrophils release reactive oxygen species (ROS) to generate superoxide, resulting in irreversible tissue damage in LTx³. To investigate neutrophils, scientists have created experimental PGD models, with most models treating neutrophils as a homogenous population⁴. However, immature neutrophils have been observed in the bronchoalveolar lavage of lung transplant recipients, and they correlate with proteolytic activation of chemokines such as CXCL8⁵. Given that neutrophil recruitment is a characteristic of ALI, these prior observations create a precedent to investigate the different neutrophil subtypes and their effects on PGD. Hence, Klein et al investigated different neutrophil populations in LTx and their effects in IRI.⁶ They first showed that circulating immature neutrophils increase early post-LTx based on PGD severity and positively correlated with ICU length of stay.⁶ Using a murine left pulmonary hilar clamp model, they subsequently demonstrated the link between IRI and increased G-CSF levels, as well as the mobilization and intrapulmonary infiltration of these immature neutrophils. After defining their roles ex vivo through differentiation of monocyte progenitors, the authors show that preoperative anti-G-CSF treatment could be a viable option to reduce the severity of lung IRI by reducing immature neutrophil mobilization and recruitment to the lung. Thus, these findings not only advance the field by providing a targeted therapeutic that can be further investigated in LTx, but also open avenues for investigating how best to translate these observations into patients.

First, to what extent is G-CSF the best target for intervention? Within the post-LTx cohort, the authors identify that in addition to circulating G-CSF, IL-2R, IL-6, IL-10 and IL-13 are also elevated in allograft recipients.⁶ The expansion of immature neutrophils significantly correlates with most of these cytokines but not IL-10. To experimentally model cytokine-mediated neutrophil mobilization, the authors utilized a murine hilar clamp model. Although this model lacks cold ischemia and alloimmunity present in LTx, it provides similar innate immune and histopathological features of IRI that are observed in transplant recipients⁴. In this model, warm ischemia is induced for 1 hour, allowing accelerated cell death and production of DAMPs, followed by reperfusion for 24 hours, which allows time for neutrophil-mediated responses while avoiding the donor macrophage response to LTx. In this model, the authors report that both G-CSF and IL-6 have the highest increase post-IRI. These two cytokines also demonstrate a strong correlation with early neutrophil expansion, which led to the authors utilizing anti-G-CSF pre-treatment to ameliorate IRI. However, while the authors focus on G-CSF in this study, they acknowledge the relevance of IL-6 as a potential target, which is important because IL-6 inhibition has been shown to be safe in lung transplant recipients⁷, and is being tested for efficacy in different clinical settings post-LTx (NCT06990711, NCT06033196).

Second, if the goal is to target immature neutrophils, then how can we best identify these cell types in lung transplant recipients and what are their functional characteristics? In their murine models, the authors utilized CD101 as a marker of mature neutrophils. The authors distinguished these cells by first identifying CD45⁺ leukocytes, then neutrophils with Ly6g⁺ and CD11b⁺, followed by CD101⁻ for immature neutrophils. The authors confirmed the phenotype of immature neutrophils in vitro by extracting murine bone marrow hematopoietic progenitors, expanding them with stem cell factor (SCF) and IL-3, followed by G-CSF differentiation until maturation on Day 7. On Day 4, neutrophils began to exhibit cytotoxic ROS activity; however, they were resistant to apoptosis and demonstrated impaired phagocytosis. These observations suggest that these immature neutrophils would sustain ongoing inflammation. To subsequently identify these populations in lung transplant recipients, the authors conducted flow cytometry on fresh blood within 4 hours of collection from 20 pre-LTx patients with advanced lung disease and 30 post-LTx patients within 72 hours of surgery. Because CD101 is not expressed in humans, the authors used CD10 as a marker of mature neutrophils, a regulator of inflammatory responses that is expressed only during late-stage neutrophil differentiation. They performed a pilot validation study by quantifying the amount of CD10⁻ cells in a separate, smaller cohort of LTx recipients. The authors subsequently assessed nuclear morphology by differentiating between segmented (mature) and non-segmented (immature) nuclei. Their sorting correlated ~90% of CD10⁻ neutrophils with non-segmented nuclei, confirming CD10⁻ as a surrogate marker for immature neutrophils. Future studies should clarify the functional characteristics of these immature neutrophils in humans, and how they can be reprogrammed to potentially facilitate the resolution of inflammation.

Of note, gut microbiota produces short chain fatty acids (SCFA) that regulate the balance of neutrophil maturity. This raises interesting questions about how the microbiome can be leveraged improve outcomes in LTx recipients. It has been previously shown that significant loss to “health-promoting” species of the gut microbiome such as Bacteroides uniformis, could be an early indicator of allograft rejection⁸. The decline in this species has been associated with the increase of circulating proinflammatory cytokines, such as IL-1β and IL-12, exacerbating alloimmune responses⁸. Conversely, a healthy, mannose-producing microbiome has been associated with a reduction in alloimmune responses, forming a lung-gut axis built upon the metabolites and cytokine alterations induced by the microbial community⁸. Addressing the effects of these communities on lung IRI would be important because empiric antibiotics are often given postoperatively to reduce the risk of donor-derived infections, but they can alter the gut microbiota. This reduction in commensal metabolites not only promotes inflammation but also shifts the neutrophil balance toward more immature cells. In this current study, neither donor nor recipient microbiological cultures had any association with immature neutrophil counts.⁶ However, the authors’ work creates a precedent to investigate whether SCFAs alone can reverse the effect of G-CSF, and if a combination of SCFA and anti-G-CSF should be utilized to reduce PGD.

In summary, Klein and colleagues convincingly show that neutrophil populations in the early post-LTx period are heterogeneous. They show that immature neutrophil populations increase in response to G-CSF signaling and that these immature neutrophils can perpetuate inflammation, thus propagating IRI and consequently, worsening PGD (Figure). Their work opens various avenues for exploring neutrophil-targeted therapeutics to improve recovery in LTx recipients with the goal of improving both short- and long-term survival.

Figure. Proposed pathophysiology of immature neutrophils leading to worse outcomes post-lung transplantation.

Lung ischemia-reperfusion injury (LIRI) results in release of damage-associated molecular patterns (DAMPs), which promote the release of proinflammatory cytokines such as granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6). These proinflammatory cytokines regulate emergency granulopoeisis, resulting in the differentiation and mobilization of immature neutrophils into the circulation (which lack CD10 in humans and CD101 in mice). These immature neutrophils infiltrate the allograft lung, resulting in cytotoxic reactive oxygen species production, but demonstrate impaired apoptosis and phagocytosis, thereby perpetuating inflammation, resulting in a form of acute lung injury referred to as primary graft dysfunction (PGD), which is a major risk factor for chronic lung allograft dysfunction (CLAD).