Mucosal-associated invariant T cells from Clostridioides difficile-infected patients exhibit a distinct proinflammatory phenotype and enhanced cytotoxic activity.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells mainly found in the mucosa and peripheral blood. We have recently demonstrated that Clostridioides difficile activates MAIT cells in vitro. However, their role in the pathogenesis of C. difficile infection (CDI) in human patients remains elusive to date. In this study, we performed comprehensive immunophenotyping of MAIT cells derived from CDI patients and compared their phenotype to that of patients with inflammatory bowel diseases (IBD) and healthy controls. Our study revealed that blood MAIT cells from CDI patients exhibit an interleukin 17a (IL-17a)-dominated proinflammatory phenotype and an increased readiness to synthesize the proinflammatory cytokine interferon γ (IFN-γ) following in vitro re-stimulation. Moreover, the cytotoxic activity of MAIT cells, as measured by surface CD107a and intracellular granzyme B expression, was strongly increased in CDI. Multi epitope ligand cartography (MELC) analysis of intestinal biopsies from CDI patients revealed that MAIT cells exhibit an increased production of granzyme B and increased cytotoxicity compared to the control group. Together with previously published in vitro data from our group, our findings suggest that MAIT cells are functionally involved in the immune response against C. difficile and contribute to the pathogenesis of CDI.

TMPRSS2 Is Essential for SARS-CoV-2 Beta and Omicron Infection.

The COVID-19 pandemic remains a global health threat and novel antiviral strategies are urgently needed. SARS-CoV-2 employs the cellular serine protease TMPRSS2 for entry into lung cells, and TMPRSS2 inhibitors are being developed for COVID-19 therapy. However, the SARS-CoV-2 Omicron variant, which currently dominates the pandemic, prefers the endo/lysosomal cysteine protease cathepsin L over TMPRSS2 for cell entry, raising doubts as to whether TMPRSS2 inhibitors would be suitable for the treatment of patients infected with the Omicron variant. Nevertheless, the contribution of TMPRSS2 to the spread of SARS-CoV-2 in the infected host is largely unclear. In this study, we show that the loss of TMPRSS2 strongly reduced the replication of the Beta variant in the nose, trachea and lung of C57BL/6 mice, and protected the animals from weight loss and disease. The infection of mice with the Omicron variant did not cause disease, as expected, but again, TMPRSS2 was essential for efficient viral spread in the upper and lower respiratory tract. These results identify the key role of TMPRSS2 in SARS-CoV-2 Beta and Omicron infection, and highlight TMPRSS2 as an attractive target for antiviral intervention.

Normoxic HIF-1α Stabilization Caused by Local Inflammatory Factors and Its Consequences in Human Coronary Artery Endothelial Cells.

Knowledge about normoxic hypoxia-inducible factor (HIF)-1α stabilization is limited. We investigated normoxic HIF-1α stabilization and its consequences using live cell imaging, immunoblotting, Bio-Plex multiplex immunoassay, immunofluorescence staining, and barrier integrity assays. We demonstrate for the first time that IL-8 and M-CSF caused HIF-1α stabilization and translocation into the nucleus under normoxic conditions in both human coronary endothelial cells (HCAECs) and HIF-1α-mKate2-expressing HEK-293 cells. In line with the current literature, our data show significant normoxic HIF-1α stabilization caused by TNF-α, INF-γ, IL-1ß, and IGF-I in both cell lines, as well. Treatment with a cocktail consisting of TNF-α, INF-γ, and IL-1ß caused significantly stronger HIF-1α stabilization in comparison to single treatments. Interestingly, this cumulative effect was not observed during simultaneous treatment with IL-8, M-CSF, and IGF-I. Furthermore, we identified two different kinetics of HIF-1α stabilization under normoxic conditions. Our data demonstrate elevated protein levels of HIF-1α-related genes known to be involved in the development of atherosclerosis. Moreover, we demonstrate an endothelial barrier dysfunction in HCAECs upon our treatments and during normoxic HIF-1α stabilization comparable to that under hypoxia. This study expands the knowledge of normoxic HIF-1α stabilization and activation and its consequences on the endothelial secretome and barrier function. Our data imply an active role of HIF-1α in vivo in the vasculature in the absence of hypoxia.

Cigarette Smoke Extract Disturbs Mitochondria-Regulated Airway Epithelial Cell Responses to Pneumococci.

Mitochondrial functionality is crucial for the execution of physiologic functions of metabolically active cells in the respiratory tract including airway epithelial cells (AECs). Cigarette smoke is known to impair mitochondrial function in AECs. However, the potential contribution of mitochondrial dysfunction in AECs to airway infection and airway epithelial barrier dysfunction is unknown. In this study, we used an in vitro model based on AECs exposed to cigarette smoke extract (CSE) followed by an infection with Streptococcus pneumoniae (Sp). The levels of oxidative stress as an indicator of mitochondrial stress were quantified upon CSE and Sp treatment. In addition, expression of proteins associated with mitophagy, mitochondrial content, and biogenesis as well as mitochondrial fission and fusion was quantified. Transcriptional AEC profiling was performed to identify the potential changes in innate immune pathways and correlate them with indices of mitochondrial function. We observed that CSE exposure substantially altered mitochondrial function in AECs by suppressing mitochondrial complex protein levels, reducing mitochondrial membrane potential and increasing mitochondrial stress and mitophagy. Moreover, CSE-induced mitochondrial dysfunction correlated with reduced enrichment of genes involved in apical junctions and innate immune responses to Sp, particularly type I interferon responses. Together, our results demonstrated that CSE-induced mitochondrial dysfunction may contribute to impaired innate immune responses to Sp.

Safety and efficacy of prophylactic and therapeutic vaccine based on live-attenuated Listeria monocytogenes in hepatobiliary cancers.

Primary liver cancer (PLC) comprising hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) represents the third deadliest cancer worldwide with still insufficient treatment options. We have previously found that CD4 T helper 1 (Th1) response is indispensable for the protection against PLC. In the present research, we aimed to test the potent inducers of Th1 responses, live-attenuated Listeria monocytogenes ∆actA/∆inlB strain as preventive/therapeutic vaccine candidate in liver fibrosis, HCC, and CCA. Studies were performed using autochthonous models of HCC and CCA, highly reflecting human disease. L. monocytogenes ∆actA/∆inlB demonstrated strong safety/efficacy in premalignant and malignant liver diseases. The protective mechanism relied on the induction of strong tumor-specific immune responses that keep the development of hepatobiliary cancers under control. Combination therapy, comprising Listeria vaccination and a checkpoint inhibitor blockade significantly extended the survival of HCC-bearing mice even at the advanced stages of the disease. This is the first report on the safety and efficacy of Listeria-based vaccine in liver fibrosis, as well as the first proof of principle study on Listeria-based vaccines in CCA. Our study paves the way for the use of live-attenuated Listeria as safe and efficient vaccine and a potent inducer of protective immune responses in liver fibrosis and hepatobiliary malignancies.

Antiviral Activity of Influenza A Virus Defective Interfering Particles against SARS-CoV-2 Replication In Vitro through Stimulation of Innate Immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) emerged in late 2019 and resulted in a devastating pandemic. Although the first approved vaccines were already administered by the end of 2020, worldwide vaccine availability is still limited. Moreover, immune escape variants of the virus are emerging against which the current vaccines may confer only limited protection. Further, existing antivirals and treatment options against COVID-19 show only limited efficacy. Influenza A virus (IAV) defective interfering particles (DIPs) were previously proposed not only for antiviral treatment of the influenza disease but also for pan-specific treatment of interferon (IFN)-sensitive respiratory virus infections. To investigate the applicability of IAV DIPs as an antiviral for the treatment of COVID-19, we conducted in vitro co-infection experiments with cell culture-derived DIPs and the IFN-sensitive SARS-CoV-2 in human lung cells. We show that treatment with IAV DIPs leads to complete abrogation of SARS-CoV-2 replication. Moreover, this inhibitory effect was dependent on janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling. Further, our results suggest boosting of IFN-induced antiviral activity by IAV DIPs as a major contributor in suppressing SARS-CoV-2 replication. Thus, we propose IAV DIPs as an effective antiviral agent for treatment of COVID-19, and potentially also for suppressing the replication of new variants of SARS-CoV-2.

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo.

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation. Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (αDEC-205) for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting αDEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to αDEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA). We have successfully used chemically crosslinked αDEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.

Mitochondria: at the crossroads of regulating lung epithelial cell function in chronic obstructive pulmonary disease.

Disturbances in mitochondrial structure and function in lung epithelial cells have been implicated in the pathogenesis of various lung diseases, including chronic obstructive pulmonary disease (COPD). Such disturbances affect not only cellular energy metabolism but also alter a range of indispensable cellular homeostatic functions in which mitochondria are known to be involved. These range from cellular differentiation, cell death pathways, and cellular remodeling to physical barrier function and innate immunity, all of which are known to be impacted by exposure to cigarette smoke and have been linked to COPD pathogenesis. Next to their well-established role as the first physical frontline against external insults, lung epithelial cells are immunologically active. Malfunctioning epithelial cells with defective mitochondria are unable to maintain homeostasis and respond adequately to further stress or injury, which may ultimately shape the phenotype of lung diseases. In this review, we provide a comprehensive overview of the impact of cigarette smoke on the development of mitochondrial dysfunction in the lung epithelium and highlight the consequences for cell function, innate immune responses, epithelial remodeling, and epithelial barrier function in COPD. We also discuss the applicability and potential therapeutic value of recently proposed strategies for the restoration of mitochondrial function in the treatment of COPD.

In vivo Neutralization of Pro-inflammatory Cytokines During Secondary Streptococcus pneumoniae Infection Post Influenza A Virus Infection.

An overt pro-inflammatory immune response is a key factor contributing to lethal pneumococcal infection in an influenza pre-infected host and represents a potential target for therapeutic intervention. However, there is a paucity of knowledge about the level of contribution of individual cytokines. Based on the predictions of our previous mathematical modeling approach, the potential benefit of IFN-γ- and/or IL-6-specific antibody-mediated cytokine neutralization was explored in C57BL/6 mice infected with the influenza A/PR/8/34 strain, which were subsequently infected with the Streptococcus pneumoniae strain TIGR4 on day 7 post influenza. While single IL-6 neutralization had no effect on respiratory bacterial clearance, single IFN-γ neutralization enhanced local bacterial clearance in the lungs. Concomitant neutralization of IFN-γ and IL-6 significantly reduced the degree of pneumonia as well as bacteremia compared to the control group, indicating a positive effect for the host during secondary bacterial infection. The results of our model-driven experimental study reveal that the predicted therapeutic value of IFN-γ and IL-6 neutralization in secondary pneumococcal infection following influenza infection is tightly dependent on the experimental protocol while at the same time paving the way toward the development of effective immune therapies.

Mucosal CD8+ T cell responses induced by an MCMV based vaccine vector confer protection against influenza challenge.

Cytomegalovirus (CMV) is a ubiquitous ß-herpesvirus that establishes life-long latent infection in a high percentage of the population worldwide. CMV induces the strongest and most durable CD8+ T cell response known in human clinical medicine. Due to its unique properties, the virus represents a promising candidate vaccine vector for the induction of persistent cellular immunity. To take advantage of this, we constructed a recombinant murine CMV (MCMV) expressing an MHC-I restricted epitope from influenza A virus (IAV) H1N1 within the immediate early 2 (ie2) gene. Only mice that were immunized intranasally (i.n.) were capable of controlling IAV infection, despite the greater potency of the intraperitoneally (i.p.) vaccination in inducing a systemic IAV-specific CD8+ T cell response. The protective capacity of the i.n. immunization was associated with its ability to induce IAV-specific tissue-resident memory CD8+ T (CD8TRM) cells in the lungs. Our data demonstrate that the protective effect exerted by the i.n. immunization was critically mediated by antigen-specific CD8+ T cells. CD8TRM cells promoted the induction of IFNγ and chemokines that facilitate the recruitment of antigen-specific CD8+ T cells to the lungs. Overall, our results showed that locally applied MCMV vectors could induce mucosal immunity at sites of entry, providing superior immune protection against respiratory infections.

The STING activator c-di-AMP exerts superior adjuvant properties than the formulation poly(I:C)/CpG after subcutaneous vaccination with soluble protein antigen or DEC-205-mediated antigen targeting to dendritic cells.

Vaccination is the most efficient strategy to protect from infectious diseases and the induction of a protective immune response not only depends on the nature of the antigen, but is also influenced by the vaccination strategy and the co-administration of adjuvants. Therefore, the precise monitoring of adjuvant candidates and their immune modulatory properties is a crucial step in vaccine development. Here, one central aspect is the induction of appropriate humoral and cellular effector mechanisms. In our study we performed a direct comparison of two promising candidates in adjuvant development, the STING activator bis-(3,5)-cyclic dimeric adenosine monophosphate (c-di-AMP) and the Toll-like receptor ligand formulation poly(I:C)/CpG. These were evaluated in C57BL/6 mice using the model antigen ovalbumin (OVA) in subcutaneous vaccination with soluble protein as well as in a dendritic cell (DC) targeting approach (αDEC-OVA). Strikingly, c-di-AMP as compared to poly(I:C)/CpG resulted in significantly higher antigen-specific IgG antibody levels when used in immunization with soluble OVA as well as in antigen targeting to DC. In vaccination with soluble OVA, c-di-AMP induced a significantly stronger CTL, Th1 and IFNγ-producing CD8+ memory T cell response than poly(I:C)/CpG. The response was CTL and Th1 cell dominated, a profile shared by both adjuvants. In the context of targeting OVA to DC, c-di-AMP induced significantly increased Th1 and Th2 cell responses as compared to poly(I:C)/CpG. Interestingly, the Th1 response dominated the overall T cell response only when c-di-AMP was used, indicating a distinct modulatory property of c-di-AMP when the DC targeting immunization approach was exploited. Taken together, we describe superior properties of c-di-AMP as compared to poly(I:C)/CpG in subcutaneous vaccination with soluble antigen as well as antigen targeting to DC. This indicates exceptionally effective adjuvant properties for c-di-AMP and provides compelling evidence of its potential for further adjuvant development, especially also when using DC targeting approaches.

Essential role of IκBNS for in vivo CD4+ T-cell activation, proliferation, and Th1-cell differentiation during Listeria monocytogenes infection in mice.

Acquisition of effector functions in T cells is guided by transcription factors, including NF-κB, that itself is tightly controlled by inhibitory proteins. The atypical NF-κB inhibitor, IκBNS, is involved in the development of Th1, Th17, and regulatory T (Treg) cells. However, it remained unclear to which extend IκBNS contributed to the acquisition of effector function in T cells specifically responding to a pathogen during in vivo infection. Tracking of adoptively transferred T cells in Listeria monocytogenes infected mice antigen-specific activation of CD4+ T cells following in vivo pathogen encounter to strongly rely on IκBNS . While IκBNS was largely dispensable for the acquisition of cytotoxic effector function in CD8+ T cells, IκBNS -deficient Th1 effector cells exhibited significantly reduced proliferation, marked changes in the pattern of activation marker expression, and reduced production of the Th1-cell cytokines IFN-γ, IL-2, and TNF-α. Complementary in vitro analyses using cells from novel reporter and inducible knockout mice revealed that IκBNS predominantly affects the early phase of Th1-cell differentiation while its function in terminally differentiated cells appears to be negligible. Our data suggest IκBNS as a potential target to modulate specifically CD4+ T-cell responses.

Gamma secretase dependent release of the CD44 cytoplasmic tail upregulates IFI16 in cd44-/- tumor cells, MEFs and macrophages.

The adhesion molecule and co-receptor of receptor tyrosine kinases, CD44, is expressed in all cells of the immune system, but also in numerous non-immune cells. CD44 plays roles in the cellular response to different pathogens. The molecular actions of CD44 during these processes are by and large still unknown. The CD44 molecule undergoes a sequential proteolytic cleavage which leads to the release of a soluble intracellular domain (CD44-ICD). Previous reports had shown that the CD44-ICD is taken up into the nucleus where it enhances transcription of specific target genes. By RNA profiling we identified a CD44-dependent transcriptional increase of interferon-responsive genes, among them IFI16. IFI16 is important in the innate immune response. It senses and binds pathogenic DNA and, together with cGAS, activates the cGAS-cGAMP-STING pathway and induces the expression of genes relevant for the response, e.g. IFN-ß. Our results show that the enhancement of IFI16 expression depended on CD44 cleavage. A CD44-negative tumor cell line, embryonic fibroblasts and bone marrow-derived macrophages from cd44-/- mice were reduced in their response to IFN-γ, to viral DNA fragments and to Listeria monocytogenes infection. We could rescue the deficiency of CD44 negative RPM-MC cells and cd44-/- MEFs by expressing only the soluble CD44-ICD in the absence of any other CD44 domain. Expression of the CD44-ICD carrying a mutation that prevented the uptake into the nucleus, could not rescue the absence of CD44. This molecular aspect of regulation by CD44 may explain part of the immune phenotypes of mice with cd44 gene disruption.

Proteomic definition of human mucosal-associated invariant T cells determines their unique molecular effector phenotype.

Mucosal-associated invariant T cells (MAIT) constitute the most abundant anti-bacterial CD8+ T-cell population in humans. MR1/TCR-activated MAIT cells were reported to organize cytotoxic and innate-like responses but knowledge about their molecular effector phenotype is still fragmentary. Here, we have examined the functional inventory of human MAIT cells (CD3+ Vα7.2+ CD161+ ) in comparison with those from conventional non-MAIT CD8+ T cells (cCD8+ ) and NK cells. Quantitative mass spectrometry characterized 5500 proteins of primary MAIT cells and identified 160 and 135 proteins that discriminate them from cCD8+ T cells and NK cells donor-independently. Most notably, MAIT cells showed a unique exocytosis machinery in parallel to a proinflammatory granzyme profile with high levels of the granzymes A, K, and M. Furthermore, 24 proteins were identified with highest abundances in MAIT cells, including CD26, CD98, and L-amino-oxidase (LAAO). Among those, expression of granzyme K and CD98 were validated as MAIT-specific with respect to non-MAIT CD8+ effector subsets and LAAO was found to be recruited together with granzymes, perforin, and CD107a at the immunological synapse of activated MAIT cells. In conclusion, this study complements knowledge on the molecular effector phenotype of MAIT cells and suggest novel immune regulatory functions as part of their cytotoxic responses.

c-REL and IκBNS Govern Common and Independent Steps of Regulatory T Cell Development from Novel CD122-Expressing Pre-Precursors.

Foxp3-expressing regulatory T cells (Tregs) are essential regulators of immune homeostasis and, thus, are prime targets for therapeutic interventions of diseases such as cancer and autoimmunity. c-REL and IκBNS are important regulators of Foxp3 induction in Treg precursors upon γ-chain cytokine stimulation. In c-REL/IκBNS double-deficient mice, Treg numbers were dramatically reduced, indicating that together, c-REL and IκBNS are pivotal for Treg development. However, despite the highly reduced Treg compartment, double-deficient mice did not develop autoimmunity even when aged to more than 1 y, suggesting that c-REL and IκBNS are required for T cell effector function as well. Analyzing Treg development in more detail, we identified a CD122+ subset within the CD25-Foxp3- precursor population, which gave rise to classical CD25+Foxp3- Treg precursors. Importantly, c-REL, but not IκBNS, controlled the generation of classical CD25+Foxp3- precursors via direct binding to the Cd25 locus. Thus, we propose that CD4+GITR+CD122+CD25-Foxp3- cells represent a Treg pre-precursor population, whose transition into Treg precursors is mediated via c-REL.

Targeted antigen delivery to dendritic cells elicits robust antiviral T cell-mediated immunity in the liver.

Hepatotropic viruses such as hepatitis C virus cause life-threatening chronic liver infections in millions of people worldwide. Targeted in vivo antigen-delivery to cross-presenting dendritic cells (DCs) has proven to be extraordinarily efficient in stimulating antigen-specific T cell responses. To determine whether this approach would as well be suitable to induce local antiviral effector T cells in the liver we compared different vaccine formulations based on either the targeting of DEC-205 or TLR2/6 on cross-presenting DCs or formulations not involving in vivo DC targeting. As read-outs we used in vivo hepatotropic adenovirus challenge, histology and automated multidimensional fluorescence microscopy (MELC). We show that targeted in vivo antigen delivery to cross-presenting DCs is highly effective in inducing antiviral CTLs capable of eliminating virus-infected hepatocytes, while control vaccine formulation not involving DC targeting failed to induce immunity against hepatotropic virus. Moreover, we observed distinct patterns of CD8+ T cell interaction with virus-infected and apoptotic hepatocytes in the two DC-targeting groups suggesting that the different vaccine formulations may stimulate distinct types of effector functions. Our findings represent an important step toward the future development of vaccines against hepatotropic viruses and the treatment of patients with hepatic virus infection after liver transplantation to avoid reinfection.

A Mouse Model of Post-Stroke Pneumonia Induced by Intra-Tracheal Inoculation with Streptococcus pneumoniae.

BACKGROUND: Stroke-induced immunodeficiency increases the risk of infectious complications, which adversely affects neurological outcome. Among those, pneumonia affects as many as one third of stroke patients and is the main contributor to mortality in the post-acute phase of stroke. Experimental findings on post-stroke susceptibility to spontaneous pneumonia in mice are contradictory. Here, we established a mouse model inducing standardized bacterial pneumonia and characterized the impaired pulmonary cellular and humoral immune responses after experimental stroke. METHODS: Bacterial pneumonia was induced by intra-tracheal inoculation with Streptococcus pneumoniae at different time points after transient middle cerebral artery occlusion (MCAO). Bacterial counts in lungs and blood, histological changes, and cytokine production in the lungs were assessed. Furthermore, we investigated the effect of pneumonia on stroke outcome. RESULTS: Intra-tracheal inoculation resulted in reproducible pneumonia and bacteraemia, and demonstrated post-stroke susceptibility to streptococcal pneumonia developing with a delay of at least 24 h after MCAO. Higher bacterial counts in mice infected 3 days after stroke induction correlated with reduced neutrophil and macrophage infiltration in the lungs and lower levels of pro-inflammatory cytokines in the broncho-alveolar lavage compared to sham-operated animals. Pneumonia increased mortality without affecting brain-infiltrating leukocytes. CONCLUSIONS: In this standardized mouse model of post-stroke pneumonia, we describe attenuated leukocyte infiltration and cytokine production in response to bacterial infection in the lungs that has a profound effect on outcome.