Neutrophil functions in patients with neutropenia due to glycogen storage disease type 1b treated with empagliflozin.

ABSTRACT: Neutropenia and neutrophil dysfunction in glycogen storage disease type 1b (GSD1b) are caused by the accumulation of 1,5-anhydroglucitol-6-phosphate in granulocytes. The antidiabetic drug empagliflozin reduces the concentration of 1,5-anhydroglucitol (1,5-AG), thus restoring neutrophil counts and functions, leading to promising results in previous case reports. Here, we present a comprehensive analysis of neutrophil function in 7 patients with GSD1b and 11 healthy donors, aiming to evaluate the immediate (after 3 months) and long-term (after 12 months) efficacy of empagliflozin compared with the reference treatment with granulocyte-colony stimulating factor (G-CSF). We found that most patients receiving G-CSF remained neutropenic with dysfunctional granulocytes, whereas treatment with empagliflozin increased neutrophil counts and improved functionality by inhibiting apoptosis, restoring phagocytosis and the chemotactic response, normalizing the oxidative burst, and stabilizing cellular and plasma levels of defensins and lactotransferrin. These improvements correlated with the decrease in serum 1,5-AG levels. However, neither G-CSF nor empagliflozin overcame deficiencies in the production of cathelicidin/LL-37 and neutrophil extracellular traps. Given the general improvement promoted by empagliflozin treatment, patients were less susceptible to severe infections. G-CSF injections were therefore discontinued in 6 patients (and the dose was reduced in the seventh) without adverse effects. Our systematic analysis, the most extensive reported thus far, has demonstrated the superior efficacy of empagliflozin compared with G-CSF, restoring the neutrophil population and normal immune functions. This trial was registered as EudraCT 2021-000580-78.

MCP-Induced Protein 1 Participates in Macrophage-Dependent Endotoxin Tolerance.

Endotoxin tolerance is a state of hyporesponsiveness to LPS, triggered by previous exposure to endotoxin. Such an immunosuppressive state enhances the risks of secondary infection and has been associated with the pathophysiology of sepsis. Although this phenomenon has been extensively studied, its molecular mechanism is not fully explained. Among candidates that play a crucial role in this process are negative regulators of TLR signaling, but the contribution of MCP-induced protein 1 (MCPIP1; Regnase-1) has not been studied yet. To examine whether macrophage expression of MCPIP1 participates in endotoxin tolerance, we used both murine and human primary macrophages devoid of MCPIP1 expression. In our study, we demonstrated that MCPIP1 contributes to LPS hyporesponsiveness induced by subsequent LPS stimulation and macrophage reprogramming. We proved that this mechanism revolves around the deubiquitinase activity of MCPIP1, which inhibits the phosphorylation of MAPK and NF-κB activation. Moreover, we showed that MCPIP1 controlled the level of proinflammatory transcripts in LPS-tolerized cells independently of its RNase activity. Finally, we confirmed these findings applying an in vivo endotoxin tolerance model in wild-type and myeloid MCPIP1-deficient mice. Taken together, this study describes for the first time, to our knowledge, that myeloid MCPIP1 participates in endotoxin tolerance and broadens the scope of known negative regulators of the TLR4 pathway crucial in this phenomenon.

MCPIP-1 Restricts Inflammation via Promoting Apoptosis of Neutrophils.

Monocyte chemoattractant protein-induced protein-1 (MCPIP-1) is a potent inhibitor of inflammatory response to pathogens. Acting as endonuclease against transcripts of inflammatory cytokines or transcription factors MCPIP-1 can significantly reduce the cytokine storm, thus limiting the tissue damage. As the adequate resolution of inflammation depends also on the efficient clearance of accumulated neutrophils, we focused on the role of MCPIP-1 in apoptosis and retention of neutrophils. We used peritoneal neutrophils from cell-specific MCPIP-1 knockout mice and showed prolonged survival of these cells. Moreover, we confirmed that MCPIP-1-dependent degradation of transcripts of antiapoptotic genes, including BCL3, BCL2A1, BCL2L1, and for the first time MCL-1, serves as an early event in spontaneous apoptosis of primary neutrophils. Additionally, we identified previously unknown miRNAs as potential binding partners to the MCPIP-1 transcript and their regulation suggest a role in MCPIP-1 half-life and translation. These phenomena may play a role as a molecular switch that balances the MCPIP-1-dependent apoptosis. Besides that, we determined these particular miRNAs as integral components of the GM-CSF-MCPIP-1 axis. Taken together, we identified the novel anti-inflammatory role of MCPIP-1 as a regulator of accumulation and survival of neutrophils that simultaneously promotes an adequate resolution of inflammation.

Citrullination-Resistant LL-37 Is a Potent Antimicrobial Agent in the Inflammatory Environment High in Arginine Deiminase Activity.

LL-37, the only member of the mammalian cathelicidin in humans, plays an essential role in innate immunity by killing pathogens and regulating the inflammatory response. However, at an inflammatory focus, arginine residues in LL-37 can be converted to citrulline via a reaction catalyzed by peptidyl-arginine deiminases (PAD2 and PAD4), which are expressed in neutrophils and are highly active during the formation of neutrophil extracellular traps (NETs). Citrullination impairs the bactericidal activity of LL-37 and abrogates its immunomodulatory functions. Therefore, we hypothesized that citrullination-resistant LL-37 variants would retain the functionality of the native peptide in the presence of PADs. To test this hypothesis, we synthetized LL-37 in which arginine residues were substituted by homoarginine (hArg-LL-37). Bactericidal activity of hArg-LL-37 was comparable with that of native LL-37, but neither treatment with PAD4 nor exposure to NETs affected the antibacterial and immunomodulatory activities of hArg-LL-37. Importantly, the susceptibilities of LL-37 and hArg-LL-37 to degradation by proteases did not significantly differ. Collectively, we demonstrated that citrullination-resistant hArg-LL-37 is an attractive lead compound for the generation of new agents to treat bacterial infections and other inflammatory diseases associated with enhanced PAD activity. Moreover, our results provide a proof-of-concept for synthesis of therapeutic peptides using homoarginine.

Discovery of Novel Potential Reversible Peptidyl Arginine Deiminase Inhibitor.

Citrullination, a posttranslational modification, is catalyzed by peptidylarginine deiminases (PADs), a unique family of enzymes that converts peptidyl-arginine to peptidyl-citrulline. Overexpression and/or increased PAD activity is observed in rheumatoid arthritis (RA), Alzheimer's disease, multiple sclerosis, and cancer. Moreover, bacterial PADs, such as Porphyromonas gingivalis PAD (PPAD), may have a role in the pathogenesis of RA, indicating PADs as promising therapeutic targets. Herein, six novel compounds were examined as potential inhibitors of human PAD4 and PPAD, and compared to an irreversible PAD inhibitor, Cl-amidine. Four of the tested compounds (compounds 2, 3, 4, and 6) exhibited a micromolar-range inhibition potency against PAD4 and no effect against PPAD in the in vitro assays. Compound 4 was able to inhibit the PAD4-induced citrullination of H3 histone with higher efficiency than Cl-amidine. In conclusion, compound 4 was highly effective and presents a promising direction in the search for novel RA treatment strategies.

Triggering NETosis via protease-activated receptor (PAR)-2 signaling as a mechanism of hijacking neutrophils function for pathogen benefits.

Neutrophil-derived networks of DNA-composed extracellular fibers covered with antimicrobial molecules, referred to as neutrophil extracellular traps (NETs), are recognized as a physiological microbicidal mechanism of innate immunity. The formation of NETs is also classified as a model of a cell death called NETosis. Despite intensive research on the NETs formation in response to pathogens, the role of specific bacteria-derived virulence factors in this process, although postulated, is still poorly understood. The aim of our study was to determine the role of gingipains, cysteine proteases responsible for the virulence of P. gingivalis, on the NETosis process induced by this major periodontopathogen. We showed that NETosis triggered by P. gingivalis is gingipain dependent since in the stark contrast to the wild-type strain (W83) the gingipain-null mutant strain only slightly induced the NETs formation. Furthermore, the direct effect of proteases on NETosis was documented using purified gingipains. Notably, the induction of NETosis was dependent on the catalytic activity of gingipains, since proteolytically inactive forms of enzymes showed reduced ability to trigger the NETs formation. Mechanistically, gingipain-induced NETosis was dependent on proteolytic activation of protease-activated receptor-2 (PAR-2). Intriguingly, both P. gingivalis and purified Arg-specific gingipains (Rgp) induced NETs that not only lacked bactericidal activity but instead stimulated the growth of bacteria species otherwise susceptible to killing in NETs. This protection was executed by proteolysis of bactericidal components of NETs. Taken together, gingipains play a dual role in NETosis: they are the potent direct inducers of NETs formation but in the same time, their activity prevents P. gingivalis entrapment and subsequent killing. This may explain a paradox that despite the massive accumulation of neutrophils and NETs formation in periodontal pockets periodontal pathogens and associated pathobionts thrive in this environment.

A Novel Biological Role for Peptidyl-Arginine Deiminases: Citrullination of Cathelicidin LL-37 Controls the Immunostimulatory Potential of Cell-Free DNA.

LL-37, the only human cathelicidin that is released during inflammation, is a potent regulator of immune responses by facilitating delivery of oligonucleotides to intracellular TLR-9, thereby enhancing the response of human plasmacytoid dendritic cells (pDCs) to extracellular DNA. Although important for pathogen recognition, this mechanism may facilitate development of autoimmune diseases. In this article, we show that citrullination of LL-37 by peptidyl-arginine deiminases (PADs) hindered peptide-dependent DNA uptake and sensing by pDCs. In contrast, carbamylation of the peptide (homocitrullination of Lys residues) had no effect. The efficiency of LL-37 binding to oligonucleotides and activation of pDCs was found to be inversely proportional to the number of citrullinated residues in the peptide. Similarly, preincubation of carbamylated LL-37 with PAD2 abrogated the peptide's ability to bind DNA. Conversely, LL-37 with Arg residues substituted by homoarginine, which cannot be deiminated, elicited full activity of native LL-37 regardless of PAD2 treatment. Taken together, the data showed that citrullination abolished LL-37 ability to bind DNA and altered the immunomodulatory function of the peptide. Both activities were dependent on the proper distribution of guanidinium side chains in the native peptide sequence. Moreover, our data suggest that cathelicidin/LL-37 is citrullinated by PADs during NET formation, thus affecting the inflammatory potential of NETs. Together this may represent a novel mechanism for preventing the breakdown of immunotolerance, which is dependent on the response of APCs to self-molecules (including cell-free DNA); overactivation may facilitate development of autoimmunity.

Calcium Regulates the Activity and Structural Stability of Tpr, a Bacterial Calpain-like Peptidase.

Porphyromonas gingivalis is a peptide-fermenting asaccharolytic periodontal pathogen. Its genome contains several genes encoding cysteine peptidases other than gingipains. One of these genes (PG1055) encodes a protein called Tpr (thiol protease) that has sequence similarity to cysteine peptidases of the papain and calpain families. In this study we biochemically characterize Tpr. We found that the 55-kDa Tpr inactive zymogen proteolytically processes itself into active forms of 48, 37, and 33 kDa via sequential truncations at the N terminus. These processed molecular forms of Tpr are associated with the bacterial outer membrane where they are likely responsible for the generation of metabolic peptides required for survival of the pathogen. Both autoprocessing and activity were dependent on calcium concentrations >1 mm, consistent with the protein's activity within the intestinal and inflammatory milieus. Calcium also stabilized the Tpr structure and rendered the protein fully resistant to proteolytic degradation by gingipains. Together, our findings suggest that Tpr is an example of a bacterial calpain, a calcium-responsive peptidase that may generate substrates required for the peptide-fermenting metabolism of P. gingivalis. Aside from nutrient generation, Tpr may also be involved in evasion of host immune response through degradation of the antimicrobial peptide LL-37 and complement proteins C3, C4, and C5. Taken together, these results indicate that Tpr likely represents an important pathogenesis factor for P. gingivalis.

The Janus face of α-toxin: a potent mediator of cytoprotection in staphylococci-infected macrophages.

After phagocytosis by macrophages, Staphylococcus aureus evades killing in an α-toxin-dependent manner, and then prevents apoptosis of infected cells by upregulating expression of antiapoptotic genes like MCL-1 (myeloid cell leukemia-1). Here, using purified α-toxin and a set of hla-deficient strains, we show that α-toxin is critical for the induction of MCL-1 expression and the cytoprotection of infected macrophages. Extracellular or intracellular treatment of macrophages with α-toxin alone did not induce cytoprotection conferred by increased Mcl-1, suggesting that the process is dependent on the production of α-toxin by intracellular bacteria. The increased expression of MCL-1 in infected cells was associated with enhanced NFκB activation, and subsequent IL-6 secretion. This effect was only partially inhibited by blocking TLR2, which suggests the participation of intracellular receptors in the specific recognition of S. aureus strains secreting α-toxin. Thus, S. aureus recognition by intracellular receptors and/or activation of downstream pathways leading to Mcl-1 expression is facilitated by α-toxin released by intracellular bacteria which permeabilize phagosomes, ensuring pathogen access to the cytoplasmatic compartment. Given that the intracellular survival of S. aureus depends on α-toxin, we propose a novel role for this agent in the protection of the intracellular niche, and further dissemination of staphylococci by infected macrophages.

Mirolase, a novel subtilisin-like serine protease from the periodontopathogen Tannerella forsythia.

The genome of Tannerella forsythia, an etiological factor of chronic periodontitis, contains several genes encoding putative proteases. Here, we characterized a subtilisin-like serine protease of T. forsythia referred to as mirolase. Recombinant full-length latent promirolase [85 kDa, without its signal peptide (SP)] processed itself through sequential autoproteolytic cleavages into a mature enzyme of 40 kDa. Mirolase latency was driven by the N-terminal prodomain (NTP). In stark contrast to almost all known subtilases, the cleaved NTP remained non-covalently associated with mirolase, inhibiting its proteolytic, but not amidolytic, activity. Full activity was observed only after the NTP was gradually, and fully, degraded. Both activity and processing was absolutely dependent on calcium ions, which were also essential for enzyme stability. As a consequence, both serine protease inhibitors and calcium ions chelators inhibited mirolase activity. Activity assays using an array of chromogenic substrates revealed that mirolase specificity is driven not only by the substrate-binding subsite S1, but also by other subsites. Taken together, mirolase is a calcium-dependent serine protease of the S8 family with the unique mechanism of activation that may contribute to T. forsythia pathogenicity by degradation of fibrinogen, hemoglobin, and the antimicrobial peptide LL-37.

Citrullination alters immunomodulatory function of LL-37 essential for prevention of endotoxin-induced sepsis.

Cathelicidin LL-37 plays an essential role in innate immunity by killing invading microorganisms and regulating the inflammatory response. These activities depend on the cationic character of the peptide, which is conferred by arginine and lysine residues. At inflammatory foci in vivo, LL-37 is exposed to peptidyl arginine deiminase (PAD), an enzyme released by inflammatory cells. Therefore, we hypothesized that PAD-mediated citrullination of the arginine residues within LL-37 will abrogate its immunomodulatory functions. We found that, when citrullinated, LL-37 was at least 40 times less efficient at neutralizing the proinflammatory activity of LPS due to a marked decrease in its affinity for endotoxin. Also, the ability of citrullinated LL-37 to quench macrophage responses to lipoteichoic acid and poly(I:C) signaling via TLR2 and TLR3, respectively, was significantly reduced. Furthermore, in stark contrast to native LL-37, the modified peptide completely lost the ability to prevent morbidity and mortality in a mouse model of d-galactosamine-sensitized endotoxin shock. In fact, administration of citrullinated LL-37 plus endotoxin actually exacerbated sepsis due to the inability of LL-37 to neutralize LPS and the subsequent enhancement of systemic inflammation due to increased serum levels of IL-6. Importantly, serum from septic mice showed increased PAD activity, which strongly correlated with the level of citrullination, indicating that PAD-driven protein modification occurs in vivo. Because LL-37 is a potential treatment for sepsis, its administration should be preceded by a careful analysis to ensure that the citrullinated peptide is not generated in treated patients.