Here we elaborate on the origin of low(er)-density neutrophils (LDNs) to better understand the variation found in literature. Supplemented with original data, we test the hypothesis that buoyant density of neutrophils is characterized by a spectrum that as a whole shifts to a lower density after activation. Both the 20% highest density (HDNs) and 20% lowest density (LDNs) neutrophils from healthy donors were isolated by Percoll of different densities. Using this method we found that LDNs were significantly better in T-cell suppression and bacterial containment than their 20% highest density counterparts. We found no statistically relevant differences in neutrophil survival or bacterial phagocytosis. Stimulation of healthy donor neutrophils with N-formylmethionyl-leucyl-phenylalanine induced LDNs co-segregating with peripheral blood mononuclear cells after Ficoll separation. These in vitro induced LDNs showed increased activation markers compared to HDNs and were comparable to the activation markers found on the LDN fraction seen in patients with chronic inflammatory conditions such as present in cancer patients. This all fits with the hypothesis that the density of neutrophils is distributed in a spectrum partially coupled to maturation. Additionally a shift in this spectrum can be induced by in vitro stimulation or by activation in disease. K E Y W O R D Sgranulocytes, LDG, neutrophil subsets INTRODUCTIONNeutrophils are main actors in the innate immune system. Until recently neutrophils were thought to belong to a relative homogeneous population of cells. However, an increasing number of studies now show heterogeneity in morphology, phenotype, function, or a combination of these factors. 1 One of the subtypes of neutrophils identified and studied is the low-density neutrophil (LDN). It was first recognized in 1986, when neutrophils co-segregated with mononuclear cells after density-gradient isolation in systemic lupus erythematosus (SLE) and rheumatoid arthritis patients. 2 Thereafter, the presence of LDNs have been shown in several, mostly chronic, diseases. 3

Neutrophils are important assets in defense against invading bacteria like staphylococci. However, (dysfunctioning) neutrophils can also serve as reservoir for pathogens that are able to survive inside the cellular environment. Staphylococcus aureus is a notorious facultative intracellular pathogen. Most vulnerable for neutrophil dysfunction and intracellular infection are immune-deficient patients or, as has recently been described, severely injured patients. These dysfunctional neutrophils can become hide-out spots or “Trojan horses” for S. aureus. This location offers protection to bacteria from most antibiotics and allows transportation of bacteria throughout the body inside moving neutrophils. When neutrophils die, these bacteria are released at different locations. In this review, we therefore focus on the capacity of several groups of antibiotics to enter human neutrophils, kill intracellular S. aureus and affect neutrophil function. We provide an overview of intracellular capacity of available antibiotics to aid in clinical decision making. In conclusion, quinolones, rifamycins and sulfamethoxazole-trimethoprim seem very effective against intracellular S. aureus in human neutrophils. Oxazolidinones, macrolides and lincosamides also exert intracellular antibiotic activity. Despite that the reviewed data are predominantly of in vitro origin, these findings should be taken into account when intracellular infection is suspected, as can be the case in severely injured patients.

Supplemental Digital Content is available in the text.

At homeostasis the vast majority of neutrophils in the circulation expresses CD16 and CD62L within a narrow expression range, but this quickly changes in disease. Little is known regarding the changes in kinetics of neutrophils phenotypes in inflammatory conditions. During acute inflammation more heterogeneity was found, characterized by an increase in CD16dim banded neutrophils. These cells were probably released from the bone marrow (left shift). Acute inflammation induced by human experimental endotoxemia (LPS model) was additionally accompanied by an immediate increase in a CD62Llow neutrophil population, which was not as explicit after injury/trauma induced acute inflammation. The situation in sub-acute inflammation was more complex. CD62Llow neutrophils appeared in the peripheral blood several days (>3 days) after trauma with a peak after 10 days. A similar situation was found in the blood of COVID-19 patients returning from the ICU. Sorted CD16low and CD62Llow subsets from trauma and COVID-19 patients displayed the same nuclear characteristics as found after experimental endotoxemia. In diseases associated with chronic inflammation (stable COPD and treatment naive HIV) no increases in CD16low or CD62Llow neutrophils were found in the peripheral blood. All neutrophil subsets were present in the bone marrow during homeostasis. After LPS rechallenge, these subsets failed to appear in the circulation, but continued to be present in the bone marrow, suggesting the absence of recruitment signals. Because the subsets were reported to have different functionalities, these results on the kinetics of neutrophil subsets in a range of inflammatory conditions contribute to our understanding on the role of neutrophils in health and disease.

Coronavirus disease 2019 (COVID‐19) is a rapidly emerging pandemic disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Critical COVID‐19 is thought to be associated with a hyper‐inflammatory process that can develop into acute respiratory distress syndrome, a critical disease normally mediated by dysfunctional neutrophils. This study tested the hypothesis whether the neutrophil compartment displays characteristics of hyperinflammation in COVID‐19 patients. Therefore, a prospective study was performed on all patients with suspected COVID‐19 presenting at the emergency room of a large academic hospital. Blood drawn within 2 d after hospital presentation was analyzed by point‐of‐care automated flow cytometry and compared with blood samples collected at later time points. COVID‐19 patients did not exhibit neutrophilia or eosinopenia. Unexpectedly neutrophil activation markers (CD11b, CD16, CD10, and CD62L) did not differ between COVID‐19‐positive patients and COVID‐19‐negative patients diagnosed with other bacterial/viral infections, or between COVID‐19 severity groups. In all patients, a decrease was found in the neutrophil maturation markers indicating an inflammation‐induced left shift of the neutrophil compartment. In COVID‐19 this was associated with disease severity.