Sulfasalazine is a prodrug known to be effective for the treatment of inflammatory bowel disease (IBD)-associated peripheral spondyloarthritis (pSpA), but the mechanistic role for the gut microbiome in regulating its clinical efficacy is not well understood. Here, treatment of 22 IBD-pSpA subjects with sulfasalazine identifies clinical responders with a gut microbiome enriched in Faecalibacterium prausnitzii and the capacity for butyrate production. Sulfapyridine promotes butyrate production and transcription of the butyrate synthesis gene but in F. prausnitzii in vitro, which is suppressed by excess folate. Sulfasalazine therapy enhances fecal butyrate production and limits colitis in wild-type and gnotobiotic mice colonized with responder, but not non-responder, microbiomes. F. prausnitzii is sufficient to restore sulfasalazine protection from colitis in gnotobiotic mice colonized with non-responder microbiomes. These findings reveal a mechanistic link between the efficacy of sulfasalazine therapy and the gut microbiome with the potential to guide diagnostic and therapeutic approaches for IBD-pSpA.

Abstract A central feature of intestinal mucosal immune system is the ability enable antigen specific tolerance to the gut bacteria. Genetic variants in TNFSF15 (which encodes protein TL1A) are linked to inflammatory bowel disease and have implicated roles for TL1A in regulating both T cell and innate lymphoid cells (ILC), but how these cellular pathways coordinate a response to limit intestinal inflammation is not completely understood. Deletion of TL1A receptor Tnfrsf25 (also called DR3), we demonstrate that TL1A signaling is required for efficient generation of RORγt+Foxp3+ intestinal iTregs at steady state. To investigate if this response resulted from DR3 deletion in T cells or the potential role for DR3 signaling in ILCs to regulate iTregs indirectly, we generated antigen specific transgenic T cells to H. Hepaticus (Hh) with DR3 deletion. Our results reveal that TL1A signaling is dispensable on T cells but required on ILC3s to regulate the balance between intestinal iTreg and inflammatory Th17 cells. RNA sequencing of ILC3s revealed the induction of Bhlhe40 in response to TL1A stimulation. Using conditional deletion models, we defined Bhlhe40 as a transcriptional regulator of ILC3 expression of Tnfsf4 (OX40L) and the subsequent requirement for ILC3-specific Bhlhe40 and Tnfsf4 in promoting Hh-specific Treg induction. These data reveal a novel function for TL1A and downstream transcription factor Bhlhe40 in enabling ILC3 coordination of gut commensal T cell immunity.

Environmental airborne antigens are central to the development of allergic asthma, but the cellular processes that trigger disease remain incompletely understood. In this report, Schmitt et al. (https://doi.org/10.1084/jem.20231236) identify TNF-like protein 1A (TL1A) as an epithelial alarmin constitutively expressed by a subset of lung epithelial cells, which is released in response to airborne microbial challenge and synergizes with IL-33 to drive allergic disease.

ABSTRACT Type III interferon signaling contributes to the pathogenesis of the important human pathogen Staphylococcus aureus in the airway. Little is known of the cellular factors important in this response. Using Ifnl2 -green fluorescent protein reporter mice combined with flow cytometry and cellular depletion strategies, we demonstrate that the alveolar macrophage is the primary producer of interferon lambda (IFN-λ) in response to S. aureus in the airway. Bone marrow chimeras showed reduced bacterial burden in IFN-λ receptor (IFNLR1)-deficient recipient mice, indicative that non-hematopoietic cells were important for pathogenesis, in addition to significant reductions in pulmonary inflammation. These observations were confirmed through the use of an airway epithelial-specific IFNLR knockout mouse. Our data suggest that upon entry to the airway, S. aureus activates alveolar macrophages to produce type III IFN that is subsequently sensed by the airway epithelium. Future steps will determine how signaling from the epithelium then exerts its influence on bacterial clearance. These results highlight the important, yet sometimes detrimental, role of type III IFN signaling during infection and the impact the airway epithelium plays during host–pathogen interactions. IMPORTANCE The contribution of type III interferon signaling to the control of bacterial infections is largely unknown. We have previously demonstrated that it contributes to the pathogenesis of acute Staphylococcus aureus respiratory infection. In this report, we document the importance of two cell types that underpin this pathogenesis. We demonstrate that the alveolar macrophage is the cell that is responsible for the production of type III interferon and that this molecule is sensed by airway epithelial cells, which impacts both bacterial clearance and induction of inflammation. This work sheds light on the first two aspects of this important pathogenic cascade.

Intestinal inflammation associated with chronic inflammatory bowel disease (IBD) increases the risk of developing colitis-associated cancer (CAC), but the cellular mechanisms driving CAC are not well defined. Polymorphisms in TNFSF15 (called TNF-like cytokine 1a or TL1A) are highly associated with IBD and the aim of this work is to evaluate a potential mechanistic role of TL1A in regulating CAC. Data from the human atlas protein database revealed that TL1A protein is present in 50% of colorectal cancer tissue samples and that high expression of TNFSF15 correlated with reduced survival. To test the functional role for TL1A in tumorigenesis, we used the well-established AOM/DSS model of CAC in mice deficient for the TL1A receptor (called death receptor 3 or DR3) and mice deficient for myeloid derived TL1A (CD11C cre+TNFSF15fl/fl). We found that both DR3-deficient mice and myeloid derived TL1A deficient mice had a significant reduction in tumor number compared to heterozygous littermate controls. To evaluate the contribution of innate lymphocytes, we used DR3-deficient mice on a RAG-deficient background in our AOM/DSS model. Even in the absence of B and T cells, DR3-deficient mice still showed significant reduction in tumor burden, suggesting innate lymphoid cell (ILC) contribution. DR3-deficient mice also revealed a significant reduction in neutrophil infiltration. Neutrophil depletion with α-Ly6G similarly resulted in a significant reduction of tumor numbers in our CAC model in a DR3 dependent manner. To test the direct regulation of neutrophils by ILCs, we establish a co-culture model. TL1A activated ILCs triggered neutrophil activation (CD177) and maturation (CD11B) which required GM-CSF. In vivo activation with a DR3 agonist confirmed intestinal ILC3 production of GM-CSF but also impacted bone marrow granulopoiesis by expanding both mature neutrophils and granulocyte-macrophage progenitors (GMPs). CONCLUSION: Our data reveals a new link between intestinal ILC production of GM-CSF and neutrophil activation that promotes CAC and bone marrow granulopoiesis.

Joint inflammation, or spondyloarthritis (SpA), is the most common extra-intestinal manifestation of inflammatory bowel disease (IBD), but the specific role for therapies targeting SpA is not well defined. One of the earliest medications used for the treatment of IBD is sulfasalazine (SAS). SAS is a prodrug composed of two chemical moieties, the anti-inflammatory 5-aminosalicilate and the antibiotic sulfapyridine. The efficacy of SAS in peripheral arthritis is thought to depend on its “antibacterial” properties, however the impact of SAS on the IBD-SpA microbiome and how it may improve extra-intestinal symptoms is unknown. Therefore, our study aims to diagnostically evaluate the role for the fecal microbiome in clinical response to SAS therapy and identify microbial and immunologic therapeutic targets associated with clinical response. We have designed an observational study to longitudinally follow IBD patients with SpA who have a medical indication for SAS therapy. Clinical data and fecal samples from 22 patients were collected before initiation of SAS and at week 12 after initiation of SAS. Eleven IBD-SpA patients were concomitantly enrolled as controls and followed by 12 weeks. Metagenomic sequencing was used to define the effect of SAS on the IBD-SpA fecal microbiome and to evaluate its relationship with improvement in joint symptoms. Mouse models and in vitro assays were used to test the sufficiency of the SAS effect observed in patients. The fecal microbiome of SAS-responders was distinct from that observed in non-responders and 6 pre-treatment microbial markers (including the short chain fatty acid (SCFA) producer Faecalibacterium prausnitzii) predicted SAS-response (AUC = 0.9). Fecal metabolome of SAS responders had lower thymine and higher deoxyuridine compared to non-responders consistent with evidence of a folate trap in response to SAS treatment. SAS therapy in SPF mouse-model of chemically-induced colitis alleviated colitis in GPR 109a-dependent fashion consistent with a synergistic role for SCFA. In vitro assays revealed SAS direct regulation of F. prausnitzii metabolic function and butyrate synthesis. CONCLUSIONS: Collectively, these findings highlight the potential role for microbial diagnostics to improve SAS efficacy, and drug modulation of microbial markers to potentiate therapy for IBD patients with SpA.

BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) is an emerging treatment modality for ulcerative colitis (UC). Several randomized controlled trials have shown efficacy for FMT in the treatment of UC, but a better understanding of the transferable microbiota and their immune impact is needed to develop more efficient microbiome-based therapies for UC. METHODS: Metagenomic analysis and strain tracking was performed on 60 donor and recipient samples receiving FMT for active UC. Sorting and sequencing of immunoglobulin (Ig) A-coated microbiota (called IgA-seq) was used to define immunereactive microbiota. Colonization of germ-free or genetically engineered mice with patient-derived strains was performed to determine the mechanism of microbial impact on intestinal immunity. RESULTS: Metagenomic analysis defined a core set of donor-derived transferable bacterial strains in UC subjects achieving clinical response, which predicted response in an independent trial of FMT for UC. IgA-seq of FMT recipient samples and gnotobiotic mice colonized with donor microbiota identified Odoribacter splanchnicus as a transferable strain shaping mucosal immunity, which correlated with clinical response and the induction of mucosal regulatory T cells. Colonization of mice with O splanchnicus led to an increase in Foxp3 + /RORyt + regulatory T cells, induction of interleukin (IL) 10, and production of short chain fatty acids, all of which were required for O splanchnicus to limit colitis in mouse models. CONCLUSIONS: This work provides the first evidence of transferable, donor-derived strains that correlate with clinical response to FMT in UC and reveals O splanchnicus as a key component promoting both metabolic and immune cell protection from colitis. These mechanistic features will help enable strategies to enhance the efficacy of microbial therapy for UC.

Acinetobacter baumannii is an opportunistic pathogen that has recently emerged as a global threat associated with high morbidity, mortality, and antibiotic resistance. We determined the role of type I interferon (IFN) signaling in A. baumannii infection. We report that A. baumannii can induce a type I IFN response that is dependent upon TLR4-TRIF-IRF3 and phagocytosis of the bacterium. Phase variants of A. baumannii that have a reduced capsule, lead to enhanced TLR4-dependent type I IFN induction. This was also observed in a capsule-deficient strain. However, we did not observe a role for this pathway in vivo. The enhanced signaling could be accounted for by increased phagocytosis in capsule-deficient strains that also lead to enhanced host cell-mediated killing. The increased cytokine response in the absence of the capsule was not exclusive to type I IFN signaling. Several cytokines, including the proinflammatory IL-6, were increased in cells stimulated with the capsule-deficient strain, also observed in vivo. After 4 h in our acute pneumonia model, the burden of a capsule-null strain was significantly reduced, yet we observed increases in innate immune cells and inflammatory markers compared to wild-type A. baumannii. This study underscores the role of phase variation in the modulation of host immune responses and indicates that the capsule of A. baumannii plays an important role in protection against host cell killing and evasion from activation of the innate immune response.

Extracytoplasmic function (ECF) sigma factors are key transcriptional regulators that prokaryotes have evolved to respond to environmental challenges. Streptomyces tsukubaensis harbours 42 ECFs to reprogram stress-responsive gene expression. Among them, SigG1 features a minimal conserved ECF σ2–σ4 architecture and an additional C-terminal extension that encodes a SnoaL_2 domain, which is characteristic for ECF σ factors of group ECF56. Although proteins with such domain organisation are widely found among Actinobacteria, the functional role of ECFs with a fused SnoaL_2 domain remains unknown. Our results show that in addition to predicted self-regulatory intramolecular amino acid interactions between the SnoaL_2 domain and the ECF core, SigG1 activity is controlled by the cognate anti-sigma protein RsfG, encoded by a co-transcribed sigG1-neighbouring gene. Characterisation of ∆sigG1 and ∆rsfG strains combined with RNA-seq and ChIP-seq experiments, suggests the involvement of SigG1 in the morphological differentiation programme of S. tsukubaensis. SigG1 regulates the expression of alanine dehydrogenase, ald and the WhiB-like regulator, wblC required for differentiation, in addition to iron and copper trafficking systems. Overall, our work establishes a model in which the activity of a σ factor of group ECF56, regulates morphogenesis and metal-ions homeostasis during development to ensure the timely progression of multicellular differentiation.

Acinetobacter baumannii (A. baumannii) is an extremely versatile multidrug-resistant pathogen with a very high mortality rate; therefore, it has become crucial to understand the host response during its infection. Given the importance of mice for modeling infection and their role in preclinical drug development, equal emphasis should be placed on the use of both sexes. Through our studies using a murine model of acute pneumonia with A. baumannii, we observed that female mice were more susceptible to infection. Likewise, treatment of male mice with estradiol increased their susceptibility to infection. Analysis of the airway compartment revealed enhanced inflammation and reduced neutrophil and alveolar macrophage numbers compared with male mice. Depletion of either neutrophils or alveolar macrophages was important for bacterial clearance; however, depletion of alveolar macrophages further exacerbated female susceptibility because of severe alterations in metabolic homeostasis. Our data highlight the importance of using both sexes when assessing host immune pathways.

The oxidative stress response is a key mechanism that microorganisms have to adapt to changeling environmental conditions. Adaptation is achieved by a fine-tuned molecular response that extends its influence to primary and secondary metabolism. In the past, the role of the intracellular redox status in the biosynthesis of tacrolimus in Streptomyces tsukubaensis has been briefly acknowledged. Here, we investigate the impact of the oxidative stress response on tacrolimus biosynthesis in S. tsukubaensis. Physiological characterization of S. tsukubaensis showed that the onset of tacrolimus biosynthesis coincided with the induction of catalase activity. In addition, tacrolimus displays antioxidant properties and thus a controlled redox environment would be beneficial for its biosynthesis. In addition, S. tsukubaensis ∆ahpC strain, a strain defective in the H2O2-scavenging enzyme AhpC, showed increased production of tacrolimus. Proteomic and transcriptomic studies revealed that the tacrolimus over-production phenotype was correlated with a metabolic rewiring leading to increased availability of tacrolimus biosynthetic precursors. Altogether, our results suggest that the carbon source, mainly used for cell growth, can trigger the production of tacrolimus by modulating the oxidative metabolism to favour a low oxidizing intracellular environment and redirecting the metabolic flux towards the increase availability of biosynthetic precursors.

Staphylococcus aureus small colony variants (SCVs) are frequently associated with chronic infection, yet they lack expression of many virulence determinants associated with the pathogenicity of wild-type strains. We found that both wild-type S. aureus and a Δ hemB SCV prototype potently activate glycolysis in host cells. Glycolysis and the generation of mitochondrial reactive oxygen species were sufficient to induce necroptosis, a caspase-independent mechanism of host cell death that failed to eradicate S. aureus and instead promoted Δ hemB SCV pathogenicity. To support ongoing glycolytic activity, the Δ hemB SCV induced over a 100-fold increase in the expression of fumC , which encodes an enzyme that catalyses the degradatin of fumarate, an inhibitor of glycolysis. Consistent with fumC -dependent depletion of local fumarate, the Δ hemB SCV failed to elicit trained immunity and protection from a secondary infectious challenge in the skin. The reliance of the S. aureus SCV population on glycolysis accounts for much of its role in the pathogenesis of S. aureus skin infection.

Acinetobacter baumannii is an emerging opportunistic pathogen that has risen to become a serious global threat, prevalent in health care settings and the community, which results in high morbidity and mortality rates. Its alarming expansion of antibiotic resistance is one of the most problematic traits of A. baumannii and as so, this bacterium has been classified as a serious threat and high priority target by the CDC. The most common types of infections induced by this pathogen include pneumonia (both hospital and community acquired), bacteremia, skin and soft tissue, urinary tract infections, endocarditis, and meningitis. Nosocomial pneumonia is the most prevalent of these. This review summarizes the current state of the signaling and innate immune components activated in response to A. baumannii infection in the airway.

Staphylococcus aureus is a major cause of both community- and healthcare-acquired pneumonias. Inducible costimulator (ICOS) is part of the CD28 family of proteins and is a target for immune checkpoint therapy. We found ICOS highly expressed on activated CD4 cells in response to S. aureus. In the absence of ICOS, mice had improved survival in a pneumonia model with the methicillin-resistant Staphylococcus aureus (MRSA) strain USA300 and significant reductions in bacterial burden in a nonlethal acute pneumonia model. Infected Icos-/- mice had major reductions in several proinflammatory cytokines, neutrophils, inflammatory monocytes, and eosinophils compared to infected wild-type mice, while there was improved expression of CD11c and macrophage receptor with collagenous structure on the surface of alveolar macrophages. Early during infection infected Icos-/- mice had increased numbers of alveolar macrophages and expression of several surface markers on alveolar macrophages and neutrophils. ICOS signaling also contributed to the pathogenesis of the airway pathogens Klebsiella pneumoniae, Pseudomonas aeruginosa, and Streptococcus pneumoniae, and neutralizing antibody to ICOS led to improved clearance of S. aureus from the airway. Our results indicate that ICOS plays a significant role in orchestrating the innate immune response to S. aureus and other airway pathogens, and could be a potential immunomodulatory target to attenuate S. aureus-related immunopathology.

Maintaining balanced levels of IL-1 is extremely important to avoid host tissue damage during infection. Our goal was to understand the mechanisms behind the reduced pathology and decreased bacterial burdens in Ifnlr1(-/-) mice during lung infection with Staphylococcus aureus. Intranasal infection of Ifnlr1(-/-) mice with S.aureus led to significantly improved bacterial clearance, survival and decrease of proinflammatory cytokines in the airway including IL-1. Ifnlr1(-/-) mice treated with recombinant IL-1 displayed increased bacterial burdens in the airway and lung. IL-1 levels in neutrophils from Ifnlr1(-/-) infected mice lungs were decreased when compared to neutrophils from WT mice. Mice lacking NLRP3 and caspase-1 had reduced IL-1 levels 4h after infection, due to reductions or absence of active caspase-1 respectively, but levels at 24h were comparable to WT infected mice. Ifnlr1(-/-) infected mice had decreases in both active caspase-1 and neutrophil elastase indicating an important role for the neutrophil serine protease in IL-1 processing. By inhibiting neutrophil elastase, we were able to decrease IL-1 levels by 39% in Nlrp3(-/-) infected mice when compared to WT mice. These results highlight the crucial role of both proteases in IL-1 processing, via inflammasome-dependent and -independent mechanisms.

Staphylococcus aureus is an important human pathogen, in particular the methicillin resistant S. aureus (MRSA) strain USA300 that is epidemic in the United States. Type III interferons (IFN-λ) signal through the IL-28 receptor (IFNLR) located on epithelial cells and neutrophils. Neutrophil-mediated killing is a crucial defence system against S. aureus. Compared to WT mice, Ifnlr−/− mice had significantly improved clearance of S. aureus from the airway and lung tissue (>90%; P<0.001) following a 24-hour infection. In a mortality model of infection, all Ifnlr−/− mice survived, whereas 75% of WT mice succumbed to infection (P<0.05). Despite a lack of difference in neutrophil recruitment to the airway in WT and Ifnlr−/− mice in response to S. aureus, Ifnlr−/− infected mice showed a significant increase in the neutrophil related cytokine G-CSF (38%; P<0.05). Moreover, expression of the neutrophil surface markers, Ly6C and Ly6G, were higher (39% and 20%, respectively; P<0.01) on infected Ifnlr−/− neutrophils compared to WT infected mice. Neutrophils isolated from Ifnlr−/− mice compared to WT neutrophils also displayed a 2-fold increase in MPO activity (peroxidation) (p<0.05) and a 2.1-fold increase in Ca2+ flux when compared to WT neutrophils (P<0.001), while WT neutrophils stimulated with purified IFN-λ displayed a 0.5-fold decrease in Ca2+ flux (p<0.005). In addition, stimulation of WT neutrophils with purified IFN-λ led to a 2-fold decrease of their S. aureus killing capacity (P<0.05). Controlling neutrophil recruitment and activation is important to provide a balanced response to infection. Our data suggests that type III IFN signalling can impair neutrophil activation and their killing capacity in response to S. aureus.