Inhibition of neutrophil swarming by type I interferon promotes intracellular bacterial evasion.

Listeria monocytogenes (LM) possesses the ability to breach multiple barriers and elicit intricate immune responses. However, there remains a lack of explicit understanding regarding how LM evades innate immune surveillance within the body. Here, we utilized liver intravital imaging to elucidate the dynamic process of LM during infection in the liver. We discovered that LM can rapidly escape from Kupffer cells (KCs) through listeriolysin O (LLO) and proliferate within hepatocytes. Upon LM exposure to the hepatic sinusoids, neutrophils rapidly aggregate at the site of infection. Subsequently, LM can induce type I interferon (IFN-I) production primarily in the spleen, which acts systemically on neutrophils to hamper their swarming by deactivating the ERK pathway, thus evading neutrophil-mediated eradication. Furthermore, our findings suggest that virus-induced IFN-I suppresses neutrophil swarming, and COVID-19 patients exhibit impaired neutrophil aggregation function. In conclusion, our findings provide compelling evidence demonstrating that intracellular bacteria represented by LM can hijack host defense mechanisms against viral infections to evade immune surveillance. Additionally, impaired neutrophil swarming caused by IFN-I is one of the significant factors contributing to the increased susceptibility to bacterial infections following viral infections.

IL-2 delivery to CD8+ T cells during infection requires MRTF/SRF-dependent gene expression and cytoskeletal dynamics.

Paracrine IL-2 signalling drives the CD8 + T cell expansion and differentiation that allow protection against viral infections, but the underlying molecular events are incompletely understood. Here we show that the transcription factor SRF, a master regulator of cytoskeletal gene expression, is required for effective IL-2 signalling during L. monocytogenes infection. Acting cell-autonomously with its actin-regulated cofactors MRTF-A and MRTF-B, SRF is dispensible for initial TCR-mediated CD8+ T cell proliferation, but is required for sustained IL-2 dependent CD8+ effector T cell expansion, and persistence of memory cells. Following TCR activation, Mrtfab-null CD8+ T cells produce IL-2 normally, but homotypic clustering is impaired both in vitro and in vivo. Expression of cytoskeletal structural and regulatory genes, most notably actins, is defective in Mrtfab-null CD8+ T cells. Activation-induced cell clustering in vitro requires F-actin assembly, and Mrtfab-null cell clusters are small, contain less F-actin, and defective in IL-2 retention. Clustering of Mrtfab-null cells can be partially restored by exogenous actin expression. IL-2 mediated CD8+ T cell proliferation during infection thus depends on the control of cytoskeletal dynamics and actin gene expression by MRTF-SRF signalling.

A mass cytometry method pairing T cell receptor and differentiation state analysis.

T cell antigen receptor (TCR) recognition followed by clonal expansion is a fundamental feature of adaptive immune responses. Here, we present a mass cytometric (CyTOF) approach to track T cell responses by combining antibodies for specific TCR Vα and Vß chains with antibodies against T cell activation and differentiation proteins in mice. This strategy identifies expansions of CD8+ and CD4+ T cells expressing specific Vß and Vα chains with varying differentiation states in response to Listeria monocytogenes, tumors and respiratory influenza infection. Expanded T cell populations expressing Vß chains could be directly linked to the recognition of specific antigens from Listeria, tumor cells or influenza. In the setting of influenza infection, we found that common therapeutic approaches of intramuscular vaccination or convalescent serum transfer altered the TCR diversity and differentiation state of responding T cells. Thus, we present a method to monitor broad changes in TCR use paired with T cell phenotyping during adaptive immune responses.

Suppressive effect of the anesthetic propofol on the T cell function and T cell-dependent immune responses.

General anesthesia is thought to suppress the immune system and negatively affect postoperative infection and the long-term prognosis of cancer. However, the mechanism underlying immunosuppression induced by general anesthetics remains unclear. In this study, we focused on propofol, which is widely used for sedation under general anesthesia and intensive care and examined its effects on the T cell function and T cell-dependent immune responses. We found that propofol suppressed T cell glycolytic metabolism, differentiation into effector T cells, and cytokine production by effector T cells. CD8 T cells activated and differentiated into effector cells in the presence of propofol in vitro showed reduced antitumor activity. Furthermore, propofol treatment suppressed the increase in the number of antigen-specific CD8 T cells during Listeria infection. In contrast, the administration of propofol improved inflammatory conditions in mouse models of inflammatory diseases, such as OVA-induced allergic airway inflammation, hapten-induced contact dermatitis, and experimental allergic encephalomyelitis. These results suggest that propofol may reduce tumor and infectious immunity by suppressing the T cell function and T cell-dependent immune responses while improving the pathogenesis and prognosis of chronic inflammatory diseases by suppressing inflammation.

Immunopeptidomics Mapping of Listeria monocytogenes T Cell Epitopes in Mice.

Listeria monocytogenes is a foodborne intracellular bacterial model pathogen. Protective immunity against Listeria depends on an effective CD8+ T cell response, but very few T cell epitopes are known in mice as a common animal infection model for listeriosis. To identify epitopes, we screened for Listeria immunopeptides presented in the spleen of infected mice by mass spectrometry-based immunopeptidomics. We mapped more than 6000 mouse self-peptides presented on MHC class I molecules, including 12 high confident Listeria peptides from 12 different bacterial proteins. Bacterial immunopeptides with confirmed fragmentation spectra were further tested for their potential to activate CD8+ T cells, revealing VTYNYINI from the putative cell wall surface anchor family protein LMON_0576 as a novel bona fide peptide epitope. The epitope showed high biological potency in a prime boost model and can be used as a research tool to probe CD8+ T cell responses in the mouse models of Listeria infection. Together, our results demonstrate the power of immunopeptidomics for bacterial antigen identification.

Molecular definition of the endogenous Toll-like receptor signalling pathways.

Innate immune pattern recognition receptors, such as the Toll-like receptors (TLRs), are key mediators of the immune response to infection and central to our understanding of health and disease1. After microbial detection, these receptors activate inflammatory signal transduction pathways that involve IκB kinases, mitogen-activated protein kinases, ubiquitin ligases and other adaptor proteins. The mechanisms that connect the proteins in the TLR pathways are poorly defined. To delineate TLR pathway activities, we engineered macrophages to enable microscopy and proteomic analysis of the endogenous myddosome constituent MyD88. We found that myddosomes form transient contacts with activated TLRs and that TLR-free myddosomes are dynamic in size, number and composition over the course of 24 h. Analysis using super-resolution microscopy revealed that, within most myddosomes, MyD88 forms barrel-like structures that function as scaffolds for effector protein recruitment. Proteomic analysis demonstrated that myddosomes contain proteins that act at all stages and regulate all effector responses of the TLR pathways, and genetic analysis defined the epistatic relationship between these effector modules. Myddosome assembly was evident in cells infected with Listeria monocytogenes, but these bacteria evaded myddosome assembly and TLR signalling during cell-to-cell spread. On the basis of these findings, we propose that the entire TLR signalling pathway is executed from within the myddosome.

The canonical antiviral protein oligoadenylate synthetase 1 elicits antibacterial functions by enhancing IRF1 translation.

An important property of the host innate immune response during microbial infection is its ability to control the expression of antimicrobial effector proteins, but how this occurs post-transcriptionally is not well defined. Here, we describe a critical antibacterial role for the classic antiviral gene 2'-5'-oligoadenylate synthetase 1 (OAS1). Human OAS1 and its mouse ortholog, Oas1b, are induced by interferon-γ and protect against cytosolic bacterial pathogens such as Francisella novicida and Listeria monocytogenes in vitro and in vivo. Proteomic and transcriptomic analysis showed reduced IRF1 protein expression in OAS1-deficient cells. Mechanistically, OAS1 binds and localizes IRF1 mRNA to the rough endoplasmic reticulum (ER)-Golgi endomembranes, licensing effective translation of IRF1 mRNA without affecting its transcription or decay. OAS1-dependent translation of IRF1 leads to the enhanced expression of antibacterial effectors, such as GBPs, which restrict intracellular bacteria. These findings uncover a noncanonical function of OAS1 in antibacterial innate immunity.

Sepsis Impairs IFN-γ Production in CD8 T Cells through Changes in Local Chromatin Landscape.

Sepsis is a complex condition of inflammatory and immune dysregulation, triggered by severe infection. In survivors, chronic inflammation and immune dysregulation linger, facilitating the emergence of infections. CD8 dysfunction contributes to immunosuppression in sepsis survivors. We devised an animal model that enabled us to identify and analyze CD8-intrinsic defects induced by sepsis. We adoptively transferred CD45.1 CD8 OT-I T cells into CD45.2 congenic mice and subjected them to cecal ligature and puncture, to induce abdominal sepsis. One month later, we isolated the transferred CD8 cells. Surface marker expression confirmed they had not been activated through the TCR. CD8 OT-I T cells isolated from septic (or sham-operated) mice were transferred to second recipients, which were challenged with OVA-expressing Listeria monocytogenes. We compared effector capacities between OT-I cells exposed to sepsis and control cells. Naive mice that received OT-I cells exposed to sepsis had higher bacterial burden and a shorter survival when challenged with OVA-expressing L. monocytogenes. OT-I cells isolated from septic mice produced less IFN-γ but had conserved activation, expansion potential, and cytotoxic function. We observed lower transcript levels of IFN-γ and of the long noncoding RNA Ifng-as1, a local regulator of the epigenetic landscape, in cells exposed to sepsis. Accordingly, local abundance of a histone modification characteristic of active promoter regions was reduced in sepsis-exposed CD8 T cells. Our results identify a mechanism through which inflammation in the context of sepsis affects CD8 T cell function intrinsically.

13C metabolite tracing reveals glutamine and acetate as critical in vivo fuels for CD8 T cells.

Infusion of 13C-labeled metabolites provides a gold standard for understanding the metabolic processes used by T cells during immune responses in vivo. Through infusion of 13C-labeled metabolites (glucose, glutamine, and acetate) in Listeria monocytogenes-infected mice, we demonstrate that CD8 T effector (Teff) cells use metabolites for specific pathways during specific phases of activation. Highly proliferative early Teff cells in vivo shunt glucose primarily toward nucleotide synthesis and leverage glutamine anaplerosis in the tricarboxylic acid (TCA) cycle to support adenosine triphosphate and de novo pyrimidine synthesis. In addition, early Teff cells rely on glutamic-oxaloacetic transaminase 1 (Got1)-which regulates de novo aspartate synthesis-for effector cell expansion in vivo. CD8 Teff cells change fuel preference over the course of infection, switching from glutamine- to acetate-dependent TCA cycle metabolism late in infection. This study provides insights into the dynamics of Teff metabolism, illuminating distinct pathways of fuel consumption associated with CD8 Teff cell function in vivo.

Designing a Multiepitope Vaccine against the Foodborne Pathogenic Bacteria Listeria monocytogenes Using Subtractive Immunoinformatics Approaches.

BACKGROUND: Listeria monocytogenes, a Gram-positive bacterium, is a prominent foodborne pathogen that causes listeriosis and poses substantial health hazards worldwide. The continuing risk of listeriosis outbreaks underlies the importance of designing an effective prevention strategy and developing a robust immune response by reverse vaccinology approaches. This study aimed to provide a critical approach for developing a potent multiepitope vaccine against this foodborne disease. METHODS: A chimeric peptide construct containing 5 B-cell epitopes, 16 major histocompatibility complex I (MHC-I) epitopes, and 18 MHC-II epitopes were used to create a subunit vaccination against L. monocytogenes. The vaccine safety was evaluated by several online methods, and molecular docking was performed using ClusPro to determine the binding affinity. Immune simulation was performed using the C-ImmSimm server to demonstrate the immune response. RESULTS: The results validated the antigenicity, non-allergenicity, and nontoxicity of the chimeric peptide construct, confirming its suitability as a subunit vaccine. Molecular docking showed a good score of 1276.5 and molecular dynamics simulations confirmed the construct's efficacy, demonstrating its promise as a good candidate for listeriosis prophylaxis. The population coverage was as high as 91.04% with a good immune response, indicating good antigen presentation with dendritic cells and production of memory cells. CONCLUSIONS: The findings of this study highlight the potential of the designed chimeric peptide construct as an effective subunit vaccine against Listeria, paving the way for future advances in preventive methods and vaccine design.

The Transcription Factor Zfp335 Promotes Differentiation and Survival of Effector Th1 Cells by Directly Regulating Lmna Expression.

Ag-specific effector CD4+ T cells play a crucial role in defending against exogenous pathogens. However, the mechanisms governing the differentiation and function of IFN-γ-producing effector CD4+ Th1 cells in immune responses remain largely unknown. In this study, we elucidated the pivotal role of zinc finger protein 335 (Zfp335) in regulating effector Th1 cell differentiation and survival during acute bacterial infection. Mice with Zfp335 knockout in OT-II cells exhibited impaired Ag-specific CD4+ T cell expansion accompanied by a significant reduction in resistance to Listeria infection. Furthermore, Zfp335 deficiency restricted the effector CD4+ Th1 cell population and compromised their survival upon Listeria challenge. The expression of T-bet and IFN-γ was accordingly decreased in Zfp335-deficient Th1 cells. Mechanistically, Zfp335 directly bound to the promoter region of the Lmna gene and regulated its expression. Overexpression of Lmna was able to rescue the survival and function of Zfp335-deficient effector Th1 cells. Therefore, our study provides novel insights into the mechanisms governing effector Th1 cell differentiation and survival during acute infection.

Cross-regulation of Listeria monocytogenes and the host ubiquitin system in listeriosis.

The facultative intracellular bacterium Listeria (L.) monocytogenes may cause severe diseases in humans and animals. The control of listeriosis/L. monocytogenes requires the concerted action of cells of the innate and adaptive immune systems. In this regard, cell-intrinsic immunity of infected cells, activated by the immune responses, is crucial for the control and elimination intracellular L. monocytogenes. Both the immune response against L. monocytogenes and cell intrinsic pathogen control are critically regulated by post-translational modifications exerted by the host ubiquitin system and ubiquitin-like modifiers (Ubls). In this review, we discuss our current understanding of the role of the ubiquitin system and Ubls in listeriosis, as well as future directions of research.

Intestinal tissue-resident memory T cells maintain distinct identity from circulating memory T cells after in vitro restimulation.

Resident memory T (TRM) cells have been recently established as an important subset of memory T cells that provide early and essential protection against reinfection in the absence of circulating memory T cells. Recent findings showing that TRM expand in vivo after repeated antigenic stimulation indicate that these memory T cells are not terminally differentiated. This suggests an opportunity for in vitro TRM expansion to apply in an immunotherapy setting. However, it has also been shown that TRM may not maintain their identity and form circulating memory T cells after in vivo restimulation. Therefore, we set out to determine how TRM respond to antigenic activation in culture. Using Listeria monocytogenes and LCMV infection models, we found that TRM from the intraepithelial compartment of the small intestine expand in vitro after antigenic stimulation and subsequent resting in homeostatic cytokines. A large fraction of the expanded TRM retained their phenotype, including the expression of key TRM markers CD69 and CD103 (ITGAE). The optimal culture of TRM required low O2 pressure to maintain the expression of these and other TRM-associated molecules. Expanded TRM retained their effector capacity to produce cytokines after restimulation, but did not acquire a highly glycolytic profile indicative of effector T cells. The proteomic analysis confirmed TRM profile retention, including expression of TRM-related transcription factors, tissue retention factors, adhesion molecules, and enzymes involved in fatty acid metabolism. Collectively, our data indicate that limiting oxygen conditions supports in vitro expansion of TRM cells that maintain their TRM phenotype, at least in part, suggesting an opportunity for therapeutic strategies that require in vitro expansion of TRM.

Lymph node stromal cells vary in susceptibility to infection but can support the intracellular growth of Listeria monocytogenes.

Lymph node stromal cells (LNSCs) are an often overlooked component of the immune system but play a crucial role in maintaining tissue homeostasis and orchestrating immune responses. Our understanding of the functions these cells serve in the context of bacterial infections remains limited. We previously showed that Listeria monocytogenes, a facultative intracellular foodborne bacterial pathogen, must replicate within an as-yet-unidentified cell type in the mesenteric lymph node (MLN) to spread systemically. Here, we show that L. monocytogenes could invade, escape from the vacuole, replicate exponentially, and induce a type I interferon response in the cytosol of 2 LNSC populations infected in vitro, fibroblastic reticular cells (FRCs) and blood endothelial cells (BECs). Infected FRCs and BECs also produced a significant chemokine and proinflammatory cytokine response after in vitro infection. Flow cytometric analysis confirmed that GFP+  L. monocytogenes were associated with a small percentage of MLN stromal cells in vivo following foodborne infection of mice. Using fluorescent microscopy, we showed that these cell-associated bacteria were intracellular L. monocytogenes and that the number of infected FRCs and BECs changed over the course of a 3-day infection in mice. Ex vivo culturing of these infected LNSC populations revealed viable, replicating bacteria that grew on agar plates. These results highlight the unexplored potential of FRCs and BECs to serve as suitable growth niches for L. monocytogenes during foodborne infection and to contribute to the proinflammatory environment within the MLN that promotes clearance of listeriosis.

Gut Microbiota Protects Listeria monocytogenes-Infected Mice by Reducing the Inflammatory Cytokines Storm and Cell Apoptosis.

Gut microbiota (GM) has been proven to resist pathogenic infection through nutritional competition, colonization resistance and promotion of the host immune response. However, in clinical practice, GM is mainly used in intestinal diseases, such as Clostridium difficile infection, and there are few reports on its application in the treatment of pathogenic bacterial infections. In this study, GM from healthy mice was transplanted into mice infected with Listeria monocytogenes using fecal microbiota transplantation (FMT) and the effects were observed. We found that GM from healthy mice could reduce the mortality of infected mice and decrease the counts of L. monocytogenes in their liver and spleen. In addition, FMT inhibited the expression of inflammatory factors in the liver and spleen of infected mice. In vitro cell experiments revealed that GM can reduce the count of L. monocytogenes invading Caco-2 cells and inhibit the L. monocytogenes-caused apoptosis. These results indicate that GM can be used to protect mice infected with L. monocytogenes by eliminating the amount of L. monocytogenes in the host and inhibiting the overexpression of inflammatory factors. Hence, this method can potentially replace antibiotics in the treatment of L. monocytogenes infection.

DRG2 in macrophages is crucial for initial inflammatory response and protection against Listeria monocytogenes infection.

Innate immune response is critical for the control of Listeria monocytogenes infection. Here, we identified developmentally regulated GTP-binding protein 2 (DRG2) in macrophages as a major regulator of the innate immune response against L. monocytogenes infection. Both whole-body DRG2 knockout (KO) mice and macrophage-specific DRG2 KO mice had low levels of IL-6 during early infection and increased susceptibility to L. monocytogenes infection. Following an initial impaired inflammatory response of macrophages upon i.p. L. monocytogenes infection, DRG2-/- mice showed delayed recruitment of neutrophils and monocytes into the peritoneal cavity, which led to elevated bacterial burden, inflammatory cytokine production at a late infection time point, and liver micro-abscesses. DRG2 deficiency decreased the transcriptional activity of NF-κB and impaired the inflammatory response of both bone marrow-derived and peritoneal macrophages upon L. monocytogenes stimulation. Our findings reveal that DRG2 in macrophages is critical for the initial inflammatory response and protection against L. monocytogenes infection.

Pregnancy enables antibody protection against intracellular infection.

Adaptive immune components are thought to exert non-overlapping roles in antimicrobial host defence, with antibodies targeting pathogens in the extracellular environment and T cells eliminating infection inside cells1,2. Reliance on antibodies for vertically transferred immunity from mothers to babies may explain neonatal susceptibility to intracellular infections3,4. Here we show that pregnancy-induced post-translational antibody modification enables protection against the prototypical intracellular pathogen Listeria monocytogenes. Infection susceptibility was reversed in neonatal mice born to preconceptually primed mothers possessing L. monocytogenes-specific IgG or after passive transfer of antibodies from primed pregnant, but not virgin, mice. Although maternal B cells were essential for producing IgGs that mediate vertically transferred protection, they were dispensable for antibody acquisition of protective function, which instead required sialic acid acetyl esterase5 to deacetylate terminal sialic acid residues on IgG variable-region N-linked glycans. Deacetylated L. monocytogenes-specific IgG protected neonates through the sialic acid receptor CD226,7, which suppressed IL-10 production by B cells leading to antibody-mediated protection. Consideration of the maternal-fetal dyad as a joined immunological unit reveals protective roles for antibodies against intracellular infection and fine-tuned adaptations to enhance host defence during pregnancy and early life.

The potential role and regulatory mechanism of IL-33/ST2 axis on T lymphocytes during lipopolysaccharide stimulation or perinatal Listeria infection.

BACKGROUND: Interleukin-33 (IL-33) is a member of the interleukin-1 family, which is reported to be important across a range of diseases. However, the mechanisms underlying IL-33/ST2 axis in infectious diseases have not yet been fully addressed. METHODS: We established both lipopolysaccharide (LPS)-induced injuryin T cells and Listeria monocytogenes (Lm) infection model to determine the effect of IL-33 on infectious immunity. RESULTS: The T cell proliferation was inhibited by LPS while IL-33 could reverse the outcome. Further, apoptosis was significantly promoted after serum stimulation (ST)2 knockdown, suggesting IL-33, acting through its receptor ST2, may attenuate the inhibitory effect of LPS on T cells through the apoptotic signaling pathway. In this study, we also identified an IL-33-mediated mechanism of T cell differentiation in pregnant mice infected with Lm. Here, we observed the elevated expression of IL-33 in pregnant mice infected with Lm. Furthermore, we revealed that blocking IL-33 markedly decreased the abortion rate and placental bacterial load, but weakened placental inflammatory repair, by inhibiting Th2 cell-mediated immune responses and relatively intensifying Th1-dominent immunoreaction. CONCLUSIONS: These findings reveal a previously unidentified mechanism underlying IL-33/ST2 axis. IL-33 signaling and targeting T cell-mediated immunity may present a new therapeutic strategy for the treatment of infectious diseases.

Developmentally distinct CD4+ Treg lineages shape the CD8+ T cell response to acute Listeria infection.

SignificanceThe CD4+ Treg response following acute Listeria infection is heterogeneous and deploys two distinct modes of suppression coinciding with initial pathogen exposure and resolution of infection. This bimodal suppression of CD8+ T cells during priming and contraction is mediated by separate Treg lineages. These findings make a significant contribution to our understanding of the functional plasticity inherent within Tregs, which allows these cells to serve as a sensitive and dynamic cellular rheostat for the immune system to prevent autoimmune pathology in the face of inflammation attendant to acute infection, enable expansion of the pathogen-specific response needed to control the infection, and reestablish immune homeostasis after the threat has been contained.

Protective Immunity against Listeria monocytogenes in Rats, Provided by HCl- and NaOH-Induced Listeria monocytogenes Bacterial Ghosts (LMGs) as Vaccine Candidates.

Listeria monocytogenes (Lm) bacterial ghosts (LMGs) were produced by the minimum inhibitory concentration (MIC) of HCl, H2SO4, and NaOH. Acid and alkali effects on the LMGs were compared by in vitro and in vivo analyses. Scanning electron microscope showed that all chemicals form lysis pores on the Lm cell envelopes. Real-time qPCR revealed a complete absence of genomic DNA in HCl- and H2SO4-induced LMGs but not in NaOH-induced LMGs. HCl-, H2SO4- and NaOH-induced LMGs showed weaker or missing protein bands on SDS-PAGE gel when compared to wild-type Lm. Murine macrophages exposed to the HCl-induced LMGs showed higher cell viability than those exposed to NaOH-induced LMGs or wild-type Lm. The maximum level of cytokine expression (TNF-α, iNOS, IFN-γ, and IL-10 mRNA) was observed in the macrophages exposed to NaOH-induced LMGs, while that of IL-1ß mRNA was observed in the macrophages exposed to HCl-induced LMGs. To investigate LMGs as a vaccine candidate, mice were divided into PBS buffer-injected, HCl- and NaOH-induced LMGs immunized groups. Mice vaccinated with HCl- and NOH-induced LMGs, respectively, significantly increased in specific IgG antibodies, bactericidal activities of serum, and CD4+ and CD8+ T-cell population. Antigenic Lm proteins reacted with antisera against HCl- and NOH-induced LMGs, respectively. Bacterial loads in HCl- and NaOH-induced LMGs immunized mice were significantly lower than PBS-injected mice after virulent Lm challenges. It suggested that vaccination with LMGs induces both humoral and cell-mediated immune responses and protects against virulent challenges.