Introduction: Macrophages are essential components of innate immunity, serving as a frontline defense against pathogens and maintaining tissue homeostasis. Human induced pluripotent stem cell (iPSC)–derived macrophages (iMacs) provide a powerful platform for studying human innate immunity and macrophage biology. Here, we describe a robust, reproducible, efficient serum-free and feeder-free protocol for generating functional iMacs and characterizing their innate immune properties. Methods: A 30-day monolayer culture system was utilized to continually generate hematopoietic progenitor cells (HPCs) from iPSCs starting on day 9, followed by macrophage differentiation over 21 days. Macrophage identity was assessed by flow cytometry, while functional assays evaluated phagocytosis and cytokine production, including interferons (IFNs). Transcriptomic profiling was performed by RNAseq across differentiation stages and following IFN stimulation. Results: The optimized protocol consistently yielded iMacs with >99% purity, expressing canonical macrophage markers including CD14, CD16, CD163, HLA-DR, and CD11b. iMacs demonstrated robust phagocytic capacity and cytokine production in response to microbial stimuli. RNA sequencing revealed distinct gene signatures during differentiation, highlighting key transitions from pluripotency to progenitors, then to mature macrophages. iMac transcriptomes aligned with tissue-resident macrophage profiles, supporting their relevance for modelling tissue-specific immunity. iMacs displayed differential interferon responses, with a strong response to type I IFNs. Conclusion: This study establishes a highly efficient and robust protocol for generating functional human iPSC-derived macrophages, providing a versatile model for investigating innate immunity, host–pathogen interactions, and interferon signaling.
INTRODUCTION: Lambda interferons signal through the interferon lambda receptor 1 (IFNLR1) and IL10RB heterodimer to induce interferon stimulated genes (ISGs). We previously showed that proteins derived from distinct IFNLR1 splice isoforms uniquely influence gene expression and hepatitis B virus replication in stem cell-derived hepatocytes (iHeps). Here, we evaluated signal transduction mechanisms of full-length IFNLR1 (variant 1) and a truncated variant missing part of the cytoplasmic JAK1-interacting domain (variant 2). METHODS: We evaluated HEK293T cells, wild-type (WT), and IFNLR1 knockout (KO) iHeps with doxycycline-inducible expression of FLAG-tagged IFNLR1 variants using the Duolink proximity ligation assay, ImageStream flow cytometry, Western blotting, susceptibility to JAK1 and TYK2 inhibitors, and gene expression profiling. RESULTS: Each variant colocalized with IL10RB after IFNL3 treatment, but variant 1 was more rapidly and extensively internalized. In WT iHeps, overexpression of variant 1 or variant 2 enabled higher IFNL-induced JAK1, TYK2, STAT1, and STAT2 phosphorylation, yet variant 2 supported less robust ISG induction. In KO iHeps, variant 2 supported less JAK1 and TYK2 phosphorylation and ISG induction than variant 1, yet supported comparable STAT1 and STAT2 phosphorylation. In iHeps expressing variant 1, WT iHeps were more resistant than KO iHeps to TYK2 inhibition of antiviral ISG induction, yet were more susceptible to TYK2 inhibition of proinflammatory ISG induction, suggesting endogenous noncanonical variants influence TYK2-signaling dependence. CONCLUSION: IFNLR1 variants promote differential utilization of signaling mediators to influence IFNL-induced gene expression patterns, indicating a putative role in pathway regulation.
INTRODUCTION:Bruton Tyrosine kinase inhibitors (BTKi) impair anti-aspergillus immune responses and increase the risk of invasive fungal infections (IFI). More selective second generation BTKi have fewer off-target effects thus a better tolerance than ibrutinib. Though clinical data suggest the incidence of IFI is lower with second generation BTKi, whether they have less impact on anti-fungal immunity remain unclear. METHODS:We studied the activation phenotype and behavior of human neutrophils in vitro with fungal extracts or living Aspergillus fumigatus in the presence of either ibrutinib or zanubrutinib, a more selective second generation BTKi, using various approaches, including live microscopy. RESULTS:Both molecules inhibited the activation-induced phenotypic changes in neutrophils following exposure to hyphae. Similarly, ibrutinib and zanubrutinib impaired neutrophil-mediated inhibition of conidia germination. By contrast, live neutrophil imaging showed that neutrophils exposed to ibrutinib but not zanubrutinib kept a round morphology, suggesting a defective activation, were less mobile and showed impaired conidia phagocytosis. CONCLUSION:We confirm here that ibrutinib and zanubrutinib inhibit neutrophil antifungal activity, strongly suggesting that BTK plays a central role. However, subtle differences between ibrutinib and zanubrutinib were evidenced using live microscopy, which might indicate additional off-target effects, and potentially partially explain the lower incidence of IFI with zanubrutinib; the clinical relevance remains to be determined.
Introduction: Small ubiquitin-like modifiers (SUMOs) are small peptides conjugated to proteins during post-translational modification, which have been reported to modulate several aspects of the immune system, notably in autoimmune disorders. Methods: We used a SUMO-based bacterial expression system to create a recombinant protein putatively expressed by Toxocara canis, which we hypothesised might antagonise responses via the chemokine receptor CXCR1. Results: Although our recombinant T. canis protein was devoid of antagonist activity, we serendipitously observed that recombinant SUMO-3 protein had chemotactic activity for CXCR1 transfectants. Further study found that SUMO-3 acted as a full agonist of CXCR1 and the closely related receptor CXCR2, the latter responses ablated by a CXCR2 antagonist. SUMO-3 showed similar efficacy at both receptors but reduced potency when compared to CXCL8, with chemotaxis observed at high nanomolar to micromolar concentrations. In receptor endocytosis assays, SUMO-3 induced internalisation of CXCR1 and CXCR2, with inferior potency and efficacy to CXCL8. Translating our findings to primary cells, a broad range of SUMO-3 concentration gradients were shown to induce the chemotaxis of human neutrophils. Finally, SUMO-3 was found to be released by necrotic cells into the extracellular milieu. Conclusions: Collectively, our findings suggest that SUMO-3 can induce the chemotaxis of neutrophils via CXCR1 and CXCR2. We postulate that in vivo, release of SUMO-3 from necrotic cells may serve to recruit neutrophils, contributing to tissue homeostasis and the resolution of inflammation.
Background: Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are proteins that mediate intercellular adhesion, intracellular signaling, and microbial recognition. Of the CEACAMs made by human neutrophils, CEACAMs 1, 3, and 6 are receptors for opacity-associated (Opa) outer membrane proteins of the bacterial pathogen Neisseria gonorrhoeae, which drive their non-opsonic phagocytosis. Summary: This review describes what is currently known about CEACAM subcellular localization, bacterial recognition, and downstream signaling events in human neutrophils in response to N. gonorrhoeae and the experimental systems used to elucidate them. It highlights the consequences of these activities on neutrophil activation and killing of N. gonorrhoeae, particularly those downstream of CEACAM3. The potential cross-talk between CEACAMs and with other neutrophil receptors, and how these signals may impact responses to N. gonorrhoeae, are also covered. Key Messages: Most Opa proteins of N. gonorrhoeae bind CEACAM1, with a subset also binding CEACAM3. Neutrophil CEACAM binding stimulates signaling events that drive phagocytosis of Opa-expressing N. gonorrhoeae, accompanied by degranulation, oxidant production, and proinflammatory signaling and transcriptional responses. In particular, CEACAM3 ligation leads to rapid phagocytic killing of N. gonorrhoeae. N. gonorrhoeae can avoid CEACAM3-mediated clearance by phase variation to turn off Opa protein expression or producing non-CEACAM3-binding Opa proteins. It can also bind host-derived complement C4b-binding protein (C4BP) and sialic acid, to impede CEACAM3-Opa interaction and engage sialic acid-binding immunoglobulin-like lectins (Siglecs) that transduce inhibitory signals to dampen CEACAM3 signaling, respectively.
INTRODUCTION:Dysregulated neutrophil functions play a crucial role in the onset and progression of periodontitis. Peptoanaerobacter stomatis is an emerging oral bacterium associated with periodontitis and is known to activate neutrophils through TLR2/6, leading to ROS production, granule release, and neutrophil extracellular trap (NET) formation. In this study, we characterized MAPK kinases that regulate these neutrophil responses and determined the mechanism responsible for bacterial killing. METHODS:Human neutrophils were challenged with P. stomatis, and kinase activation was assessed by immunoblot. Functional relevance was tested using selective kinase inhibitors prior to bacterial exposure. Neutrophil responses measured included ROS production by kinetic fluorometric assay, degranulation by flow cytometry, and NET formation using immunofluorescence and immunoblotting. Bacterial killing was determined by a CFU assay. RESULTS:P. stomatis activated ERK and p38 MAPKs, PI3K/AKT, and Src and Syk tyrosine kinases in neutrophils. Inhibition of all kinases except AKT significantly reduced ROS production. Because TAK1 is an upstream regulator of MAPK signaling, its role was also evaluated. Degranulation was mainly regulated by TAK1 and p38, with partial involvement of ERK and Src. NET formation required TAK1, ROS, and PAD4. TAK1 and degranulation inhibition markedly decreased bacterial killing, whereas inhibition of ROS or NET had no effect. CONCLUSION:These findings demonstrate that although P. stomatis evades ROS- and NET-mediated killing, it remains susceptible to TAK1-mediated neutrophil degranulation, identifying degranulation as the primary mechanism for bacterial clearance.
Objective: The cGAS-STING pathway is a critical sensor in the innate immune response to intracellular pathogens, yet its therapeutic potential for augmenting macrophage-mediated control of Mycobacterium tuberculosis (Mtb) remains incompletely understood. This study investigated whether pharmacological activation of the STING pathway could enhance autophagy to promote Mtb clearance in human macrophages. Methods: Human THP-1 monocytes were differentiated into macrophages and infected with Mtb. The effects of the STING agonist MIW815 (ADU-S100) on Mtb phagocytosis, intracellular bacterial survival, and autophagic flux were assessed using a combination of molecular and cellular techniques, including qRT-PCR, western blotting, colony-forming unit (CFU) assays, and confocal immunofluorescence microscopy. The dependency on the cGAS-STING pathway was confirmed using siRNA-mediated gene silencing. Results: Pharmacological activation of STING with ADU-S100 significantly enhanced Mtb phagocytosis and subsequent intracellular clearance. This enhanced bactericidal activity was mechanistically linked to an increase in autophagic flux, as evidenced by elevated LC3-II protein levels and significantly increased colocalization of Mtb with lysosomal compartments. Importantly, treatment with the autophagy inhibitor hydroxychloroquine or silencing of cGAS significantly reversed these phenotypes, confirming the pivotal role of the STING-autophagy axis. Conclusion: Activating the STING pathway with ADU-S100 is a potent host-directed strategy to bolster macrophage autophagy and enhance the elimination of intracellular Mtb. This provides a strong rationale for exploring STING agonists as a novel therapeutic intervention for tuberculosis, addressing a significant and clinically relevant challenge in infectious disease.
Background: Neutrophils are essential components of host defense, traditionally recognized for their well-described antimicrobial functions in bacterial infections. Far from being merely short-lived and functionally limited, emerging evidence demonstrates that neutrophils are also key players in viral infections. Through mechanisms such as degranulation and formation of NETs, neutrophils play an important role in virus-induced inflammation and pathogenesis. However, their persistence and hyperactivated states also contribute to tissue damage and failed resolution of acute inflammation. Summary: Here, we summarize recent data regarding the role of neutrophils in respiratory and arboviral infections, from clinical evidence to mechanistic insights available from the last ten years. Finally, we discuss emerging therapeutic strategies that target neutrophil fate and function to promote the resolution of inflammation. Key messages: Overall, although well characterized in some viral infections, for a few other viral diseases the role of neutrophils remains elusive. We highlight the importance of modulating neutrophils death in a timely and non-inflammatory manner, as some viruses influence apoptosis for their own benefit, favoring viral replication. For those infections, and also due to non-resolving inflammation provoked by these viruses, pro-resolving mediators certainly display a beneficial effect. Rather than broadly suppressing antiviral immunity, pro-resolving approaches aimed at recalibrating neutrophil activation, lifespan, and clearance may represent a promising and safer avenue for the treatment of viral diseases characterized by excessive and non-resolving inflammation.
Introduction: During acute inflammation, such as experimental human endotoxemia, circulating neutrophil counts initially decrease, followed by pronounced neutrophilia. Banded (CD16dim/CD62Lbright) and hypersegmented (CD16bright/CD62Ldim) neutrophils, which are absent in the blood during homeostasis, are rapidly mobilized. Banded and mature neutrophils are thought to be recruited from the bone marrow, while the origin of hypersegmented neutrophils remains disputed and underlying recruitment mechanisms remain to be established. Methods: Fifteen volunteers received an intravenous bolus of bacterial lipopolysaccharide (LPS) followed by continuous infusion for 3 h. Seven were randomly assigned to additional cytokine adsorption therapy, enabling analysis of neutrophil subsets across a broad range of circulating cytokine concentrations. Blood samples were obtained at baseline and 3 h post-LPS bolus to assess neutrophil subset quantities, neutrophil surface activation markers, and plasma cytokine levels. Results: Circulating concentrations of pro-inflammatory cytokines (TNF, interleukin (IL)-6, CXCL8, CCL3, and CCL2) were negatively associated with circulating mature and banded neutrophil numbers, suggesting a role for these cytokines in their migration. The absence of a correlation between plasma cytokines with hypersegmented neutrophils indicates an alternative mobilization signal. In contrast to cell numbers, neutrophil activation markers positively correlated with concentrations of the various pro-inflammatory cytokines, indicating involvement of cytokines in neutrophil activation. Conclusion: Circulating cytokines appear to be key drivers of mature and banded neutrophil migration, whereas this complex signal does not influence the abundance of CD62Ldim cells in peripheral blood.
INTRODUCTION:Progression to end-stage lung diseases is influenced by both environmental and genetic factors, yet predictive markers remain limited. The CD32-131H>R polymorphism in the FCGR2A gene alters the affinity of the FcγRIIa receptor for IgG and may impair immune clearance, potentially influencing immune responses involved in chronic lung disease. In our study, we investigated the association between the CD32-131H>R polymorphism and end-stage pulmonary diseases requiring LTX. METHODS:The CD32-131H>R polymorphism was determined in 985 lung transplant recipients and 2,116 matched controls via TaqMan genotyping assays. RESULTS:The low-affinity CD32-131R allele was significantly more prevalent among lung transplant recipients with chronic obstructive pulmonary disease (COPD) and interstitial lung diseases (ILD) compared to controls (p < 0.0001 and p = 0.01, respectively). CONCLUSION:Our study demonstrates that the CD32-131R allele is enriched among patients with end-stage COPD and ILD requiring LTX, highlighting a possible association between Fcγ receptor-mediated immune responses and end-stage pulmonary disease.
INTRODUCTION:Neutrophils are the most abundant leukocytes in human blood and a key component of host defense against Streptococcus pyogenes (group A streptococci [GAS]). They are rapidly recruited to the site of infection, where they mediate phagocytosis, degranulation, and release of neutrophil extracellular traps, thereby influencing both bacterial clearance and tissue damage. Traditionally considered short-lived effector cells, neutrophils are increasingly recognized for their immunomodulatory functions, including regulation of adaptive immunity. METHODS:Human neutrophils were infected with GAS wild-type 5448 or isogenic mutants lacking streptolysin O (Δslo) or streptolysin S (ΔsagA) and analyzed either after infection alone or after infection followed by co-culture with autologous CD4+ T cells. Neutrophil activation and T cell responses were evaluated via flow cytometry. RESULTS:GAS infection robustly activated neutrophils in a dose-dependent manner, driving expansion of the CD15bright/CD66bbright population and heparin-binding-protein release. Infection prompted neutrophils to acquire antigen-presenting cell (APC)-like characteristics, including HLA-DR, CD40, and CD86 expression, particularly when exposed to autologous CD4+ T cells. In 5448ΔsagA infections, neutrophils showed lower expression of costimulatory markers, and bacteria were more susceptible to intracellular killing. In co-cultures, CD4+ T cells were partially activated, as indicated by CD25 upregulation and Th1- and Th17-cell-associated cytokine release. CONCLUSION:GAS-infected neutrophils acquire an APC-like phenotype and partially modulate CD4+ T cell activation, revealing a previously unrecognized role for infected neutrophils in shaping adaptive immunity in streptococcal infections.
INTRODUCTION:This study investigates METTL3's function and mechanisms in hemorrhoidal disease-related inflammation. METHODS:A rat model of hemorrhoidal disease was induced in vivo using croton oil. Human monocytic leukemia cells THP-1 were induced into macrophages using phorbol myristate acetate, and then lipopolysaccharide (LPS) was used to induce an inflammatory phenotype in the macrophages. The pathological changes in the rectal and anal tissues of rats were evaluated using the rectoanal coefficient and HE staining. Key gene and protein expression levels were detected using real-time quantitative PCR, Western blotting, immunofluorescence, and immunohistochemistry. RESULTS:METTL3 expression was significantly upregulated in hemorrhoidal tissues and LPS-induced macrophages, and its knockdown alleviated rectal-anal lesions in hemorrhoidal rats. Silencing METTL3 suppressed the expression of M1 polarization markers (iNOS, CD80, CD86) while increasing interleukin-10 and reducing tumor necrosis factor-α, IL-1β, and IL-6 levels in hemorrhoidal tissues and LPS-stimulated macrophages. Furthermore, LPS treatment enhanced Smad2/3 and IL-6R expression in macrophages, and positive correlations were observed between the expression of Smad2, Smad3, and METTL3, as well as between METTL3 and IL-6R. Mechanistically, nuclear-translocated Smad2/3 bound to METTL3 to promote its expression. The resulting elevated METTL3 then promoted IL-6R expression via m6A modification, driving macrophage activation and ultimately exacerbating the inflammatory response in hemorrhoidal disease. CONCLUSION:This work demonstrates that the Smad2/3-induced METTL3 drives hemorrhoidal inflammation via m6A modification of IL-6R, presenting a novel intervention target.
Background: Cystic fibrosis (CF) is a hereditary disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The major causes of morbidity and mortality in CF are related to lung disease, involving neutrophil-dominated lung inflammation. Summary: Whether the altered inflammatory response of neutrophils in patients with CF is an intrinsic defect due to a lack of CFTR protein expression, or alternatively, exacerbated by chronic exposure to infection and inflammation, is extensively debated. Fuelling this dispute are conflicting past studies on CFTR expression by neutrophils, with opposing results described at both the gene and protein level. This is pertinent in the era of CFTR modulator therapies, with clinicians and scientists exploring the impact of different CFTR mutation classes and CFTR modulators on neutrophil function. Key Messages: The focus of this article was to uncover the cause for the described disparity of data on neutrophil CFTR expression, by investigating methods utilised for CFTR detection and drawing consensus on the optimal protocol for identifying CFTR protein in neutrophils.
Introduction: Neutrophils are the most abundant innate immune cells in the peripheral blood and eliminate bacteria through phagocytosis and antimicrobial mechanisms. Early in infection, they often encounter high bacterial loads before full recruitment. Individual neutrophils can ingest many bacteria, but it remains unclear how high bacterial loads per neutrophil affect intracellular killing. Methods: Neutrophils were isolated from healthy donor blood by fluorescence-activated cell sorting. Intracellular bacterial load was quantified using imaging flow cytometry to measure spot counts and green fluorescent protein (GFP) intensity after exposure to GFP-expressing Staphylococcus aureus. A single-cell killing assay assessed intracellular killing across bacterial load categories by sorting individual GFP+ neutrophils into 384-well plates and counting wells with outgrowth after 100 h. Phagolysosomal acidification was measured using dual-labeled (pH-sensitive pHrodo and pH-insensitive PromoFluor 520 LSS NHS ester [PF520]) S. aureus bioparticles. Results: Bacterial uptake by neutrophils was highly heterogeneous in vivo and in vitro. GFP spot counts strongly correlated with GFP intensity (R2 = 0.66), allowing stratification into GFP fluorescence intensity categories. In the single-cell killing assay, higher bacterial loads per neutrophil were associated with reduced intracellular killing (χ2(4) = 11.72, p = 0.0003). Higher bacterial loads per neutrophil corresponded with diminished phagolysosomal acidification capacity (χ2(4) = 24.00, p < 0.0001). Conclusion: Neutrophils ingesting higher bacterial loads exhibit reduced intracellular killing, likely due to decreased phagolysosomal acidification. These findings highlight how bacterial load per neutrophil shapes antimicrobial capacity and early infection control.
Introduction: Bothrops jararacussu venom contains snake venom metalloproteinases (SVMPs) that contribute to inflammation and tissue damage. BjussuMP-II, a PI class SVMP, lacks hemorrhagic activity but retains proteolytic and immunomodulatory properties. Methods: Here, we uncover a previously unrecognized function of BjussuMP-II in triggering NLRP3 inflammasome activation in human neutrophils, leading to IL-1β release and pyroptosis. Inflammasome activation was investigated through gene expression analysis by quantitative PCR and protein expression by immunoblotting. Caspase-1 activation and gasdermin D (GSDMD) cleavage were assessed to confirm pyroptotic signaling. Pharmacological inhibition assays were performed to dissect the involvement of specific signaling pathways, and IL-1β secretion was quantified by enzyme immunoassay. This discovery reveals a direct molecular link between SVMP activity and inflammasome-mediated inflammation, a fundamental mechanism with implications beyond snakebite pathology. It highlights inflammasomes as potential therapeutic targets to mitigate severe inflammatory responses in envenomed patients. Results: Mechanistically, BjussuMP-II increased expression of NLRP3, ASC, caspase-1, NEK7, and HIF-1α, as well as IL-1β and GSDMD cleavage, confirmed by immunoblotting and immunofluorescence. It promoted the release of IL-1β, LTB4, LDH, and dsDNA, consistent with pyroptosis, and this release was reduced by MCC950 or disulfiram. Studies in Gsdmd−/− and HIF-1α-deficient neutrophils further demonstrated the requirement of these pathways for cytokine release. Conclusion: Collectively, these results indicate that BjussuMP-II modulates inflammasome-associated signaling pathways in neutrophils, contributing to the inflammatory responses triggered by SVMPs.
Introduction: The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is a critical sensor in the innate immune response to intracellular pathogens, yet its therapeutic potential for augmenting macrophage-mediated control of Mycobacterium tuberculosis (Mtb) remains incompletely understood. This study investigated whether pharmacological activation of the STING pathway could enhance autophagy to promote Mtb clearance in human macrophages. Methods: Human THP-1 monocytes were differentiated into macrophages and infected with Mtb. The effects of the STING agonist MIW815 (ADU-S100) on Mtb phagocytosis, intracellular bacterial survival, and autophagic flux were assessed using a combination of molecular and cellular techniques, including quantitative real-time polymerase chain reaction, Western blotting, colony-forming unit (CFU) assays, and confocal immunofluorescence microscopy. The dependency on the cGAS-STING pathway was confirmed using small interfering RNA-mediated gene silencing. Results: Pharmacological activation of STING with ADU-S100 significantly enhanced Mtb phagocytosis and subsequent intracellular clearance. This enhanced bactericidal activity was mechanistically linked to an increase in autophagic flux, as evidenced by elevated LC3-II protein levels and significantly increased colocalization of Mtb with lysosomal compartments. Importantly, treatment with the autophagy inhibitor hydroxychloroquine or silencing of cGAS significantly reversed these phenotypes, confirming the pivotal role of the STING-autophagy axis. Conclusion: Activating the STING pathway with ADU-S100 is a potent host-directed strategy to bolster macrophage autophagy and enhance the elimination of intracellular Mtb. This provides a strong rationale for exploring STING agonists as a novel therapeutic intervention for tuberculosis, addressing a significant and clinically relevant challenge in infectious disease.
Introduction: Interleukin (IL)-6 has an important role in limiting urinary tract infection (UTI). Mice lacking IL-6 are more susceptible to uropathogenic Escherichia coli (UPEC), including increased formation of intracellular bacterial communities (IBCs). How IL-6 promotes UPEC clearance is unknown. We hypothesize IL-6 reduces UTI susceptibility by limiting IBC formation through an early mechanism of infection. Methods: Female mice were treated with vehicle or neutralizing antibodies to inhibit IL-6 or the IL-6 receptor (IL-6R) prior to transurethral UPEC infection. In rescue experiments, murine recombinant (r)IL-6 was administered to IL-6 knockout (KO) mice. Bladder IBCs, urinary and bladder bacterial burden, and UPEC expulsion were quantified. For clinical translation, human urothelial cells were pretreated with human rIL-6 and infected with UPEC. Bacterial attachment, invasion, and expulsion were quantified. Results: Neutralization of IL-6 or IL-6R increased bladder IBC counts compared to isotype controls. Similarly, while IL-6 KO mice exhibited higher IBC counts than wild-type controls, this phenotype was reversed by rIL-6 administration. Gentamicin protection assays confirmed increased intracellular UPEC burden and reduced bacterial expulsion in IL-6 KO bladders. rIL-6 treatment enhanced UPEC expulsion in human urothelial cells without impacting bacterial attachment or invasion. Conclusion: IL-6 facilitates UPEC expulsion, limiting intracellular UPEC early in infection and thus the initial formation of IBCs. Since IBC formation is a bottleneck in UPEC survival during UTI, these findings identify a mechanism whereby IL-6 reduces early UPEC urothelial infectivity.
Introduction: Pseudomonas aeruginosa is a significant pathogen associated with acute and chronic infections, particularly in immunocompromised individuals. Its capacity for biofilm formation, combined with antibiotic resistance, plays a critical role in the persistence of these infections. Peptidyl-arginine deiminases (PADs), including PAD2 and PAD4 isoforms, are involved in neutrophil phagocytic killing of P. aeruginosa. This study aimed to investigate the impact of PAD enzymes on biofilm development and virulence factor production in P. aeruginosa, with focus on the multidrug resistant strain, PGO2330. Methods: Biofilm formation was assessed using crystal violet assays and confocal scanning laser microscopy. Quorum sensing (QS) gene expression and QS-related virulence factor production were quantified using qPCR and virulence factor assays. Results: Exposure to 20 nm of PAD2 or PAD4 reduced PGO2330 surface attachment (p < 0.0001) and biofilm formation to 67.9 ± 5.6% (p < 0.0001) and 68.2 ± 4.2% (p = 0.0004), respectively. Moreover, rPAD2 and rPAD4 citrullinated multiple protein substrates of P. aeruginosa, yet citrullination activity was not required by rPADs to reduce P. aeruginosa biofilm formation. PGO2330 exposed to PAD2 and PAD4 showed reduced lasR, lasI, rhlR, rhlI, and mvfR gene expression and reduced levels of extracellular DNA, rhamnolipids, pyocyanin, and protease activity. Conclusion: These results demonstrate that PADs inhibit P. aeruginosa biofilm formation and decrease the production of QS-related virulence factors, highlighting their potential as novel antimicrobials and supporting further research into the development of PAD-based therapeutics.
Background: Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by dysregulated immune responses in the gut. Macrophages, as key innate immune cells in the intestinal mucosa, play a central role in both maintaining homeostasis and driving pathology in UC. Summary: Under physiological conditions, intestinal macrophages exhibit a unique “inflammatory anergy” phenotype, supporting epithelial integrity and immune tolerance. In UC, however, persistent inflammatory signals promote monocyte recruitment and their polarization into pro-inflammatory M1-like macrophages. These cells secrete cytokines such as TNF-α, IL-1β, IL-6, and IL-12/23, produce reactive oxygen species and reactive nitrogen species, and release matrix metalloproteinases, collectively driving epithelial barrier disruption, tissue damage, and sustained inflammation. This review comprehensively discusses the origin, heterogeneity, and functional plasticity of intestinal macrophages, their dynamic interactions with other cells, and key regulatory signaling pathways – such as NF-κB, JAK-STAT, and the NLRP3 inflammasome – in UC. Key Messages: We evaluate current and emerging macrophage-targeted therapies, including cytokine blockade, chemokine receptor antagonism, phenotypic reprogramming, nanomedicine, and cell-based interventions. Furthermore, we highlight the limitations of the M1/M2 dichotomy and emphasize the need for single-cell and spatial transcriptomic approaches to better define macrophage subsets in human disease. Advancing the understanding of macrophage biology in UC will facilitate the development of precise immunomodulatory strategies and biomarker-based diagnostics, ultimately aiming to bridge the gap between mechanistic discovery and improved patient care.
