Objectives:Rapid diagnosis and intervention are critical for sepsis patient outcomes. However, diagnosis is challenging because of a heterogenic patient group as well as sometimes vague symptoms when the patient presents at the emergency department. Mucosal-associated invariant T (MAIT) cells are rapid responders to infection, but their role and characteristics in the early course of sepsis remain unknown. Here, we evaluate the early MAIT cell characteristics in the blood of patients triggering a clinical sepsis alert system at the emergency department. Methods:Peripheral blood mononuclear cells were isolated from freshly drawn blood and immediately stained. MAIT cell phenotyping analyses were conducted using multiparameter flow cytometry. All analyses were completed prior to the stratification of patients into sepsis or non-sepsis groups. Soluble factors in plasma were measured using a multiplex assay. Results:Unsupervised high-dimensional phenotyping identified distinct MAIT cell activation profiles in sepsis and non-sepsis groups. Among sepsis patients, hierarchical clustering of MAIT cell phenotypes separated clinical endotypes into three groups with different infection focus, severity and aetiology. A prominent characteristic of sepsis severity was high expression of CD69 on MAIT cells, which was associated with organ dysfunction, lymphopenia and poor outcome. Plasma levels of IL-12, IL-15, TNF, IFNγ and CXCL10 correlated with the magnitude of MAIT cell activation in sepsis patients. Conclusions:These clinical endotype-specific MAIT cell phenotypes presenting already in the emergency department are of interest for early patient identification and prognostication in sepsis.

Staphylococcus aureus is an important human pathogen causing severe invasive infections. Pathogenesis is attributed to a wide array of virulence factors, including several potent exotoxins such as the pore-forming α-toxin. In this study, we found that patients with S. aureus respiratory tract infections had elevated CX3CL1 levels in airway fluid and plasma. Using human-organotypic lung models, we observed that stimulation of lung epithelium with α-toxin induces an intensified CX3CL1 expression apically in the epithelium as well as the release of CX3CL1. Blocking α-toxin or ADAM10 activity in organotypic lung using an α-toxin-blocking antibody or a specific ADAM10 inhibitor confirmed their role in modulating CX3CL1 cleavage and release. Analyses of CD14+ human monocytes in combination with a CX3CR1 inhibitor revealed that α-toxin-mediated CX3CL1 release induces CX3CL1-dependent chemotaxis. In line with these data, lung tissue from patients with S. aureus respiratory tract infection showed elevated CX3CL1 and CD14 staining as compared with tissue from patients with non-infectious lung diseases. Functional studies of monocytes showed that CX3CL1 released by lung models resulted in upregulated CD83 and downregulated CD86, as well as impaired killing of phagocytosed S. aureus. Furthermore, stimulation of monocytes with soluble CX3CL1 hampered their reactive-oxygen and nitric-oxide production. Taken together our data show that S. aureus triggers the release of lung epithelial CX3CL1, and we identify an immunomodulatory effect of α-toxin involving its cytotoxic and ADAM10-interacting properties, inducing CX3CL1 release leading to impaired monocyte effector function.IMPORTANCEExotoxins are essential virulence factors for the pathobiont S. aureus and contribute toward severe invasive infections such as pneumonia. S. aureus α-toxin is a pore-forming exotoxin that causes host cell lysis and severe lung pathology. We found that α-toxin drives the release of membrane-bound chemokine CX3CL1 by involving ADAM10-mediated proteolytic activity. Furthermore, the release of CX3CL1 modulated immune responses locally, as demonstrated by enhanced monocyte migration and reduced capacity of monocytes to kill ingested bacteria. CX3CL1-induced reduction in bacterial killing coincided with impaired production of reactive oxygen and nitric oxide species. This reveals a novel mechanism in the pathogenesis of S. aureus lung infections involving α-toxin-induced release of CX3CL1, leading to impaired bacterial killing by monocytes.

Pain in rheumatoid arthritis (RA) often persists despite effective control of inflammation, suggesting distinct mechanisms driving nociception. In both patients and animal models, pain severity does not strongly correlate with the degree of inflammation1,2. Sensory neurons, with cell bodies located in the dorsal root ganglia (DRG), innervate peripheral tissues, including joints, and transmit pain signals to the central nervous system. Crosstalk between sensory neurons and immune cells occurs at all of these sites. While sensory neurons can be directly activated by immune mediators, it remains unclear whether pain-like behaviour in antibody-induced arthritis models arises independently of immune cell activity, or which immune cell populations and mediators are required to activate pronociceptive mechanisms. Through temporal profiling of the CAIA joint-DRG transcriptomic axis, we identified SEMA4D and OSM signalling as candidate molecular mediators of neutrophil-neuron communication and neuronal sprouting. The joint-DRG atlas also revealed persistent changes in the fibroblast-immune cellular composition of the joint, along with molecular changes in DRG neurons. We showed that mechanical and cold hypersensitivity, as well as sprouting of CGRP+ nociceptive fibers in synovial tissue of mice with collagen antibody-induced arthritis (CAIA), require neutrophils but not macrophages. Analysis of publicly available datasets showed that neutrophils from the synovium of RA patients express high levels of SEMA4D and OSM, and corresponding expression of their receptors, PLEXINB1 and OSMR, in human DRG neurons, underscoring the translational relevance of this axis. Both murine and human-derived DRG neurons sprout in response to OSM. Our findings demonstrate that neutrophils produce molecules that act as cues for nociceptor sensitization and structural remodelling. Targeting these molecules could improve the efficacy of RA treatments by reducing pain while simultaneously preventing disease progression. ### Competing Interest Statement The authors have declared no competing interest. Swedish Research Council, 2023-02139, 2024- 02509 Knut and Alice Wallenberg Foundation, https://ror.org/004hzzk67 European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme, 866075, 764860 Swedish Society for Medicine KI Foundation Grants for doctoral studies KI Foundation Grants for Rheumatology Research, 2023-02725, 2024-03817 KI Faculty Funded Assistant Professorship Generous donation from Leif Lundblad and family.

The M protein is an essential virulence factor of Streptococcus pyogenes, or group A streptococcus (GAS), one of the most common and dangerous human pathogens. Molecular and functional characterization of M protein variants and their interactions with host components is crucial for understanding streptococcal pathogenesis and vaccine development. The M3 protein is produced by the prevalent emm3 GAS serotype, which is frequently associated with severe invasive diseases. Here, we structurally and biochemically characterize the interaction of M3 with human collagens. High-resolution structures of the N-terminal M3 domain in the free state as well as bound to a collagen peptide derived from the Collagen Ligands Collection reveal a novel T-shaped protein fold that presents binding sites complementing the characteristic topology of collagen triple helices. The structure of the M3/collagen peptide complex explains how emm3 GAS and related streptococci, such as Streptococcus dysgalactiae subsp. equisimilis, can target collagens to enable colonization of various tissues. In line with this, we demonstrate that the M3/collagen interaction promotes enhanced biofilm formation of emm3 GAS in an emm type-specific manner, which can be inhibited with the recombinant N-terminal M3 domain. Further, emm3 GAS are shown to colocalize with collagen in tissue biopsies from patients with necrotizing soft tissue infections, where GAS biofilms are common. This observation is reproduced in infected organotypic skin models. Together, these data provide detailed molecular insights into an important streptococcal virulence mechanism with implications for the understanding of invasive infections, strategies for treating biofilm and M-protein-based vaccine design.

The human blood proteome provides a holistic readout of health states through the assessment of thousands of circulating proteins. In this study, we present a pan-disease resource to enable the study of diverse disease phenotypes within a harmonized proteomics dataset. By profiling protein concentrations across 59 diseases and healthy cohorts, we identified proteins associated with age, sex, and body mass index, as well as disease-specific signatures. This study highlights shared and distinct protein patterns across conditions, demonstrating the power of a unified proteomics approach to uncover biological insights. The dataset, covering 8262 individuals and up to 5416 proteins, serves as an online resource for exploring disease-specific protein profiles and advancing precision medicine research.

Degradation of immunoglobulin (Ig) represents an important bacterial immune evasion strategy. For mucosal colonization, degradation of IgA is of particular importance, and many bacteria secrete specific IgA proteases that typically target the extended hinge region of IgA1. Such a specialized IgA protease has not yet been reported in Group A Streptococcus (GAS), despite its ability to successfully colonize human mucosal surfaces. In this study, we focused on the cysteine protease SpeB secreted by GAS and analyzed the interaction of SpeB with IgA. Assays using bacterial supernatants from wild-type and speB-deficient isogenic mutant strains, as well as recombinant SpeB, showed a SpeB-dependent IgA-modifying activity. SpeB resulted in the degradation of multimeric IgA, including the dimeric form, which was most notable in IgA2. The modification products were smaller in size than the heavy chain, suggesting a modification different from the classical hinge cleavage. Mass spectrometry analysis and glycosylation profiles indicated a putative cleavage in the C-terminal region, affecting the tailpiece and resulting in the loss of higher molecular weight multimeric/dimeric forms of IgA. Given the importance of dimeric IgA at mucosal surfaces, future studies are warranted to address whether IgA modification by SpeB represents a GAS immune evasion mechanism at this site.IMPORTANCEGroup A Streptococcus (GAS) is an important human pathogen with the ability to efficiently colonize mucosal surfaces and cause a wide spectrum of diseases ranging from pharyngotonsillitis to severe invasive infections or post-streptococcal sequelae. Immunoglobulins (Ig), in particular IgA, are critical effector molecules in the defense against pathogen colonization at mucosal surfaces. In this study, we focused on the cysteine protease SpeB, secreted by GAS, and investigated its interaction with human IgA. We report a SpeB-dependent IgA modification that involved the loss of multimeric/dimeric forms of IgA, predominantly affecting IgA2. The putative modification region is the C-terminus of IgA, which differs from the cleavage site of specialized IgA proteases targeting the hinge region. These findings suggest that IgA modification by SpeB might represent an immune evasion strategy utilized by GAS to colonize human mucosal tissue.

Objectives Bioengineered artificial skin substitutes (BASS) are an advanced therapy for treating extensively burned patients. Pseudomonas aeruginosa (P. aeruginosa) infections represent a major challenge in these patients as formation of biofilms impede wound healing and perpetuate a chronic inflammatory state. Here we assessed antibiotics (alone or in combination) with respect to cytotoxicity, as well as antimicrobial efficacy in P. aeruginosa biofilm formed on infection of BASS.Methods Cell viability, structure and functionality were evaluated using microscopy and trans-epidermal water loss analyses, respectively. BASS were established and infected for 24 h to allow P. aeruginosa biofilm formation, after which two antimicrobial approaches, treatment and prevention, were tested. In the latter, antibiotics were added to BASS before infection. The antimicrobial effect was determined using real-time calorimetry.Results In dose-response experiments, 1.25 mg/mL amikacin, 0.02 mg/mL ciprofloxacin, 0.051 mg/mL colistin, 1 mg/mL meropenem and colistin in combination with either amikacin, ciprofloxacin and meropenem did not affect BASS' viability, structure and functionality. All antibiotics, except colistin, showed effective antimicrobial activity at these non-cytotoxic concentrations. For concentrations below the highest non-cytotoxic ones, successive treatments resulted in higher bacterial metabolic rates. Only the combinations managed to eradicate the infection with repeated treatments. With respect to prevention of infection, all antibiotics at the highest non-cytotoxic concentrations and the combinations were effective. This preventive capacity was maintained for at least 5 days.Conclusion The findings highlight the potential for developing BASS with antimicrobial properties that can prevent infections during wound healing in burn patients.

Abstract Background Activated fibroblast-like synoviocytes (FLS) are drivers of synovitis and structural joint damage in rheumatoid arthritis (RA). Despite the use of disease-modifying drugs, only about 50% of RA patients reach remission in real-world settings. We used an unbiased approach to investigate the effects of standard-of-care methotrexate (MTX) and a Janus kinase inhibitor, tofacitinib (TOFA), on gene expression in RA-FLS, in order to identify untargeted disease mediators. Methods Primary RA-FLS were activated by stimulation with interleukin-1β (IL-1β) or platelet-derived growth factor + IL-1β in the presence or absence of MTX or TOFA, with or without additional inhibitors. Co-cultures of synovial cells were performed in direct and indirect systems. Cells were collected for RNA sequencing or qPCR, and supernatants were analyzed for protein concentrations. Results Six thousand three hundred fifty genes were differentially expressed, the majority being upregulated, in MTX-treated activated RA-FLS and 970 genes, the majority being downregulated, in TOFA-treated samples. Pathway analysis showed that MTX had largest effects on ‘Molecular mechanisms of cancer’ and TOFA on ‘Interferon signaling’. Targeted analysis of disease-associated genes revealed that MTX increased the expression of cell cycle-regulating genes but also of pro-inflammatory mediators like IL-1α (IL1A) and granulocyte–macrophage colony-stimulating factor, GM-CSF (CSF2). The MTX-promoted expression of CSF2 in activated RA-FLS peaked at 48 h, could be mediated via either NF-κB or AP-1 transcription factors, and was abrogated by IL-1 inhibitors (IRAK4 inhibitor and anakinra). In a co-culture setting, MTX-treatment of activated RA-FLS induced IL1B expression in macrophages. Conclusions MTX treatment induces secretion of IL-1 from activated RA-FLS which by autocrine signaling augments their release of GM-CSF. This unexpected effect of MTX might contribute to the persistence of synovitis.

Pseudomonas aeruginosa is one of the most common microorganisms causing infections of severe skin wounds. Antibiotic or antiseptic treatments are crucial to prevent and curb these infections. Antiseptics have been reported to be cytotoxic to skin cells and few studies evaluate the impact of commonly used antibiotics. This study evaluates how clinical antibiotics affect skin cells' viability, proliferation, migration, and cytokine secretion and defines the highest non-cytotoxic concentrations that maintain antibacterial activity. Cell proliferation, viability, and migration were evaluated on cell monolayers. Cytokines related to the wound healing process were determined. The minimum inhibitory concentrations and the impact on bacterial biofilm were assessed. Results showed that 0.02 mg/mL ciprofloxacin and 1 mg/mL meropenem are the highest non-cytotoxic concentrations for fibroblasts and keratinocytes while 1.25 mg/mL amikacin and 0.034 mg/mL colistin do not affect fibroblasts' viability and cytokine secretion but have an impact on keratinocytes. These concentrations are above the minimum inhibitory concentration but only amikacin could eradicate the biofilm. For the other antibiotics, cytotoxic concentrations are needed to eradicate the biofilm. Combinations with colistin at non-cytotoxic concentrations effectively eliminate the biofilm. These results provide information about the concentrations required when administering topical antibiotic treatments on skin lesions, and how these antibiotics affect wound management therapies. This study set the basis for the development of novel antibacterial wound healing strategies such as antibiotic artificial skin substitutes.

AbstractThe M protein is an essential virulence factor ofStreptococcus pyogenes, or group A streptococci (GAS), one of the most common and dangerous human pathogens. Molecular and functional characterization of M protein variants and their interactions with host components is crucial for understanding streptococcal pathogenesis and vaccine development. The M3 protein is produced by the prevalentemm3 GAS serotype, which is frequently associated with severe invasive diseases. Here we characterize the interaction of M3 with human collagens through detailed structural and biochemical binding analysis. High-resolution structures of the N-terminal M3 domain in the free state as well as bound to a collagen peptide derived from the Collagen Ligands Collection reveal a novel T-shaped protein fold that presents binding sites complementing the characteristic topology of collagen triple helices. The structure of the M3/collagen peptide complex explains howemm3 GAS and related streptococci, such as the emerging human pathogenStreptococcus dysgalactiaesubsp.equisimilis, can target collagens to enable colonization of various tissues. In line with this, we demonstrate that the M3/collagen interaction promotes enhanced biofilm formation ofemm3 GAS in anemmtype specific manner, which can be inhibited with the recombinant M3 N-terminal domain fragment. Further,emm3 GAS, but not anemm1 strain, are shown to colocalize with collagen in tissue biopsies from patients with necrotizing soft tissue infections, where GAS biofilms are common. This observation is reproduced in organotypic skin models. Together, these data provide detailed molecular insights into an important streptococcal virulence mechanism with implications for the understanding of invasive infections, strategies for treating biofilm and M-protein based vaccine design.

Stromal cells support epithelial cell and immune cell homeostasis and play an important role in inflammatory bowel disease (IBD) pathogenesis. Here, we quantify the stromal response to inflammation in pediatric IBD and reveal subset-specific inflammatory responses across colon segments and intestinal layers. Using data from a murine dynamic gut injury model and human ex vivo transcriptomic, protein and spatial analyses, we report that PDGFRA + CD142 − /low fibroblasts and monocytes/macrophages co-localize in the intestine. In primary human fibroblast-monocyte co-cultures, intestinal PDGFRA + CD142 − /low fibroblasts foster monocyte transition to CCR2 + CD206 + macrophages through granulocyte-macrophage colony-stimulating factor (GM-CSF). Monocyte-derived CCR2 + CD206 + cells from co-cultures have a phenotype similar to intestinal CCR2 + CD206 + macrophages from newly diagnosed pediatric IBD patients, with high levels of PD-L1 and low levels of GM-CSF receptor. The study describes subset-specific changes in stromal responses to inflammation and suggests that the intestinal stroma guides intestinal macrophage differentiation.

Objective: Early stages with streptococcal necrotizing soft tissue infections (NSTIs) are often difficult to discern from cellulitis. Increased insight into inflammatory responses in streptococcal disease may guide correct interventions and discovery of novel diagnostic targets. Methods: Plasma levels of 37 mediators, leucocytes and CRP from 102 patients with 0-hemolytic streptococcal NSTI derived from a prospective Scandinavian multicentre study were compared to those of 23 cases of streptococcal cellulitis. Hierarchical cluster analyses were also performed. Results: Differences in mediator levels between NSTI and cellulitis cases were revealed, in particular for IL-10, TNF alpha and CXCL8 (AUC >0.90). Across streptococcal NSTI etiologies, eight biomarkers separated cases with septic shock from those without, and four mediators predicted a severe outcome. Conclusion: Several inflammatory mediators and wider profiles were identified as potential biomarkers of NSTI. Associations of biomarker levels to type of infection and outcomes may be utilized to improve patient care and outcomes.

Disease-modifying drugs have improved the treatment for autoimmune joint disorders, such as rheumatoid arthritis, but inflammatory flares are a common experience. This work reports the development and application of flare-modulating poly(lactic-co-glycolic acid)-poly(ethylene glycol)-maleimide (PLGA-PEG-MAL)-based nanoparticles conjugated with joint-relevant peptide antigens, aggrecan70-84 and type 2 bovine collagen256-270. Peptide-conjugated PLGA-PEG-MAL nanoparticles encapsulated calcitriol, which acted as an immunoregulatory agent, and were termed calcitriol-loaded nanoparticles (CLNP). CLNP had a ∼200 nm hydrodynamic diameter with a low polydispersity index. In vitro, CLNP induced phenotypic changes in bone marrow derived dendritic cells (DC), reducing the expression of costimulatory and major histocompatibility complex class II molecules, and proinflammatory cytokines. Bulk RNA sequencing of DC showed that CLNP enhanced expression of Ctla4, a gene associated with downregulation of immune responses. In vivo, CLNP accumulated in the proximal lymph nodes after intramuscular injection. Administration of CLNP was not associated with changes in peripheral blood cell numbers or cytokine levels. In the collagen-induced arthritis and SKG mouse models of autoimmune joint disorders, CLNP reduced clinical scores, prevented bone erosion, and preserved cartilage proteoglycan, as assessed by high-resolution microcomputed tomography and histomorphometry analysis. The disease protective effects were associated with increased CTLA-4 expression in joint-localized DC and CD4+ T cells but without generalized suppression of T cell-dependent immune response. The results support the potential of CLNP as modulators of disease flares in autoimmune arthropathies.

ABSTRACT Staphylococcus aureus is an important human pathogen causing severe invasive infections. Pathogenesis is attributed to a wide array of virulence factors, including several potent exotoxins such as the pore-forming alpha-toxin. In this study, we found that patients with S. aureus respiratory tract infections had elevated CX 3 CL1 levels in airway fluid and plasma. Using humanized organotypic lung models, we observed that stimulation of lung epithelium with alpha-toxin induce an intensified CX 3 CL1 expression apically in the epithelium as well as the release of CX 3 CL1. Blocking alpha-toxin or ADAM10 activity in organotypic lung using an alpha-toxin-blocking antibody or a specific ADAM-10 inhibitor confirmed their role in modulating CX 3 CL1 cleavage and release. Analyses of CD14 + human monocytes in combination with a CX 3 CR1 inhibitor revealed that alpha-toxin-mediated CX 3 CL1 release induce CX 3 CL1-dependent chemotaxis. In line with these data, lung tissue from patients with S. aureus respiratory tract infection showed elevated CX 3 CL1 and CD14 staining as compared to tissue from patients with non-infectious lung diseases. Functional studies of monocytes showed that CX 3 CL1 released by lung models resulted in upregulated CD83 and downregulated CD86, as well as impaired killing of phagocytosed S. aureus . Furthermore, stimulation of monocytes with soluble CX 3 CL1 hampered their reactive-oxygen and nitric-oxide production. Taken together, our data show that S. aureus triggers the release of lung epithelial CX 3 CL1; a process found to be dependent on the alpha-toxin’s effect on ADAM10 mediating cytotoxicity and resulting in impaired monocyte phagocytic killing. Hence, we identify an immunomodulatory effect of alpha-toxin involving the CX 3 CL1-ADAM10 axis extending beyond the cytolysis function.

Disease modifying antirheumatic drugs (DMARDs) have improved the prognosis of autoimmune inflammatory arthritides but a large fraction of patients display partial or nonresponsiveness to front-line DMARDs. Here, an immunoregulatory approach based on sustained joint-localized release of all-trans retinoic acid (ATRA), which modulates local immune activation and enhances disease-protective T cells and leads to systemic disease control is reported. ATRA imprints a unique chromatin landscape in T cells, which is associated with an enhancement in the differentiation of naïve T cells into anti-inflammatory regulatory T cells (Treg ) and suppression of Treg destabilization. Sustained release poly-(lactic-co-glycolic) acid (PLGA)-based biodegradable microparticles encapsulating ATRA (PLGA-ATRA MP) are retained in arthritic mouse joints after intra-articular (IA) injection. IA PLGA-ATRA MP enhance migratory Treg which in turn reduce inflammation and modify disease in injected and uninjected joints, a phenotype that is also reproduced by IA injection of Treg . PLGA-ATRA MP reduce proteoglycan loss and bone erosions in the SKG and collagen-induced arthritis mouse models of autoimmune arthritis. Strikingly, systemic disease modulation by PLGA-ATRA MP is not associated with generalized immune suppression. PLGA-ATRA MP have the potential to be developed as a disease modifying agent for autoimmune arthritis.