Discovery Immunology

IntroductionT cell activation is central to the adaptive immune response. In vitro studies on T cell activation often utilize two distinct approaches: first, engaging T cell receptors (TCR) using plate-bound CD3 together with soluble CD28 (TCR-dependent). Second, triggering intracellular signalling cascades using phorbol 12-myristate 13-acetate (PMA) and Ionomycin or P+I (TCR-independent). Both methods are widely used; however, a systematic comparison of the activation methods across a range of stimulation strengths to evaluate their effects on T cell function and metabolism has not been investigated in great detail. In this study, we compared the consequences of engaging T cells using TCR-dependent and TCR-independent activation pathways across varying signal strengths. MethodsT cells from BALB/c mice were isolated and activated under four conditions: CD3, CD3+CD28, PMA with low Ionomycin (P+IL) and PMA with high Ionomycin (P+IH). We studied differences with respect to several parameters: morphology, flow analysis, metabolic activities, cytokines. The roles of Protein kinase C (PKC) and Ca{superscript 2} pathways were addressed by supplementing CD3+CD28 cultures with different doses of exogenous PMA or Ionomycin. ResultsP+I activation outperformed the CD3+CD28 activation system across most readouts by displaying enhanced blasts, higher cycling, greater glucose uptake, increased lactate and ROS production, together with higher upregulation of CD25 and CD44 activation markers. P+IH activation dampened several responses including CD69 expression. CD4 co-receptor was downregulated greatly with P+I activation but not CD3+CD28. Most cytokines followed signal strength comparably between both systems; however, differences were observed with others: P+I stimulation favoured IL-6 and IL-12 induction whereas CD3+CD28 activation preferentially induced CCL2 and IL-1{beta}. Importantly, PKC activity was substantially lower upon CD3+CD28 stimulation and the addition of PMA, but not Ionomycin, to CD3+CD28 cultures enhanced proliferation, metabolism and expression of activation markers. DiscussionTCR-dependent and TCR-independent T cell activation models have clear functional and metabolic differences. The observation that PKC signalling can boost T cell activation with CD3+CD28 is likely to be significant and may have translational implications such as CAR-T cell anti-tumor therapy where CD3+CD28 stimulation is widely used. The implications of our findings with regard to augmenting T cell mediated immunotherapies are discussed.

Cannabinoid receptor 2 (CB2R) is highly expressed on immune cells, but its role in T cell metabolism remains unclear. Here, we show that CB2R activation rapidly increases glucose uptake in human Jurkat T cells and drives a broader metabolic reprogramming away from glycolysis toward oxidative metabolism and the pentose phosphate pathway. Pharmacological CB2R activation increased mitochondrial mass, spare respiratory capacity, proton leak, and NADPH production, while CB2R inverse agonism produced the opposite effects. These metabolic changes were accompanied by upregulation of key pentose phosphate pathway enzymes, including GALT and TALDO1, and were abolished in CNR2-deficient cells, confirming receptor dependence. In primary human lamina propria mononuclear cells, CB2R signalling also influenced memory and gut-homing-associated T cell phenotypes, including integrin 4{beta}7 expression. Together, these findings identify CB2R as a regulator of T cell bioenergetics and suggest that cannabinoid signalling may promote metabolic states linked to memory and tissue-homing functions in chronic intestinal inflammation.

Dysregulated gut homing of leukocytes drives chronic inflammation in Crohns disease (CD). We employed phage display selection campaigns with libraries of stabilized, constrained peptides against endogenous conformation states of the cannabinoid receptor CB2R on human T cells, to discover novel receptor antagonists with potential to inhibit gut homing. Cluster and frequency analysis of 50,000 enriched sequences resulted in expression and functional characterisation of 10 protein candidates using assays of glucose uptake, ERK phosphorylation (pERK) and beta-arrestin recruitment. Each candidate antagonised CB2R activity with recorded IC50 values of between 5-10 nM. Cannabinoid receptor nanodisc binding experiments and SPR confirmed CB2R selectivity. SLKC_09 with an IC50 of 5.4 nM, was studied in a mouse model of chronic ileitis where it significantly inhibited gut homing of CD4+ & CD8+ naive, effector and memory cell types. Our findings highlight an alternative route to therapeutic inhibition of leukocyte trafficking in CD with a biologic inhibitor of CB2R.

Macrophages in the tumor microenvironment are known to upregulate PD-L1 expression, thereby suppressing T cells through PD-1 ligation. However, the manner in which PD-L1 expression intrinsically impacts macrophages and their immunomodulatory phenotype is less clear. Clarifying this knowledge gap would yield insight into the mechanisms of immunosuppression within the tumor microenvironment. To characterize the macrophage intrinsic role of PD-L1, we used complementary genetic and pharmacological approaches by analyzing primary murine bone marrow-derived macrophages (BMDMs) with complete genetic PD-L1 deletion and wildtype BMDMs treated with anti-PD-L1 blocking antibodies. Macrophages were evaluated across naive, pro-inflammatory (M1), and tumor conditioned (TCM) polarization states in vitro. Unlike prior reports, neither genetic deletion nor antibody blockade dramatically altered the expression of macrophage polarization markers or in vitro phagocytic capacity. Both conditions consistently reduced surface levels of the M1-associated costimulatory molecule CD80, prompting further analysis of T cell interacting and antigen presenting proteins, in which we revealed disparate effects of genetic deletion and antibody blockade on the surface levels of MHCI, MHCII, PD-1, and PD-L2. These findings suggest that PD-L1 deletion and antibody-mediated blockade contribute to macrophage immune regulatory profiles in distinct manners. This difference supports a model in which PD-L1 functions in macrophages beyond its canonical role as a ligand for PD-1, influencing antigen presentation and checkpoint molecule levels and playing a broader role in immune regulation in the tumor microenvironment.

Glycosylphosphatidylinositol (GPI) anchors are involved in the organization of membrane microdomains that support T cell receptor (TCR) signaling. However, their role in regulating expression of TCR-related proteins and downstream functional output remains unclear. This study aimed to characterize the effects of GPI-deficiency on TCR, cluster of differentiation 3 (CD3), and CD28 expression as well as interleukin-2 (IL-2) production using a GPI-deficient Jurkat T cell line (S12). Flow cytometry confirmed the complete loss of GPI anchors and GPI-anchored proteins (GPI-APs) in the S12 cell line. Compared to GPI-producing parental Jurkat, S12 had significantly higher expression of CD3 and TCR{beta} while CD28 had similar expression. IL-2 production by S12 was assessed following stimulation with anti-CD3/anti-CD28 beads and following stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Neither S12 nor parental Jurkat produced detectable IL-2 in response to anti-CD3/anti-CD28 bead-mediated stimulation. Both parental Jurkat and S12 produced IL-2 following PMA/ionomycin-mediated stimulation. No significant difference in IL-2 production was observed between S12 and parental Jurkat following PMA/ionomycin-mediated stimulation. These findings demonstrate that GPI-deficiency influences surface receptor expression but does not significantly impair downstream IL-2 production under PMA/ionomycin stimulation. This finding suggests that GPI anchors and GPI-APs contribute to proximal signaling organization but are not required for cytokine production when downstream pathways are directly activated.

Naturally occurring variation in the bovine mannose receptor C-type 1 gene (MRC1) may shape macrophage responses to Mycobacterium (M.) bovis, a key driver of bovine tuberculosis (bTB). We identified four coding region SNPs in MRC1 across Bos taurus (Holstein Friesian, Brown Swiss) and Bos indicus (Boran, Sahiwal) cattle breeds, including a non-synonymous variant, rs380943118 (c.2963G>A; Ser988Asn) in C-type lectin-like domain (CTLD) 6, most prevalent in Sahiwal cattle. Structural modelling suggested that the S988N substitution, which is spatially separated from the monosaccharide binding site of CTLD4, might indirectly affect glycan binding, perhaps through a conformational change in the receptor. Monocyte-derived macrophages upregulated MR expression during differentiation, with heterozygous (G/A) animals showing higher MR expression and increased uptake of GFP-M. bovis BCG, although differences were not statistically significant. Anti-CD206 blockade did not inhibit BCG internalization, either indicating that this specific antibody did not bind to a CTLD involved in ligand binding or that MR is not the sole entry receptor. These results highlight naturally occurring MRC1 polymorphisms that may influence MR structure and macrophage function, providing a foundation for future studies to assess their role in bTB susceptibility.

Aberrant communication between cells of the immune system can drive disease progression. Cytokines form the central pilar of immune cell communication and are well established factors in lymphomagenesis. An increasing body of evidence suggests that immunometabolism is tightly connected to cytokine production. However, the exact link between metabolism and cytokine responses during lymphomagenesis remains largely unknown. Here, we used established cell models representing the most common form of B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), to study the effect of metabolism on cytokine production. We found that stimulation or inhibition of the glycolysis pathway could attenuate IL-6, IL-10 and TNFa; production by DLBCL. Furthermore, we found that two different subtypes of DLBCL displayed distinct metabolic responses to IL-4. In summary, our work suggests that metabolic pathways could be involved in controlling cytokine production in DLBCL, and paves the road for further research aimed at finding specific metabolic targets that can be exploited for therapeutic intervention.

Receptor activator of NF{kappa}B ligand (RANKL) is important for bone metabolism, but also modulates immune processes. We showed that mast cells (MCs) are involved in RANKL regulation, but the importance of MC-derived RANKL in skin inflammation has not yet been investigated. In contact hypersensitivity (CHS), the absence of MC-derived RANKL led to reduced skin inflammation due to impaired leukocyte infiltration and blood lymphopenia. Surprisingly, we observed a massive hyperplasia of the distant inguinal lymph nodes in the absence of MC-RANKL. Using adoptive transfers, flow cytometry and whole-mount 3D imaging, we demonstrated that this was not caused by structural maladaptation, but rather by the inability of lymphocytes to exit in a timely manner. Importantly, RANKL deletion in skin MCs only replicated the effect of LN hyperplasia and blood lymphopenia. Moreover, MCs were involved in serum sphingosine-1-phosphate (S1P) regulation during sensitization and challenge. Intravascular administration of S1P restored timely lymphocyte egress, demonstrating a MC-induced organ-spanning RANKL-S1P axis. Consequently, peripheral skin MC-derived RANKL is essential for the timely lymphocyte egress from distant LNs, which may have important implications for the targeted treatment of inflammatory skin diseases.

The IgG Fc chain carries a single N-linked glycan which may undergo changes. Increased agalactosylated N-glycans are associated with rheumatoid arthritis (RA) and regarded as pro-inflammatory. Dysregulated neutrophils can make important contributions to host tissue damage. In RA, immune complexes (ICs) that have precipitated onto synovial joint surfaces activate neutrophils via Fc receptors, promoting localised inflammation. We engineered recombinant human monoclonal IgG with agalactosylated or galactosylated N-glycans, generated immobilised ICs and stimulated healthy donor and RA patient blood-derived neutrophils, comparing reactive oxygen species (ROS) production as read-out of neutrophilic inflammation. Both healthy donor and RA patient neutrophils generated less ROS when stimulated with ICs made from agalactosylated IgG. Mechanistically this was due to poorer binding of agalactosylated ICs to neutrophil FcgammaRs, causing lower activation of Akt and p38 MAPK. Both are required for immobilised IC-mediated stimulation of the neutrophil NADPH oxidase. Taken together, this suggests that disease-associated, agalactosylated IgG does not in fact promote inflammation and host tissue injury, at least not by acting on neutrophils. We propose that rather than promoting inflammation, agalactosylated IgG N-glycans that accompany inflammatory disease may arise as part of a compensatory mechanism that is aimed at reducing excessive inflammation and host tissue injury.

Inflammatory arthritis and uveitis frequently co-exist, yet the mechanisms linking joint and ocular inflammation remain ill-defined. Here, we investigated how inflammatory arthritis influences ocular immune homeostasis using murine models of antigen-induced arthritis and collagen-induced arthritis. Arthritis promoted the accumulation of T cells, myeloid cells, and neutrophils within the vitreoretinal compartment, without progression to overt clinical uveitis. Ocular leukocyte recruitment was dynamically coupled to arthritis activity, resolving with remission of joint inflammation and recurring during arthritic flares. The magnitude of ocular immune perturbation correlated with arthritis severity, being enhanced in IL-27R-deficient mice and markedly reduced in IL-6R-deficient mice. Mechanistically, arthritis increased blood-retinal barrier permeability, demonstrating that systemic inflammation perturbs ocular immune privilege even in the absence of apparent ocular disease. While arthritis alone was insufficient to induce uveitis, it established a permissive ocular microenvironment that selectively enhanced the recruitment of adoptively transferred uveitogenic CD4+ T cells. These findings identify inflammatory arthritis as a systemic driver of subclinical ocular immune dysregulation and reveal a mechanism by which inflammation at a distant site may promote vulnerability to ocular autoimmunity. These data provide a framework for understanding immune dysregulation at the joint-eye axis and highlight cytokine pathways that may be targeted to preserve ocular immune homeostasis.

BackgroundSevere eosinophilic asthma (SEA), eosinophilic granulomatosis with polyangiitis (EGPA) and nasal polyposis (NP) are immune-mediated diseases characterised by eosinophilic inflammation. However, there is also increasing interest in the potential pathological roles of autoantibodies in these diseases. Understanding their B cell receptor (BCR) repertoires may provide valuable insights into disease mechanisms, and potential role of B cells in their pathology. MethodsWe conducted BCR repertoire sequencing using peripheral blood from 43 patients, comprising SEA with nasal polyps (SEA+NP), SEA without nasal polyps (SEA-NP), and EGPA, along with 16 healthy controls (HCs). ResultsCompared to HCs, patients with EGPA exhibited increased relative proportions of IgA1, IgG1, IgG2, and IgG4 subclasses. Similarly, SEA-NP patients demonstrated significantly high proportion of IgG2 sequences. Notably, the IgG4 subclass was significantly elevated across all patient groups compared to HCs. Patients receiving anti-IL-5/5R biologic treatments showed increased relative proportions of IgA2 and IgG2 subclasses compared to untreated patients. Some variation across participant groups in mean somatic hypermutation and mutation frequency was evident. 1,508 clones shared across patients, but not healthy controls, were evident though the majority showed low clonal expansion. Nevertheless, a few shared clones did show either high prevalence across patients and/or higher clonal expansion. ConclusionChanges in BCR repertoires in SEA/EGPA are consistent with a pattern of a more mature B cell component in the periphery and with the T2 inflammatory response observed in SEA and EGPA. BCR clonotypes shared across patients were evident, however, whether such clonotypes are pathological in SEA/EGPA requires further investigation.

Functionally competent, highly purified neutrophils were isolated from healthy human donors. Neutrophil necroptosis and neutrophil extracellular trap (NET) formation were induced using TNF- in combination with the pan-caspase inhibitor zVAD-FMK and the IAP antagonist BV6. NETs were visualized, quantified, and morphologically characterized by fluorescence microscopy following staining with Hoechst 33342 and Sytox Green. Levels of extracellular, cell-free neutrophil elastase (NE), myeloperoxidase (MPO), and DNA were also measured as indicators of NET release. The ability of TACT507, a proprietary RIPK3 antagonist, to efficiently block NETs formation and NET related necroptosis was evaluated. TACT507 demonstrated concentration dependent, significant inhibition of neutrophil necroptosis and NETs formation. Summary SentenceIsolated human neutrophils treated ex vivo by TNF- in combination with apoptosis inhibitors, underwent necroptosis causing formation of neutrophil extracellular traps. This process was inhibited by the proprietary antagonist of RIPK3.

Gasdermins (GSDMs) are a family of pore forming protein that trigger pyroptosis by permeabilizing cell membranes. Pyroptotic cells often release the proinflammatory cytokines interleukin-1{beta} (IL-1{beta}), and IL-18, thereby promoting an inflammatory response. GSDMs are typically cleaved by caspases or granzymes, which enable their translocation to the membrane. Here, we showed GSDMA and GSMDB localize to the cytoskeletal fraction of keratinocytes. Disruption of cell junctions causes gasdermin A and B (GSDMA and GSDMB) to translocate to the membrane fraction in the absence of cleavage. Cell junction disrupted keratinocytes release post-translationally modified keratins, but not IL-1{beta} or IL-18. These events depend on endocytic mechanisms associated with recycling of cell junctional proteins. Our study suggests that cell junction disruption can drive translocation of GSDMA and GSDMB from cytoskeleton to plasma membrane in keratinocytes, however there may be a subsequent trigger that causes the confirmational change allowing these gasdermins to form open pores.

Streptococcus pneumoniae (pneumococcus) are asymptomatic colonizers of the nasopharynx but can progress to pulmonary and systemic pathogens upon influenza A virus (IAV) infection. Polymorphonuclear cells (PMNs) are required to control bacterial numbers, but we previously found that IAV infection impairs their ability to kill S. pneumoniae. Here, using a model that allows transition of pneumococci from colonizers to disease-causing pathogens upon IAV co-infection, we examined the signaling pathways impairing PMN responses. When we investigated the effect of type I interferons (IFN) produced upon IAV infection on PMN antibacterial activity, we found that PMNs treated with IFN were unable to kill S. pneumoniae ex vivo, and that in vivo blocking of IFN receptor 1 (IFNAR1) in IAV infected mice rescued PMN antibacterial function. In exploring what controls PMN responsiveness to IFN, we examined CD73, an ectonucleotidase that is known to regulate PMN function in primary pneumococcal pneumonia. To test if there is an intersection between CD73 and IFN signaling, we examined receptor levels and IFN production in wildtype versus CD73KO mice and found no difference in IFNAR expression on PMNs or IFN[a] and IFN levels in the lungs and circulation. However, CD73KO PMNs expressed significantly lower levels of the interferon stimulated protein IFIT1. When we looked at ex vivo PMN responsiveness to IFNs, CD73KO PMNs were less responsive to IFN-mediated inhibition of antimicrobial activity. In exploring mechanisms, we found that CD73 expressing PMNs had elevated production of reactive oxygen species in response to IAV challenge, that paradoxically impaired their ability to kill S. pneumoniae. Importantly, despite similar pathogen loads in the respiratory tract, co-infected CD73KO mice cleared bacteremia and survived significantly better than wildtype controls. These findings suggest that CD73 impairs host defense against IAV/S. pneumoniae co-infection in part by sensitizing PMNs to type I IFN-mediated inhibition of antibacterial function. Author SummaryDespite available therapeutics and vaccines, secondary bacterial pneumonia following influenza A virus (IAV) infection remains a major cause of disease. A common cause of secondary bacterial pneumonia are Streptococcus pneumoniae (pneumococcus), bacteria that resides asymptomatically in the nasopharynx, but upon viral infection can transition to cause severe disease in susceptible hosts. In this study we examined how host responses change during single versus polymicrobial infections. We focused on neutrophils, which are innate immune cells that are required for effective clearance of S. pneumoniae, and proper control of IAV. We found that the immune response to IAV, mediated by type I interferons (IFN), impair the ability of neutrophils to kill bacteria. We identified an enzyme called CD73 to be required for the ability of neutrophils to respond to IFN. In exploring mechanisms, we found that IFN and CD73 result in dysregulated reactive oxygen species production by neutrophils. Importantly, this impairs the ability of the host to clear bacteria that spread from the lungs to the blood upon viral co-infection and results in overall worse host outcome. This study describes a novel interaction between CD73 and type I interferons and provides a new therapeutic target to treat secondary pneumococcal pneumonia.

To establish chronic infection in the vasculature of their infected host, schistosomes have developed multifaceted strategies of immune subversion. Extracellular parasite proteins are believed to play immunomodulatory functions, but their mode of action remains largely elusive. To investigate whether proteins secreted by the Schistosoma mansoni parasite have the potential to directly interact with host immune receptors, we performed a large-scale protein:protein interaction study between selected parasite proteins sharing structural similarities with known host immune effectors and a protein array of over 750 full-length human ectodomains mostly expressed by immune cells. We identified CD177 as a neutrophil receptor for S. mansoni Granulin (SmGrn). SmGrn exclusively bound the surface of CD177+ human neutrophils and led to cellular hyporesponsiveness following stimulation with LPS as evidenced by decreases in surface markers of activation, delayed reactive oxygen species production and reduced IL-8 release. In addition, human neutrophils exposed to SmGrn showed delayed apoptosis and morphological changes compatible with a more quiescent state as well as transcriptional upregulation of negative regulators of interferon signalling. These data suggest that SmGrn dampens human neutrophil response to stimulation and may lead to suboptimal function during schistosome infection.

Hematopoietic stem and progenitor cells (HSPCs) sustain the production of hundreds of billions of new cells per day to maintain our blood and immune system. In this process, HSPCs regulate the hematopoietic output by sensing and integrating diverse physiological cues. Thus, HSPCs express many receptors traditionally studied for their functions in the immune system, and this allows HSPCs to directly detect microbial compounds, endogenous danger signals, cytokines, and other inflammatory mediators. However, how the expression levels of such receptors on HSPCs change under chronic inflammation and how such changes alter HSPC functions and immune cell production remains unexplored. Working in a murine model of rheumatoid arthritis, we demonstrate the induction of microbial sensors TLR2 and CD14, orphan inflammatory receptor TREM1, and checkpoint receptor PD-L1 on HSPCs and particularly the myeloid progenitor cells in the arthritis-afflicted mice. Furthermore, we demonstrate that the stimulation of HSPCs through these receptors in culture can significantly alter the dynamics of cell expansion and differentiation, with distinct responses from HSPCs of arthritis-afflicted versus healthy control mice. We hypothesize that the induction and stimulation of HSPCs through these immune receptors under chronic inflammation may impact the output and functional properties of their immune cell progeny, positing HSPCs as central players in the pathogenic inflammatory responses of rheumatoid arthritis and potentially other chronic inflammatory diseases. HIGHLIGHTSO_LIHematopoietic progenitor cells in murine models of rheumatoid arthritis show an upregulation of immune receptors TREM1, PD-L1, TLR2, and CD14. C_LIO_LIStimulation of murine hematopoietic stem and progenitor cells through these receptors in culture alters the dynamics of their expansion and differentiation. C_LIO_LIIn such cultures, hematopoietic stem and progenitor cells from mice afflicted with rheumatoid arthritis show altered responses to stimulation as compared to healthy controls. C_LI

Chitin is a highly abundant poly- N-acetyl-glucosamine (GlcNAc) and linked to immune recognition of fungal infections and asthma in humans. Ubiquitous in fungi and insects, in mammals and plants chitin represents a microbe-associated molecular pattern (MAMP) and whereas highly polymeric chitin is insoluble and immunologically inert, soluble chitin oligomers of 6 to 15 GlcNAc activate immediate pro-inflammatory cytokine release via TLR2 in human immune cells. However, TLR2 ligands do not the most typical activators of the NLRP3 inflammasome pathway or innate immune training, a phenomenon of long-term immunological remodeling. Here we show that especially 16-20 GlcNAc long chitin oligomers activate NLRP3-dependent IL-1{beta} and IL-18 release in human myeloid immune cells in an atypical, phagocytosis-dependent manner. Moreover, phagocytosis and methyl transferase activity were essential for innate immune training, by which the same chito-oligomer-training enhanced TNF release in primary murine and human immune cells. Collectively, this suggests that oligomer length impacts on the immune features of chitin which can be customized using glycan assembly.

Regulatory T cells (Tregs) are a suppressive subset of CD4 T cells that maintain immune homeostasis. Most Tregs develop in the thymus but how thymic selection impacts peripheral Treg fate remains unknown. Here we show that the strength of TCR signal experienced during positive selection in the thymic cortex impacts how CD4 SP thymocytes respond to Treg inducing signals in the medulla. Thymocytes that experienced weak signals during positive selection (identified as CD5LO CD4 SP) tend to produce Foxp3+ cells that lack the classic Treg marker CD25 and show a greater dependence on TGF{beta} than IL-2 for Treg induction in vitro. Moreover, CD4 clones that give rise to CD25- thymic Tregs also produce CD25- peripheral Tregs. These data indicate that positive selection provides critical context for how CD4 T cells respond to TCR agonist and cytokine signals, leading to an alternative lineage of Tregs that lacks constitutive CD25 expression.

Glucose metabolism is pivotal in regulating innate immune responses in primary human monocyte-derived macrophages (MDMs). Lipopolysaccharide (LPS) stimulation induces both inflammatory and antiviral programs; however, despite the established importance of glucose metabolism in these responses, its precise role in coordinating them remains poorly defined. Here, we identify the STAT1/NF-{kappa}B/IRF5 signaling axis as a key mediator linking glucose metabolism to inflammatory responses through the upregulation of the rate-limiting glycolytic enzyme PFKFB3. We found that LPS triggered delayed expression and activation of NF-{kappa}B p65, accompanied by increased expression of inflammatory target genes, including CD38 and CD40. Using complementary pharmacological and genetic approaches, we demonstrate that glycolysis and PFKFB3 activity are required for NF-{kappa}B p65 expression and activation. Strikingly, inhibition of PFKFB3 also suppressed LPS-induced STAT1 activation and nuclear translocation, revealing a glucose-dependent amplification loop that potentiates STAT1-mediated antiviral and NF-{kappa}B p65-mediated inflammatory responses. Collectively, these findings establish a mechanistic link between glycolytic metabolism and STAT1/IRF5- and NF-{kappa}B-dependent transcriptional programs in human MDMs responding to LPS, highlighting potential therapeutic targets for modulating innate immune responses in inflammatory disease.

In polymicrobial infections, how the host recognizes and responds to pathogens influences which species will persist to cause chronic infections. The human respiratory tract is a common anatomical site for viral-bacterial co-infections, where primary viral infections predispose to secondary bacterial infections, leading to increased morbidity and mortality. Additionally, co-infections are disproportionately prevalent in people with chronic lung diseases, such as chronic obstructive pulmonary disease and cystic fibrosis. We previously reported that primary viral infections and antiviral interferon (IFN) signaling stimulate Pseudomonas aeruginosa (PA) biofilm formation on airway epithelial cells (AECs). IFN signaling induces aerobic glycolysis in AECs and generates lactate as a cellular byproduct. Given that innate immune systems play an integral role in co-infection dynamics, we investigated the role of host-secreted metabolites (i.e. lactate) on innate immune cell activity during respiratory co-infections. We found that exposure to the apical secretions from IFN{beta}-treated AECs significantly compromised macrophage antibacterial activity, with the soluble metabolite lactate playing an important role. Macrophages used monocarboxylate transporters and G-protein receptors to transport and/or sense lactate, respectively, and this exposure to lactate diminished their bacterial-killing activity in a time-exposure dependent manner. Lactate exposure particularly reprogrammed macrophage cellular metabolism towards an anti-inflammatory state by increasing oxidative phosphorylation and fatty acid oxidation. Collectively, these findings provide insight into metabolites as complex regulators of trans-kingdom interactions and epithelial-macrophage crosstalk during respiratory co-infections.