Assessing neutrophil subsets in autoimmune disease: Moving away from relying on density?

Neutrophils are the most abundant immune cell in circulation. However, due to a number of technical challenges for researchers, including the neutrophil's short lifespan and difficulties with preservation, they are often discarded during blood processing and thus ignored in cohort studies. As such, the contribution of neutrophils to disease and their involvement in disease mechanisms is less explored compared with other immune cell types. Systemic lupus erythematosus (SLE) is a heterogenous, multi-organ, autoimmune disease where autoantibodies against nuclear antigens are common. With the discovery of neutrophil extracellular traps (NETs), which are chromatin complexes released by neutrophils that harbour many of the autoantigens observed in SLE, the contribution of neutrophils to SLE pathology has gained much attention over the last decade. In addition, interferon (IFN)-responsive low-density neutrophils (LDN) are significantly more prominent in SLE.1 Thus, neutrophils are now considered a central player in SLE pathology. LDN are neutrophils that are isolated in the peripheral blood mononuclear cell layer during density gradient-based separation. LDN are rare in healthy individuals but can be prominent in SLE2 and a number of other autoimmune, cancerous or infectious conditions, such as multiple sclerosis3 and tuberculosis. In SLE, LDN display activation of IFN genes, upregulation of acute phase proteins and produce more reactive oxygen species and NETs compared with normal density neutrophils, which suggests that LDN may contribute to disease pathology. A recent study has also suggested that LDN display an altered morphology, leading to LDN being more easily trapped in narrow blood vessels and possibly contributing to cardiovascular manifestations that are common in SLE.4 LDN are a heterogenous population and can likely be induced through several pathways and exposures, which may impact their functional characteristics. As an example, LDN from cancer patients appear to display immunosuppressive properties and can suppress T cell activation.5 In SLE, however, LDN do not have suppressive characteristics, but rather contribute to T cell activation.6 The ratio of mature and immature LDN, where maturity is defined by expression of CD10, likely contributes to the pro- and anti-inflammatory properties of LDN. This ratio differs with pathological conditions, and may relate to the disparity in properties described for LDN.7 In SLE, mature CD10+ LDN appear to be the main producers of NETs, and are efficient in phagocytosis, but produce less myeloperoxidase compared to CD10− LDN.8 Moreover, compared with healthy individuals, LDN from SLE patients produce NETs that have a higher content of mitochondrial DNA. These NETs are oxidised and likely more inflammatory/immunogenic compared to NETs with a lower proportion of mitochondrial DNA.9 As such, LDN represent a pathological target in SLE, and better methods of identifying them in patient samples would be important tools that could have significant implications for the management of this disease. So far, no definitive marker for LDN in whole blood has been identified. Currently, expression of CD10 is used as a marker of maturation as a majority of LDN are CD10−/immature when induced in healthy individuals. However, in SLE, the majority of LDN are CD10+, and as evident from above, the ratio of immature and mature LDN is essential for the functional characteristics of this population; therefore, further markers are required that can identify LDN in whole blood. Recent work from Martin et al.10 used whole-cell proteomic analysis to identify CD98 as a potential new surface marker of LDN. CD98 is involved in cell adhesion and amino acid transportation. It is expressed across multiple cell types, including monocytes, but is not typically expressed on normal-density neutrophils. Following granulocyte-colony stimulating factor administration in healthy individuals, which upregulates neutrophil trafficking from the bone marrow to the blood, CD98 was expressed by the vast majority of circulating LDN. In SLE patients, however, approximately two-thirds of LDN were CD98+, which was lower compared with controls. This aligns with the heterogeneity observed for the SLE-LDN population. Nevertheless, the study found that the expression of CD98 on LDN in SLE patients correlated with SLE disease activity. The study further described an important role of CD98 in the metabolic flexibility of LDN, where it facilitated the uptake of amino acids that were used for mitochondrial-dependent adenosine triphosphate production. It was also involved in inhibiting apoptotic responses alongside intact necroptotic responses. As such, the study provides a potential molecular mechanism by which LDN may adapt and contribute to an inflammatory environment. CD98 may also be evaluated as a therapeutic target, where a CD98 antagonist is likely to reduce LDN survival and thus tissue damage and inflammation. In summary, the role of LDN in SLE pathogenesis continues to become clearer, and CD98 expression shows some promise of either being a useful biomarker of LDN in whole blood assays or as a potential novel therapeutic target in SLE. This is also likely to be useful for advancing knowledge in other diseases where increased LDN are observed. This work was supported by MSWA. The authors declare that they have no conflict of interest.