TLR Signaling Rescues Fungicidal Activity in Syk-Deficient Neutrophils

Abstract

An impaired neutrophil response to pathogenic fungi puts patients at risk for fungal infections with a high risk of morbidity and mortality. Acquired neutrophil dysfunction in the setting of iatrogenic immune modulators can include the inhibition of critical kinases such as spleen tyrosine kinase (Syk). In this study, we used an established system of conditionally immortalized mouse neutrophil progenitors to investigate the ability to augment Syk-deficient neutrophil function against Candida albicans with TLR agonist signaling. LPS, a known immunomodulatory molecule derived from Gram-negative bacteria, was capable of rescuing effector functions of Syk-deficient neutrophils, which are known to have poor fungicidal activity against Candida species. LPS priming of Syk-deficient mouse neutrophils demonstrates partial rescue of fungicidal activity, including phagocytosis, degranulation, and neutrophil swarming, but not reactive oxygen species production against C. albicans, in part due to c-Fos activation. Similarly, LPS priming of human neutrophils rescues fungicidal activity in the presence of pharmacologic inhibition of Syk and Bruton’s tyrosine kinase (Btk), both critical kinases in the innate immune response to fungi. In vivo, neutropenic mice were reconstituted with wild-type or Syk-deficient neutrophils and challenged i.p. with C. albicans. In this model, LPS improved wild-type neutrophil homing to the fungal challenge, although Syk-deficient neutrophils did not persist in vivo, speaking to its crucial role on in vivo persistence. Taken together, we identify TLR signaling as an alternate activation pathway capable of partially restoring neutrophil effector function against Candida in a Syk-independent manner.

Decoding the biology and clinical implication of neutrophils in intracranial aneurysm

“…S3A ). The core transcriptional regulatory network of the neutrophils included Module 1, comprising the CEBPB and FOS / JUN families that regulates neutrophil function, 46 , 47 and Module 2 comprising mainly NF‐κB that is involved in response to stimuli, function, and death. 48 , 49 Neutrophil subclusters differed in the level of activation of Module 1 and Module 2.…”

Innate immune signal transduction pathways to fungal infection: Components and regulation

“…recently identified the C. albicans small secreted cysteine-rich protein Sel1 is a novel PAMP for TLR2 and TLR4 ( Wang et al., 2019 ). Several TLRs including TLR2 and TLR4 coordinate with Dectin-1 for fungal recognition ( Gantner et al., 2003 ; Viens et al., 2022 ; Wang et al., 2019 ; Willcocks et al., 2013 ). TLRs link the downstream adaptors MyD88 (myeloid differentiation primary response 88) to activate the signaling including nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) such as extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 for the induction of proinflammatory cytokines and chemokines ( Gantner et al., 2003 ).…”

Multiparametric Profiling of Neutrophil Function via a High-Throughput Flow Cytometry-Based Assay

“…After the 30 min, the plate was placed on ice for 10 min in the dark and prepared for flow cytometry. During rescue studies, neutrophils were first treated with kinase inhibitors for 30 min [37] in an incubator. LPS (400 ng/mL) was then added to the well and the plate was returned to the incubator for an additional 45 min [37] prior to co-culturing neutrophils with C. albicans for 30 min.…”

“…Inhibition of downstream kinases (e.g., Btk, Syk, Src) within the Dectin-1 signal transduction pathway can render neutrophils unresponsive to fungal pathogens such as C. albicans or Aspergillus fumigatus [42,43]. Syk and Btk inhibition can completely block neutrophil functions, including swarming, phagocytosis, oxidative burst, and cytokine production, even when challenged with C. albicans or A. fumigatus [37,44,45]. In the context of our assay, the inhibition of Btk, Syk, and Src, prevented phagocytosis, ROS production, ectodomain shedding, and secondary granule release (Figure 7A-D Neutrophil inhibition by ibrutinib (IBT) or R406 can be overcome by alternate pathway stimulation (e.g., TLR stimulation using LPS) before challenging with C. albicans [37].…”

“…Syk and Btk inhibition can completely block neutrophil functions, including swarming, phagocytosis, oxidative burst, and cytokine production, even when challenged with C. albicans or A. fumigatus [37,44,45]. In the context of our assay, the inhibition of Btk, Syk, and Src, prevented phagocytosis, ROS production, ectodomain shedding, and secondary granule release (Figure 7A-D Neutrophil inhibition by ibrutinib (IBT) or R406 can be overcome by alternate pathway stimulation (e.g., TLR stimulation using LPS) before challenging with C. albicans [37]. LPS stimulation alone did not trigger ROS production in pre-treatment conditions when C. albicans was not present (Figure 7B).…”