Dimethyl fumarate and 4-octyl itaconate are anticoagulants that suppress Tissue Factor in macrophages via inhibition of Type I Interferon.

Excessive inflammation-associated coagulation is a feature of infectious diseases, occurring in such conditions as bacterial sepsis and COVID-19. It can lead to disseminated intravascular coagulation, one of the leading causes of mortality worldwide. Recently, type I interferon (IFN) signaling has been shown to be required for tissue factor (TF; gene name F3) release from macrophages, a critical initiator of coagulation, providing an important mechanistic link between innate immunity and coagulation. The mechanism of release involves type I IFN-induced caspase-11 which promotes macrophage pyroptosis. Here we find that F3 is a type I IFN-stimulated gene. Furthermore, F3 induction by lipopolysaccharide (LPS) is inhibited by the anti-inflammatory agents dimethyl fumarate (DMF) and 4-octyl itaconate (4-OI). Mechanistically, inhibition of F3 by DMF and 4-OI involves suppression of Ifnb1 expression. Additionally, they block type I IFN- and caspase-11-mediated macrophage pyroptosis, and subsequent TF release. Thereby, DMF and 4-OI inhibit TF-dependent thrombin generation. In vivo, DMF and 4-OI suppress TF-dependent thrombin generation, pulmonary thromboinflammation, and lethality induced by LPS, E. coli, and S. aureus, with 4-OI additionally attenuating inflammation-associated coagulation in a model of SARS-CoV-2 infection. Our results identify the clinically approved drug DMF and the pre-clinical tool compound 4-OI as anticoagulants that inhibit TF-mediated coagulopathy via inhibition of the macrophage type I IFN-TF axis.

Bystander activation of Bordetella pertussis-induced nasal tissue-resident memory CD4 T cells confers heterologous immunity to Klebsiella pneumoniae.

Tissue-resident memory CD4 T (TRM ) cells induced by infection with Bordetella pertussis persist in respiratory tissues and confer long-term protective immunity against reinfection. However, it is not clear how they are maintained in respiratory tissues. Here, we demonstrate that B. pertussis-specific CD4 TRM cells produce IL-17A in response to in vitro stimulation with LPS or heat-killed Klebsiella pneumoniae (HKKP) in the presence of dendritic cells. Furthermore, IL-17A-secreting CD4 TRM cells expand in the lung and nasal tissue of B. pertussis convalescent mice following in vivo administration of LPS or HKKP. Bystander activation of CD4 TRM cells was suppressed by anti-IL-12p40 but not by anti-MHCII antibodies. Furthermore, purified respiratory tissue-resident, but not circulating, CD4 T cells from convalescent mice produced IL-17A following direct stimulation with IL-23 and IL-1ß or IL-18. Intranasal immunization of mice with a whole-cell pertussis vaccine induced respiratory CD4 TRM cells that were reactivated following stimulation with K. pneumoniae. Furthermore, the nasal pertussis vaccine conferred protective immunity against B. pertussis but also attenuated infection with K. pneumoniae. Our findings demonstrate that CD4 TRM cells induced by respiratory infection or vaccination can undergo bystander activation and confer heterologous immunity to an unrelated respiratory pathogen.

The circadian clock influences T cell responses to vaccination by regulating dendritic cell antigen processing.

Dendritic cells play a key role in processing and presenting antigens to naïve T cells to prime adaptive immunity. Circadian rhythms are known to regulate many aspects of immunity; however, the role of circadian rhythms in dendritic cell function is still unclear. Here, we show greater T cell responses when mice are immunised in the middle of their rest versus their active phase. We find a circadian rhythm in antigen processing that correlates with rhythms in both mitochondrial morphology and metabolism, dependent on the molecular clock gene, Bmal1. Using Mdivi-1, a compound that promotes mitochondrial fusion, we are able to rescue the circadian deficit in antigen processing and mechanistically link mitochondrial morphology and antigen processing. Furthermore, we find that circadian changes in mitochondrial Ca2+ are central to the circadian regulation of antigen processing. Our results indicate that rhythmic changes in mitochondrial calcium, which are associated with changes in mitochondrial morphology, regulate antigen processing.

Suppressive effects of the obese tumor microenvironment on CD8 T cell infiltration and effector function.

Obesity is one of the leading preventable causes of cancer; however, little is known about the effects of obesity on anti-tumor immunity. Here, we investigated the effects of obesity on CD8 T cells in mouse models and patients with endometrial cancer. Our findings revealed that CD8 T cell infiltration is suppressed in obesity, which was associated with a decrease in chemokine production. Tumor-resident CD8 T cells were also functionally suppressed in obese mice, which was associated with a suppression of amino acid metabolism. Similarly, we found that a high BMI negatively correlated with CD8 infiltration in human endometrial cancer and that weight loss was associated with a complete pathological response in six of nine patients. Moreover, immunotherapy using anti-PD-1 led to tumor rejection in lean and obese mice and partially restored CD8 metabolism and anti-tumor immunity. These findings highlight the suppressive effects of obesity on CD8 T cell anti-tumor immunity, which can partially be reversed by weight loss and/or immunotherapy.

Evaluation of Whole-Cell and Acellular Pertussis Vaccines in the Context of Long-Term Herd Immunity.

After the pertussis vaccine had been introduced in the 1940s and was shown to be very successful in reducing the morbidity and mortality associated with the disease, the possibility of improving both vaccine composition and vaccination schedules has become the subject of continuous interest. As a result, we are witnessing a considerable heterogeneity in pertussis vaccination policies, which remains beyond universal consensus. Many pertussis-related deaths still occur in low- and middle-income countries; however, these deaths are attributable to gaps in vaccination coverage and limited access to healthcare in these countries, rather than to the poor efficacy of the first generation of pertussis vaccine consisting in inactivated and detoxified whole cell pathogen (wP). In many, particularly high-income countries, a switch was made in the 1990s to the use of acellular pertussis (aP) vaccine, to reduce the rate of post-vaccination adverse events and thereby achieve a higher percentage of children vaccinated. However the epidemiological data collected over the past few decades, even in those high-income countries, show an increase in pertussis prevalence and morbidity rates, triggering a wide-ranging debate on the causes of pertussis resurgence and the effectiveness of current pertussis prevention strategies, as well as on the efficacy of available pertussis vaccines and immunization schedules. The current article presents a systematic review of scientific reports on the evaluation of the use of whole-cell and acellular pertussis vaccines, in the context of long-term immunity and vaccines efficacy.

IL-17 mediates protective immunity against nasal infection with Bordetella pertussis by mobilizing neutrophils, especially Siglec-F+ neutrophils.

Understanding the mechanism of protective immunity in the nasal mucosae is central to the design of more effective vaccines that prevent nasal infection and transmission of Bordetella pertussis. We found significant infiltration of IL-17-secreting CD4+ tissue-resident memory T (TRM) cells and Siglec-F+ neutrophils into the nasal tissue during primary infection with B. pertussis. Il17A-/- mice had significantly higher bacterial load in the nasal mucosae, associated with significantly reduced infiltration of Siglec-F+ neutrophils. Re-infected convalescent mice rapidly cleared B. pertussis from the nasal cavity and this was associated with local expansion of IL-17-producing CD4+ TRM cells. Depletion of CD4 T cells from the nasal tissue during primary infection or after re-challenge of convalescent mice significantly delayed clearance of bacteria from the nasal mucosae. Protection was lost in Il17A-/- mice and this was associated with significantly less infiltration of Siglec-F+ neutrophils and antimicrobial peptide (AMP) production. Finally, depletion of neutrophils reduced the clearance of B. pertussis following re-challenge of convalescent mice. Our findings demonstrate that IL-17 plays a critical role in natural and acquired immunity to B. pertussis in the nasal mucosae and this effect is mediated by mobilizing neutrophils, especially Siglec-F+ neutrophils, which have high neutrophil extracellular trap (NET) activity.

Caspase-11 promotes allergic airway inflammation.

Activated caspase-1 and caspase-11 induce inflammatory cell death in a process termed pyroptosis. Here we show that Prostaglandin E2 (PGE2) inhibits caspase-11-dependent pyroptosis in murine and human macrophages. PGE2 suppreses caspase-11 expression in murine and human macrophages and in the airways of mice with allergic inflammation. Remarkably, caspase-11-deficient mice are strongly resistant to developing experimental allergic airway inflammation, where PGE2 is known to be protective. Expression of caspase-11 is elevated in the lung of wild type mice with allergic airway inflammation. Blocking PGE2 production with indomethacin enhances, whereas the prostaglandin E1 analog misoprostol inhibits lung caspase-11 expression. Finally, alveolar macrophages from asthma patients exhibit increased expression of caspase-4, a human homologue of caspase-11. Our findings identify PGE2 as a negative regulator of caspase-11-driven pyroptosis and implicate caspase-4/11 as a critical contributor to allergic airway inflammation, with implications for pathophysiology of asthma.

A Pertussis Outer Membrane Vesicle-Based Vaccine Induces Lung-Resident Memory CD4 T Cells and Protection Against Bordetella pertussis, Including Pertactin Deficient Strains.

Pertussis is a respiratory infectious disease that has been resurged during the last decades. The change from the traditional multi-antigen whole-cell pertussis (wP) vaccines to acellular pertussis (aP) vaccines that consist of a few antigens formulated with alum, appears to be a key factor in the resurgence of pertussis in many countries. Though current aP vaccines have helped to reduce the morbidity and mortality associated with pertussis, they do not provide durable immunity or adequate protection against the disease caused by the current circulating strains of Bordetella pertussis, which have evolved in the face of the selection pressure induced by the vaccines. Based on the hypothesis that a new vaccine containing multiple antigens could overcome deficiencies in the current aP vaccines, we have designed and characterized a vaccine candidate based on outer membrane vesicle (OMVs). Here we show that the OMVs vaccine, but not an aP vaccine, protected mice against lung infection with a circulating pertactin (PRN)-deficient isolate. Using isogenic bacteria that in principle only differ in PRN expression, we found that deficiency in PRN appears to be largely responsible for the failure of the aP vaccine to protect against this circulating clinical isolates. Regarding the durability of induced immunity, we have already reported that the OMV vaccine is able to induce long-lasting immune responses that effectively prevent infection with B. pertussis. Consistent with this, here we found that CD4 T cells with a tissue-resident memory (TRM) cell phenotype (CD44+CD62LlowCD69+ and/or CD103+) accumulated in the lungs of mice 14 days after immunization with 2 doses of the OMVs vaccine. CD4 TRM cells, which have previously been shown to play a critical role sustained protective immunity against B. pertussis, were also detected in mice immunized with wP vaccine, but not in the animals immunized with a commercial aP vaccine. The CD4 TRM cells secreted IFN-γ and IL-17 and were significantly expanded through local proliferation following respiratory challenge of mice with B. pertussis. Our findings that the OMVs vaccine induce respiratory CD4 TRM cells may explain the ability of this vaccine to induce long-term protection and is therefore an ideal candidate for a third generation vaccine against B. pertussis.

Retinoic acid-induced autoantigen-specific type 1 regulatory T cells suppress autoimmunity.

Regulatory T (Treg) cells help to maintain tolerance and prevent the development of autoimmune diseases. Retinoic acid (RA) can promote peripheral conversion of naïve T cells into Foxp3+ Treg cells. Here, we show that RA can act as an adjuvant to induce antigen-specific type 1 Treg (Tr1) cells, which is augmented by co-administration of IL-2. Immunization of mice with the model antigen KLH in the presence of RA and IL-2 induces T cells that secrete IL-10, but not IL-17 or IFN-γ, and express LAG-3, CD49b and PD-1 but not Foxp3, a phenotype typical of Tr1 cells. Furthermore, immunization of mice with the autoantigen MOG in the presence of RA and IL-2 induces Tr1 cells, which suppress pathogenic Th1 and Th17 cells that mediate the development of experimental autoimmune encephalomyelitis (EAE), an autoimmune disease of the CNS. Furthermore, immunization with a surrogate autoantigen, RA and IL-2 prevents development of spontaneous autoimmune uveitis. Our findings demonstrate that the induction of autoantigen-specific Tr1 cells can prevent the development of autoimmunity.

Immunization with whole cell but not acellular pertussis vaccines primes CD4 TRM cells that sustain protective immunity against nasal colonization with Bordetella pertussis.

Protective immunity wanes rapidly after immunization of children with acellular pertussis (aP) vaccines and these vaccines do not prevent nasal colonization or transmission of Bordetella pertussis in baboons. In this study, we examined the role of tissue-resident memory T (TRM) cells in persistent protective immunity induced by infection or immunization with aP and whole-cell pertussis (wP) vaccines in mice. Immunization of mice with a wP vaccine protected against lung and nasal colonization, whereas an aP vaccine failed to protect in the nose. IL-17 and IFN-γ-secreting CD69+CD4+ TRM cells were expanded in the lung and nasal tissue after B. pertussis challenge of mice immunized with wP, but not aP vaccines. However, previous infection induced the most persistent protection against nasal colonization and this correlated with potent induction of nasal tissue TRM cells, especially IL-17-secreting TRM cells. Blocking T cell migration to respiratory tissue during immunization with a wP vaccine impaired bacterial clearance, whereas transfer of TRM cells from convalescent or wP-immunized mice conferred protection to naïve mice. Our findings reveal that previous infection or wP vaccination are significantly more effective than aP vaccination in conferring persistent protective immunity against B. pertussis and that this is mediated by respiratory TRM cells.

Sustained protective immunity against Bordetella pertussis nasal colonization by intranasal immunization with a vaccine-adjuvant combination that induces IL-17-secreting TRM cells.

Current acellular pertussis (aP) vaccines induce strong antibody and Th2 responses but fail to protect against nasal colonization and transmission of Bordetella pertussis. Furthermore, immunity wanes rapidly after immunization. We have developed a novel adjuvant combination (called LP-GMP), comprising c-di-GMP, an intracellular receptor stimulator of interferon genes (STING) agonist, and LP1569, a TLR2 agonist from B. pertussis, which synergistically induces production of IFN-ß, IL-12 and IL-23, and maturation of dendritic cells. Parenteral immunization of mice with an experimental aP vaccine formulated with LP-GMP promoted Th1 and Th17 responses and conferred protection against lung infection with B. pertussis. Intranasal immunization with the same aP vaccine-induced potent B. pertussis-specific Th17 responses and IL-17-secreting respiratory tissue-resident memory (TRM) CD4 T cells, and conferred a high level of protection against nasal colonization as well as lung infection, which was sustained for at least 10 months. Furthermore, long-term protection against nasal colonization with B. pertussis correlated with the number of IL-17-secreting TRM cells in nasal tissue. Our study has identified an approach for inducing IL-17-secreting TRM cells that sustain sterilizing immunity against nasal colonization of mice with B. pertussis, and could form the basis of a third generation pertussis vaccine for humans.

CD4 TRM Cells Following Infection and Immunization: Implications for More Effective Vaccine Design.

The induction of immunological memory, which is mediated by memory T and B cells, is central to adaptive protective immunity to pathogens induced by previous infection and is the cornerstone of effective vaccine design. Recent studies in mice have suggested that memory T cells that accumulate in tissues, termed tissue-resident memory T (TRM) cells, play a crucial role in maintaining long-term protective immunity to mucosal pathogens. CD4 and CD8 TRM cells can be induced following infection at mucosal sites or the skin, where they are maintained and poised to respond rapidly to reinfection with the same pathogen. TRM cells can also be generated by vaccination, but their induction is influenced by a number of factors, including the type of vaccine, the adjuvant, and the route of immunization. Live attenuated vaccines appear to be more effective than killed or subunit vaccines at inducing TRM cells and mucosal immunization, especially by intranasal route, is more effective than parenteral delivery. However, evidence is emerging that formulation of killed or subunit vaccines with novel adjuvants, especially those that generate Th1 and Th17 responses, can promote the induction of TRM cells. While TRM cells are also present at high number in mucosal tissues in humans, one of the challenge will be to develop methodologies for routine quantification of these cells in humans. Nevertheless, the identification of approaches for optimum induction of TRM cells in mice should assist in the design of more effective vaccines that sustain protective immunity against a range of human pathogens.

Azithromycin Clears Bordetella pertussis Infection in Mice but Also Modulates Innate and Adaptive Immune Responses and T Cell Memory.

Treatment with the macrolide antibiotic azithromycin (AZM) is an important intervention for controlling infection of children with Bordetella pertussis and as a prophylaxis for preventing transmission to family members. However, antibiotics are known to have immunomodulatory effects independent of their antimicrobial activity. Here, we used a mouse model to examine the effects of AZM treatment on clearance of B. pertussis and induction of innate and adaptive immunity. We found that treatment of mice with AZM either 7 or 14 days post challenge effectively cleared the bacteria from the lungs. The numbers of innate immune cells in the lungs were significantly reduced in antibiotic-treated mice. Furthermore, AZM reduced the activation status of macrophages and dendritic cells, but only in mice treated on day 7. Early treatment with antibiotics also reduced the frequency of tissue-resident T cells and IL-17-producing cells in the lungs. To assess the immunomodulatory effects of AZM independent of its antimicrobial activity, mice were antibiotic treated during immunization with a whole cell pertussis (wP) vaccine. Protection against B. pertussis induced by immunization with wP was slightly reduced in AZM-treated mice. Antibiotic-treated wP-immunized mice had reduced numbers of lung-resident memory CD4 T cells and IL-17-production and reduced CD49d expression on splenic CD4 T cells after challenge, suggestive of impaired CD4 T cell memory. Taken together these results suggest that AZM can modulate the induction of memory CD4 T cells during B. pertussis infection, but this may in part be due to the clearance of B. pertussis and resulting loss of components that stimulate innate and adaptive immune response.

FTY720 Attenuates Infection-Induced Enhancement of Aß Accumulation in APP/PS1 Mice by Modulating Astrocytic Activation.

It is well established that infection has a significant detrimental effect on patients with Alzheimer's disease (AD), accelerating cognitive decline and, even in healthy ageing individuals, increasing amyloid-ß (Aß) accumulation in the brain. In animal models of AD infection can also cause damage, with evidence of increased neuroinflammation, amyloid pathology and deterioration of cognitive function. These changes are against a backdrop of an age- and AD-related increase in susceptibility to infection. Here we set out to determine whether FTY720, a molecule that binds sphingosine-1-phosphate (S1P) receptors and with known immunosuppressant effects mediating its therapeutic action in multiple sclerosis (MS), might modulate the impact of infection in a mouse model of AD. Transgenic mice that overexpress amyloid precursor protein (APP) and presenilin 1 (PS1; APP/PS1 mice) and their littermates were/were not infected with Bordetella pertussis and were treated orally with FTY720 or vehicle beginning 3 days before infection. Infection increased astrocytic activation and enhanced blood brain barrier (BBB) permeability and these changes were attenuated in FTY720-treated B. pertussis-infected mice. Significantly, infection increased Aß containing plaques and soluble Aß and these infection-related changes were also attenuated in FTY720-treated B. pertussis-infected mice. The data suggest that this effect results from an FTY720-induced increase in Aß phagocytosis by astrocytes. FTY720 did not impact on genotype-related changes in the absence of an infection indicating that its potential usefulness is restricted to reducing the impact of acute inflammatory stimuli in AD.

Lung CD4 Tissue-Resident Memory T Cells Mediate Adaptive Immunity Induced by Previous Infection of Mice with Bordetella pertussis.

Th1 and Th17 cells have an established role in protective immunity to Bordetella pertussis, but this evidence is based largely on peripheral T cells. There is emerging evidence that local tissue-resident memory T (TRM) cells that accumulate in tissue following mucosal infection may be crucial for long-term immunity. In this study, we examined the role of respiratory CD4 TRM cells in immunity to B. pertussis Natural immunity to B. pertussis induced by infection is considered long lasting and effective at preventing reinfection. Consistent with this, we found that convalescent mice rapidly cleared the bacteria after reinfection. Furthermore, CD4 T cells with a TRM cell phenotype (CD44+CD62L-CD69+ or CD44+CD62L-CD69+CD103+) accumulated in the lungs of mice during infection with B. pertussis and significantly expanded through local proliferation following reinfection. These CD4 TRM cells were B. pertussis specific and secreted IL-17 or IL-17 and IFN-γ. Treatment of mice with FTY720, which prevented migration of T and B cells from lymph nodes to the circulation, significantly exacerbated B. pertussis infection. This was associated with significantly reduced infiltration of central memory T cells and B cells into the lungs. However, the local expansion of TRM cells and the associated rapid clearance of the secondary infection were not affected by treatment with FTY720 before rechallenge. Moreover, adoptive transfer of lung CD4 TRM cells conferred protection in naive mice. Our findings reveal that Ag-specific CD4 TRM cells play a critical role in adaptive immunity against reinfection and memory induced by natural infection with B. pertussis.

IL-17-Producing Innate and Pathogen-Specific Tissue Resident Memory γδ T Cells Expand in the Lungs of Bordetella pertussis-Infected Mice.

γδ T cells play a role in protective immunity to infection at mucosal surface, but also mediate pathology in certain autoimmune diseases through innate IL-17 production. Recent reports have suggested that γδ T cells can have memory analogous to conventional αß T cells. In this study we have examined the role of γδ T cells in immunity to the respiratory pathogen Bordetella pertussis γδ T cells, predominantly Vγ4-γ1- cells, produced IL-17 in the lungs as early as 2 h after infection. The bacterial burden during primary infection was significantly enhanced and the induction of antimicrobial peptides was reduced in the absence of early IL-17. A second peak of γδ T cells is detected in the lungs 7-14 d after challenge and these γδ T cells were pathogen specific. γδ T cells, exclusively Vγ4, from the lungs of infected but not naive mice produced IL-17 in response to heat-killed B. pertussis in the presence of APC. Furthermore, γδ T cells from the lungs of mice reinfected with B. pertussis produced significantly more IL-17 than γδ T cells from infected unprimed mice. γδ T cells with a tissue resident memory T cell phenotype (CD69+CD103+) were expanded in the lungs during infection with B. pertussis and proliferated rapidly after rechallenge of convalescent mice. Our findings demonstrate that lung γδ T cells provide an early source of innate IL-17, which promotes antimicrobial peptide production, whereas pathogen-specific Vγ4 cells function in adaptive immunological memory against B. pertussis.

Anti-PD-1 inhibits Foxp3+ Treg cell conversion and unleashes intratumoural effector T cells thereby enhancing the efficacy of a cancer vaccine in a mouse model.

The co-inhibitory molecule PD-1 suppresses T cell responses and has been targeted in the treatment of cancer. Here, we examined the role of PD-1 in regulating the balance between regulatory and effector T cells and whether blocking PD-1 could enhance tumour vaccine-induced protective immunity. A significantly higher proportion of tumour-resident T cells expressed PD-1 and Foxp3 compared with T cells in the tumour circulation or draining lymph nodes, and this correlated with a lower frequency of IFN-γ- and TNF-secreting CD8 T cells. Blocking PD-1 with a specific antibody reduced Foxp3+ regulatory T (Treg) cell induction and enhanced proliferation, cytokine production, and tumour killing by CD8 T cells. Treatment of CT26 tumour-bearing mice with anti-PD-1 in combination with a vaccine, comprising heat-shocked irradiated tumour cells and a TLR 7/8 agonist, significantly reduced tumour growth and enhanced survival. Furthermore, surviving mice resisted tumour re-challenge. The rejection of tumours in mice treated with the anti-PD-1 vaccine combination was associated with a reduction in tumour-infiltrating Treg cells and enhancement of IFN-γ-secreting CD8 T cells. Our findings demonstrate that high PD-1 expression correlates with increased tumour-infiltrating Treg cells and reduced effector T cells and that when combined with a potent antigen-adjuvant combination, blocking PD-1 effectively enhances anti-tumour immunity.