Interaction of kindlin-3 and β2-integrins differentially regulates neutrophil recruitment and NET release in mice

Abstract

• Kindlin-3-b 2 -integrin signaling in neutrophils is involved in regulation of both neutrophil recruitment and NET release.• Disrupting the crosstalk between kindlin-3 and b 2 -integrins in neutrophils with a blocking peptide preferentially attenuates NET release.Kindlin-3 essentially supports integrin activation in blood cells. Absence of kindlin-3 in humans causes leukocyte adhesion deficiency-III characterized with severe bleeding disorder and recurrent infections. Previously, we generated kindlin-3 knock-in (K3KI) mice carrying an integrin-interaction disrupting mutation in kindlin-3 and verified the functional significance of the binding of kindlin-3 to integrin aIIbb3 in platelets. Here, using K3KI mice, we functionally evaluate the crosstalk between kindlin-3 and b 2 -integrins in neutrophils. Although the kindlin-3 mutant in K3KI neutrophils is normally expressed, its binding ability to b 2 -integrins in neutrophils is disabled. In vitro and in vivo analyses disclose that b 2 -integrin-mediated K3KI neutrophil adhesion and recruitment are significantly suppressed. Interestingly, the ability of releasing neutrophil extracellular traps (NETs) from K3KI neutrophils is also compromised. Substantially, a peptide derived from the integrin b 2 cytoplasmic tail that can inhibit the interaction between kindlin-3 and b 2 -inegrins significantly jeopardizes NET release without affecting neutrophil adhesion and recruitment under the experimental conditions. These findings suggest that crosstalk between kindlin-3 and b 2 -integrins in neutrophils is required for supporting both neutrophil recruitment and NET release, but the involved regulatory mechanisms in these two cellular events might be differential, thus providing a novel therapeutic concept to treat innate immune-related diseases. (Blood. 2015;126(3):373-377) IntroductionNeutrophil recruitment and neutrophil extracellular trap (NET) release are hallmarks of innate immune responses. The engagement of b 2 -integrins on neutrophils with their ligands mediates firm adhesion of neutrophils to inflamed endothelium 1,2 and may also be involved in regulating NET release.3,4 Ligand-binding capability of b 2 -integrins on leukocytes is tightly regulated by a process of integrin activation, which is triggered by extracellular agonists and eventually implemented by the integrin a/b cytoplasmic tail (CT) binding proteins. 5,6 Kindlins are recently identified integrin b CT binding proteins, and kindlin-3, one of the 3 kindlin family members, is mainly expressed in hematopoietic cells.7-9 Kindlin-3 mutants in humans are responsible for type III leukocyte adhesion deficiency (LAD-III) with severe bleeding tendency and recurrent infections due to the dysfunction of integrins in both leukocytes and platelets. 10,11 Since the first case was described in 1997, a list of LAD-III patients from more than 20 families have been reported so far. The identified kindlin-3 mutations in these patients vary but all result in undetectable expression of kindlin-3 protein in blood cells...

Neutrophil extracellular traps — the dark side of neutrophils

“…These pathways include activation by integrins (complement receptor) and Toll-like receptors (14)(15)(16). Chemokine receptors are necessary for integrin activation and signaling (10,17). Integrins generate signals that elicit NETosis in response to bacteria, and NETs are not produced when integrins are deficient, such as in leukocyte adhesion deficiency (18).…”

The role of neutrophils and NETosis in autoimmune and renal diseases

“…Inhibition of neutrophil recruitment and NET release by disrupting the cross-talk between kindlin-3 and β2-integrin, is also an interesting therapeutic avenue 159 . CXCL5 could also be targeted to restrict pathogenic neutrophil infiltration in T H 17-mediated autoimmune diseases, especially crescentic glomerulonephritis, while retaining the protective function of neutrophils against invading pathogens 132 .…”

Regulation of tissue infiltration by neutrophils

“…Neutrophil recruitment to necrotic tissues is mediated by high-mobility group box 1 (HMGB1) and its receptor, receptor for advanced glycation end products (RAGE) [31]; here, a potential link to integrin-dependent neutrophil recruitment may exist, as RAGE interacts with the β2-integrin Mac-1 [32, 33]. An important player in leukocyte integrin activation is kindlin-3, which interacts with the cytoplasmic tail of β2-integrins [34], and the absence of which causes leukocyte adhesion deficiency (LAD) Type III [35]. In genetically modified mice that express a mutant form of kindlin-3 that is incapable of interacting with integrins, neutrophil adhesion and recruitment in the course of systemic inflammation is decreased [34].…”