Chemokines are an indispensable component of our immune system through the regulation of directional migration and activation of leukocytes. CXCL8 is the most potent human neutrophil-attracting chemokine and plays crucial roles in the response to infection and tissue injury. CXCL8 activity inherently depends on interaction with the human CXC chemokine receptors CXCR1 and CXCR2, the atypical chemokine receptor ACKR1, and glycosaminoglycans. Furthermore, (hetero)dimerization and tight regulation of transcription and translation, as well as post-translational modifications further fine-tune the spatial and temporal activity of CXCL8 in the context of inflammatory diseases and cancer. The CXCL8 interaction with receptors and glycosaminoglycans is therefore a promising target for therapy, as illustrated by multiple ongoing clinical trials. CXCL8-mediated neutrophil mobilization to blood is directly opposed by CXCL12, which retains leukocytes in bone marrow. CXCL12 is primarily a homeostatic chemokine that induces migration and activation of hematopoietic progenitor cells, endothelial cells, and several leukocytes through interaction with CXCR4, ACKR1, and ACKR3. Thereby, it is an essential player in the regulation of embryogenesis, hematopoiesis, and angiogenesis. However, CXCL12 can also exert inflammatory functions, as illustrated by its pivotal role in a growing list of pathologies and its synergy with CXCL8 and other chemokines to induce leukocyte chemotaxis. Here, we review the plethora of information on the CXCL8 structure, interaction with receptors and glycosaminoglycans, different levels of activity regulation, role in homeostasis and disease, and therapeutic prospects. Finally, we discuss recent research on CXCL12 biochemistry and biology and its role in pathology and pharmacology.

Neutrophils are recognized as important circulating effector cells in the pathophysiology of severe coronavirus disease 2019 (COVID-19). However, their role within the inflamed lungs is incompletely understood. Here, we collected bronchoalveolar lavage (BAL) fluids and parallel blood samples of critically ill COVID-19 patients requiring invasive mechanical ventilation and compared BAL fluid parameters with those of mechanically ventilated patients with influenza, as a non–COVID-19 viral pneumonia cohort. Compared with those of patients with influenza, BAL fluids of patients with COVID-19 contained increased numbers of hyperactivated degranulating neutrophils and elevated concentrations of the cytokines IL-1 β , IL-1RA, IL-17A, TNF- α , and G-CSF; the chemokines CCL7, CXCL1, CXCL8, CXCL11, and CXCL12 α ; and the protease inhibitors elafin, secretory leukocyte protease inhibitor, and tissue inhibitor of metalloproteinases 1. In contrast, α -1 antitrypsin levels and net proteolytic activity were comparable in COVID-19 and influenza BAL fluids. During antibiotic treatment for bacterial coinfections, increased BAL fluid levels of several activating and chemotactic factors for monocytes, lymphocytes, and NK cells were detected in patients with COVID-19 whereas concentrations tended to decrease in patients with influenza, highlighting the persistent immunological response to coinfections in COVID-19. Finally, the high proteolytic activity in COVID-19 lungs suggests considering protease inhibitors as a treatment option.

Objectives: Emerging evidence of dysregulation of the myeloid cell compartment urges investigations on neutrophil characteristics in coronavirus disease 2019 (COVID‐19). We isolated neutrophils from the blood of COVID‐19 patients receiving general ward care and from patients hospitalised at intensive care units (ICUs) to explore the kinetics of circulating neutrophils and factors important for neutrophil migration and activation.Methods: Multicolour flow cytometry was exploited for the analysis of neutrophil differentiation and activation markers. Multiplex and ELISA technologies were used for the quantification of protease, protease inhibitor, chemokine and cytokine concentrations in plasma. Neutrophil polarisation responses were evaluated microscopically. Gelatinolytic and metalloproteinase activity in plasma was determined using a fluorogenic substrate. Co‐culturing healthy donor neutrophils with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) allowed us to investigate viral replication in neutrophils.Results: Upon ICU admission, patients displayed high plasma concentrations of granulocyte–colony‐stimulating factor (G‐CSF) and the chemokine CXCL8, accompanied by emergency myelopoiesis as illustrated by high levels of circulating CD10⁻, immature neutrophils with reduced CXCR2 and C5aR expression. Neutrophil elastase and non‐metalloproteinase‐derived gelatinolytic activity were increased in plasma from ICU patients. Significantly higher levels of circulating tissue inhibitor of metalloproteinase 1 (TIMP‐1) in patients at ICU admission yielded decreased total MMP proteolytic activity in blood. COVID‐19 neutrophils were hyper‐responsive to CXCL8 and CXCL12 in shape change assays. Finally, SARS‐CoV‐2 failed to replicate inside human neutrophils.Conclusion: Our study provides detailed insights into the kinetics of neutrophil phenotype and function in severe COVID‐19 patients, and supports the concept of an increased neutrophil activation state in the circulation.

Patients with severe COVID-19 develop acute respiratory distress syndrome (ARDS) that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that complement component 5a (C5a), through its cellular receptor C5aR1, has potent proinflammatory actions and plays immunopathological roles in inflammatory diseases, we investigated whether the C5a/C5aR1 pathway could be involved in COVID-19 pathophysiology. C5a/C5aR1 signaling increased locally in the lung, especially in neutrophils of critically ill patients with COVID-19 compared with patients with influenza infection, as well as in the lung tissue of K18-hACE2 Tg mice (Tg mice) infected with SARS-CoV-2. Genetic and pharmacological inhibition of C5aR1 signaling ameliorated lung immunopathology in Tg-infected mice. Mechanistically, we found that C5aR1 signaling drives neutrophil extracellular traps-dependent (NETs-dependent) immunopathology. These data confirm the immunopathological role of C5a/C5aR1 signaling in COVID-19 and indicate that antagonists of C5aR1 could be useful for COVID-19 treatment.

Although glycosaminoglycan (GAG)–protein interactions are important in many physiological and pathological processes, the structural requirements for binding are poorly defined. Starting with GAG-binding peptide CXCL9(74-103), peptides were designed to elucidate the contribution to the GAG-binding affinity of different: (1) GAG-binding motifs (i.e., BBXB and BBBXXB); (2) amino acids in GAG-binding motifs and linker sequences; and (3) numbers of GAG-binding motifs. The affinity of eight chemically synthesized peptides for various GAGs was determined by isothermal fluorescence titration (IFT). Moreover, the binding of peptides to cellular GAGs on Chinese hamster ovary (CHO) cells was assessed using flow cytometry with and without soluble GAGs. The repetition of GAG-binding motifs in the peptides contributed to a higher affinity for heparan sulfate (HS) in the IFT measurements. Furthermore, the presence of Gln residues in both GAG-binding motifs and linker sequences increased the affinity of trimer peptides for low-molecular-weight heparin (LMWH), partially desulfated (ds)LMWH and HS, but not for hyaluronic acid. In addition, the peptides bound to cellular GAGs with differential affinity, and the addition of soluble HS or heparin reduced the binding of CXCL9(74-103) to cellular GAGs. These results indicate that the affinity and specificity of peptides for GAGs can be tuned by adapting their amino acid sequence and their number of GAG-binding motifs.

To improve outcomes following lung transplantation, it is essential to understand the immunological mechanisms that result in chronic graft failure. The associated clinical syndrome is termed chronic lung allograft dysfunction (CLAD), which is known to be induced by alloimmune-dependent (i.e., rejection) and alloimmune-independent factors (e.g., infections, reflux and environmental factors). We aimed to explore the alloimmune-related mechanism, i.e., pulmonary rejection. In this study, we use a murine orthotopic left lung transplant model using isografts and allografts (C57BL/6 or BALB/c as donors to C57BL/6 recipients), with daily immunosuppression (10 mg/kg cyclosporin A and 1.6 mg/kg methylprednisolone). Serial sacrifice was performed at days 1, 7 and 35 post-transplantation (n = 6 at each time point for each group). Left transplanted lungs were harvested, a single-cell suspension was made and absolute numbers of immune cells were quantified using multicolor flow cytometry. The rejection process followed the principles of a classic immune response, including innate but mainly adaptive immune cells. At day 7 following transplantation, the numbers of interstitial macrophages, monocytes, dendritic cells, NK cells, NKT cells, CD4+ T cells and CD8+ T and B cells were increased in allografts compared with isografts. Only dendritic cells and CD4+ T cells remained elevated at day 35 in allografts. Our study provides insights into the immunological mechanisms of true pulmonary rejection after murine lung transplantation. These results might be important in further research on diagnostic evaluation and treatment for CLAD.