Secretory leukocyte protease inhibitor (SLPI) is described as a potent regulator of inflammation and tissue homeostasis with pleiotropic functions. It has been shown to inhibit pro-inflammatory responses in myeloid cells. However, its expression patterns and specific functions in different monocyte and macrophage populations remain poorly understood. Therefore, we investigated its expression patterns in murine tissue macrophage populations by analysis of publicly available datasets and flow cytometry. Among various tissues, peritoneal macrophages were identified as a major source of SLPI, suggesting the highest impact of this inhibitor on their physiological and pathophysiological functions. To elucidate the role of SLPI in the inflammatory response, SLPI-deficient mice were used. First, the response to LPS was compared in resident and thioglycolate-recruited peritoneal macrophages. Moreover, we evaluated the role of SLPI in an in vivo mouse model of LPS-induced septic shock. Results demonstrated that while the lack of SLPI did not affect pro-inflammatory cytokine production in activated resident macrophages, it regulated the production of matrix metalloproteinase-9 (MMP-9). Similar results were observed in thioglycolate-elicited and LPS-activated peritoneal macrophage populations, further highlighting the link between SLPI and MMP-9. Furthermore, in vivo LPS-induced changes in SLPI expression were evident among various myeloid populations, including monocytes. Loss of SLPI also influenced the frequency of blood monocyte populations in this model. Overall, these findings highlight a specific role for SLPI in regulating MMP-9 in response to LPS both in vitro and in vivo and suggest that SLPI might play a role in tissue remodeling orchestrated by macrophages.
Copyright © 2025 Tyshchenko, Pocałuń, Kwiecińska, Cichy, Wilk and Oleszycka.

The notion that neutrophils exist as a homogeneous population is being replaced with the knowledge that neutrophils adopt different functional states. Neutrophils can have a pro-inflammatory phenotype or an anti-inflammatory state, but how these states are regulated remains unclear. Here, we demonstrated that the neutrophil-expressed G-protein-coupled receptor (GPCR) Mrgpra1 is a negative regulator of neutrophil bactericidal functions. Mrgpra1-mediated signaling was driven by its ligand, neuropeptide FF (NPFF), which dictated the balance between pro- and anti-inflammatory programming. Specifically, the Mrgpra1-NPFF axis counter-regulated interferon (IFN) γ-mediated neutrophil polarization during acute lung infection by favoring an alternative-like polarization, suggesting that it may act to balance overzealous neutrophilic responses. Distinct, cross-regulated populations of neutrophils were the primary source of NPFF and IFNγ during infection. As a subset of neutrophils at steady state expressed NPFF, these findings could have broad implications in various infectious and inflammatory diseases. Therefore, a neutrophil-intrinsic pathway determines their cellular fate, function, and magnitude of infection.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

Respiratory syncytial virus (RSV) infection in infants and toddlers is a major public health problem. Here, we provide a protocol for neonatal RSV infection in mice and immune analysis of infected lungs and bronchoalveolar lavage (BAL) fluid. We describe steps for anesthesia and intranasal inoculation, weight monitoring, and whole lung collection. We then detail BAL fluid immune and whole lung analyses. This protocol can be used for neonatal pulmonary infection with other viruses or bacteria.
Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.

In severe bacterial infections, there is a pro-inflammatory response to promote bacterial clearance but this response can cause tissue injury. Later, the immune system becomes dysregulated and the host is unable to clear a secondary or a pre-existing infection. Specialized Pro-resolving Mediators (SPMs) such as resolvin D2 (RvD2) have been shown to be beneficial for inflammation/infection resolution in animal models of sepsis but in vivo mechanisms by which RvD2 may promote bacterial clearance and/or attenuate deleterious effects of a secondary infection have not been fully established. In this study, we used the 2-hit model of cecal ligation and puncture (CLP) induced infectious peritonitis and secondary lung infection with Pseudomonas aeruginosa to find possible antimicrobial and immunomodulatory mechanisms of RvD2. We show that RvD2 given as late as 48h after CLP surgery reduced blood bacterial load without altering plasma cytokines compared to mice given saline vehicle. RvD2 increased splenic neutrophil accumulation as well as average reactive oxygen species (ROS) production. There was also an increase in an immature leukocyte population the myeloid derived suppressor cells (MDSCs) in the spleen of RvD2 treated mice. RvD2 reduced lung lavage bacterial load 24h after P. aeruginosa administration and significantly decreased lung lavage levels of IL-23, a cytokine essential in the Th-17 inflammatory response. In addition, we show that RvD2 increased the number of non-inflammatory alveolar macrophages after P. aeruginosa administration compared to saline treated mice. The study uncovered an antimicrobial mechanism of RvD2 where RvD2 increases mature neutrophil and MDSC accumulation into the spleen to promote blood bacterial clearance. The study showed that in this 2-hit model, RvD2 promotes lung bacterial clearance, increased non-inflammatory alveolar macrophage number and inhibits an adaptive immune pathway providing evidence of its resolution mechanism in secondary pulmonary infection.
Copyright © 2022 Sundarasivarao, Walker, Rodriguez, Spur and Yin.

Persistent neutrophil-dominated lung inflammation contributes to lung damage in cystic fibrosis (CF). However, the mechanisms that drive persistent lung neutrophilia and tissue deterioration in CF are not well characterized. Starting from the observation that, in patients with CF, c-c motif chemokine receptor 2 (CCR2)+ monocytes/macrophages are abundant in the lungs, we investigate the interplay between monocytes/macrophages and neutrophils in perpetuating lung tissue damage in CF. Here we show that CCR2+ monocytes in murine CF lungs drive pathogenic transforming growth factor β (TGF-β) signaling and sustain a pro-inflammatory environment by facilitating neutrophil recruitment. Targeting CCR2 to lower the numbers of monocytes in CF lungs ameliorates neutrophil inflammation and pathogenic TGF-β signaling and prevents lung tissue damage. This study identifies CCR2+ monocytes as a neglected contributor to the pathogenesis of CF lung disease and as a therapeutic target for patients with CF, for whom lung hyperinflammation and tissue damage remain an issue despite recent advances in CF transmembrane conductance regulator (CFTR)-specific therapeutic agents.Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.