BCG infection dose guides dendritic cell migration and T cell priming in the draining lymph node.

In contrast to delayed-type hypersensitivity (DTH) and other hallmark reactions of cell-mediated immunity that correlate with vaccine-mediated protection against Mycobacterium tuberculosis, the contribution of vaccine dose on responses that emerge early after infection in the skin with Bacille Calmette-Guérin (BCG) is not well understood. We used a mouse model of BCG skin infection to study the effect of BCG dose on the relocation of skin Dendritic cells (DCs) to draining lymph node (DLN). Mycobacterium antigen 85B-specific CD4+ P25 T cell-receptor transgenic (P25 TCRTg) cells were used to probe priming to BCG in DLN. DC migration and T cell priming were studied across BCG inocula that varied up to 100-fold (104 to 106 Colony-forming units-CFUs). In line with earlier results in guinea pigs, DTH reaction in our model correlated with BCG dose. Importantly, priming of P25 TCRTg cells in DLN also escalated in a dose-dependent manner, peaking at day 6 after infection. Similar dose-escalation effects were seen for DC migration from infected skin and the accompanying transport of BCG to the DLN. BCG-triggered upregulation of co-stimulatory molecules on migratory DCs was restricted to the first 24 hour after infection and was independent of BCG dose over a 10-fold range (105 to 106 CFUs). The dose seemed to be a determinant of the number of total skin DCs that move to the DLN. In summary, our results support the use of higher BCG doses to detect robust DC migration and T cell priming.

Cyclooxygenase-Derived Prostaglandin E2 Drives IL-1-Independent Mycobacterium bovis Bacille Calmette-Guérin-Triggered Skin Dendritic Cell Migration to Draining Lymph Node.

Inoculation of Mycobacterium bovis Bacille Calmette-Guérin (BCG) in the skin mobilizes local dendritic cells (DC) to the draining lymph node (dLN) in a process that remains incompletely understood. In this study, a mouse model of BCG skin infection was used to investigate mechanisms of skin DC migration to dLNs. We found enhanced transcription of cyclooxygenase (COX)-2 and production of COX-derived PGE2 early after BCG infection in skin. Animals treated with antagonists for COX or the PGE2 receptors EP2 and EP4 displayed a marked reduction in the entry of skin DCs and BCG to dLNs, uncovering an important contribution of COX-derived PGE2 in this migration process. In addition, live BCG bacilli were needed to invoke DC migration through this COX-PGE2 pathway. Having previously shown that IL-1R partially regulates BCG-induced relocation of skin DCs to dLNs, we investigated whether PGE2 release was under control of IL-1. Interestingly, IL-1R ligands IL-1α/ß were not required for early transcription of COX-2 or production of PGE2 in BCG-infected skin, suggesting that the DC migration-promoting role of PGE2 is independent of IL-1α/ß in our model. In DC adoptive transfer experiments, EP2/EP4, but not IL-1R, was needed on the moving DCs for full-fledged migration, supporting different modes of action for PGE2 and IL-1α/ß. In summary, our data highlight an important role for PGE2 in guiding DCs to dLNs in an IL-1-independent manner.

Detection and isolation of airborne SARS-CoV-2 in a hospital setting.

Transmission mechanisms for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are incompletely understood. In particular, aerosol transmission remains unclear, with viral detection in air and demonstration of its infection potential being actively investigated. To this end, we employed a novel electrostatic collector to sample air from rooms occupied by COVID-19 patients in a major Swedish hospital. Electrostatic air sampling in conjunction with extraction-free, reverse-transcriptase polymerase chain reaction (hid-RT-PCR) enabled detection of SARS-CoV-2 in air from patient rooms (9/22; 41%) and adjoining anterooms (10/22; 45%). Detection with hid-RT-PCR was concomitant with viral RNA presence on the surface of exhaust ventilation channels in patients and anterooms more than 2 m from the COVID-19 patient. Importantly, it was possible to detect active SARS-CoV-2 particles from room air, with a total of 496 plaque-forming units (PFUs) being isolated, establishing the presence of infectious, airborne SARS-CoV-2 in rooms occupied by COVID-19 patients. Our results support circulation of SARS-CoV-2 via aerosols and urge the revision of existing infection control frameworks to include airborne transmission.

Intestinal helminth infection transforms the CD4+ T cell composition of the skin.

Intestinal helminth parasites can alter immune responses to vaccines, other infections, allergens and autoantigens, implying effects on host immune responses in distal barrier tissues. We herein show that the skin of C57BL/6 mice infected with the strictly intestinal nematode Heligmosomoides polygyrus contain higher numbers of CD4+ T cells compared to the skin of uninfected controls. Accumulated CD4+ T cells were H. polygyrus-specific TH2 cells that skewed the skin CD4+ T cell composition towards a higher TH2/TH1 ratio which persisted after worm expulsion. Accumulation of TH2 cells in the skin was associated with increased expression of the skin-homing chemokine receptors CCR4 and CCR10 on CD4+ T cells in the blood and mesenteric lymph nodes draining the infected intestine and was abolished by FTY720 treatment during infection, indicating gut-to-skin trafficking of cells. Remarkably, skin TH2 accumulation was associated with impaired capacity to initiate IFN-γ recall responses and develop skin-resident memory cells to mycobacterial antigens, both during infection and months after deworming therapy. In conclusion, we show that infection by a strictly intestinal helminth has long-term effects on immune cell composition and local immune responses to unrelated antigens in the skin, revealing a novel process for T cell colonisation and worm-mediated immunosuppression in this organ.

Vaccinia Virus Infection Inhibits Skin Dendritic Cell Migration to the Draining Lymph Node.

There is a paucity of information on dendritic cell (DC) responses to vaccinia virus (VACV), including the traffic of DCs to the draining lymph node (dLN). In this study, using a mouse model of infection, we studied skin DC migration in response to VACV and compared it with the tuberculosis vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG), another live attenuated vaccine administered via the skin. In stark contrast to BCG, skin DCs did not relocate to the dLN in response to VACV. Infection with UV-inactivated VACV or modified VACV Ankara promoted DC movement to the dLN, indicating that interference with skin DC migration requires replication-competent VACV. This suppressive effect of VACV was capable of mitigating responses to a secondary challenge with BCG in the skin, ablating DC migration, reducing BCG transport, and delaying CD4+ T cell priming in the dLN. Expression of inflammatory mediators associated with BCG-triggered DC migration were absent from virus-injected skin, suggesting that other pathways invoke DC movement in response to replication-deficient VACV. Despite adamant suppression of DC migration, VACV was still detected early in the dLN and primed Ag-specific CD4+ T cells. In summary, VACV blocks skin DC mobilization from the site of infection while retaining the ability to access the dLN to prime CD4+ T cells.

Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing.

Operative and Technical Modifications to the Coriolis® µ Air Sampler That Improve Sample Recovery and Biosafety During Microbiological Air Sampling.

Detecting infectious aerosols is central for gauging and countering airborne threats. In this regard, the Coriolis® µ cyclonic air sampler is a practical, commercial collector that can be used with various analysis methods to monitor pathogens in air. However, information on how to operate this unit under optimal sampling and biosafety conditions is limited. We investigated Coriolis performance in aerosol dispersal experiments with polystyrene microspheres and Bacillus globigii spores. We report inconsistent sample recovery from the collector cone due to loss of material when sampling continuously for more than 30 min. Introducing a new collector cone every 10 min improved this shortcoming. Moreover, we found that several surfaces on the device become contaminated during sampling. Adapting a high efficiency particulate air-filter system to the Coriolis prevented contamination without altering collection efficiency or tactical deployment. A Coriolis modified with these operative and technical improvements was used to collect aerosols carrying microspheres released inside a Biosafety Level-3 laboratory during simulations of microbiological spills and aerosol dispersals. In summary, we provide operative and technical solutions to the Coriolis that optimize microbiological air sampling and improve biosafety.

A fieldable electrostatic air sampler enabling tuberculosis detection in bioaerosols.

Tuberculosis (TB) infects about 25% of the world population and claims more human lives than any other infectious disease. TB is spread by inhalation of aerosols containing viable Mycobacterium tuberculosis expectorated or exhaled by patients with active pulmonary disease. Air-sampling technology could play an important role in TB control by enabling the detection of airborne M. tuberculosis, but tools that are easy to use and scalable in TB hotspots are lacking. We developed an electrostatic air sampler termed the TB Hotspot DetectOR (THOR) and investigated its performance in laboratory aerosol experiments and in a prison hotspot of TB transmission. We show that THOR collects aerosols carrying microspheres, Bacillus globigii spores and M. bovis BCG, concentrating these microparticles onto a collector piece designed for subsequent detection analysis. The unit was also successfully operated in the complex setting of a prison hotspot, enabling detection of a molecular signature for M. tuberculosis in the cough of inmates. Future deployment of this device may lead to a measurable impact on TB case-finding by screening individuals through the aerosols they generate.

Atrophy of skin-draining lymph nodes predisposes for impaired immune responses to secondary infection in mice with chronic intestinal nematode infection.

Intestinal nematodes suppress immune responses in the context of allergy, gut inflammation, secondary infection and vaccination. Several mechanisms have been proposed for this suppression including alterations in Th2 cell differentiation and increased Treg cell suppressive function. In this study, we show that chronic nematode infection leads to reduced peripheral responses to vaccination because of a generalized reduction in the available responsive lymphocyte pool. We found that superficial skin-draining lymph nodes (LNs) in mice that are chronically infected with the intestinal nematode Heligmosomides polygyrus, do not reach the same cellularity as worm-free mice upon subsequent BCG infection in the skin. B cells and T cells, all declined in skin-draining LN of H. polygyrus-infected mice, resulting in LNs atrophy and altered lymphocyte composition. Importantly, anti-helminthic treatment improved lymphocyte numbers in skin-draining LN, indicating that time after de-worming is critical to regain full-scale LN cellularity. De-worming, and time for the skin LN to recover cellularity, also mended responses to Bacille Calmette-Guerin (BCG) in the LN draining the footpad injection site. Thus, our findings show that chronic nematode infection leads to a paucity of lymphocytes in peripheral lymph nodes, which acts to reduce the efficacy of immune responses at these sites.

Immunogenicity is preferentially induced in sparse dendritic cell cultures.

We have previously shown that human monocyte-derived dendritic cells (DCs) acquired different characteristics in dense or sparse cell cultures. Sparsity promoted the development of IL-12 producing migratory DCs, whereas dense cultures increased IL-10 production. Here we analysed whether the density-dependent endogenous breaks could modulate DC-based vaccines. Using murine bone marrow-derived DC models we show that sparse cultures were essential to achieve several key functions required for immunogenic DC vaccines, including mobility to draining lymph nodes, recruitment and massive proliferation of antigen-specific CD4+ T cells, in addition to their TH1 polarization. Transcription analyses confirmed higher commitment in sparse cultures towards T cell activation, whereas DCs obtained from dense cultures up-regulated immunosuppressive pathway components and genes suggesting higher differentiation plasticity towards osteoclasts. Interestingly, we detected a striking up-regulation of fatty acid and cholesterol biosynthesis pathways in sparse cultures, suggesting an important link between DC immunogenicity and lipid homeostasis regulation.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Guérin.

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Guérin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

BCG Skin Infection Triggers IL-1R-MyD88-Dependent Migration of EpCAMlow CD11bhigh Skin Dendritic cells to Draining Lymph Node During CD4+ T-Cell Priming.

The transport of antigen from the periphery to the draining lymph node (DLN) is critical for T-cell priming but remains poorly studied during infection with Mycobacterium bovis Bacille Calmette-Guérin (BCG). To address this we employed a mouse model to track the traffic of Dendritic cells (DCs) and mycobacteria from the BCG inoculation site in the skin to the DLN. Detection of BCG in the DLN was concomitant with the priming of antigen-specific CD4+ T cells at that site. We found EpCAMlow CD11bhigh migratory skin DCs to be mobilized during the transport of BCG to the DLN. Migratory skin DCs distributed to the T-cell area of the LN, co-localized with BCG and were found in close apposition to antigen-specific CD4+ T cells. Consequently, blockade of skin DC traffic into DLN dramatically reduced mycobacterial entry into DLN and muted T-cell priming. Interestingly, DC and mycobacterial entry into the DLN was dependent on IL-1R-I, MyD88, TNFR-I and IL-12p40. In addition, we found using DC adoptive transfers that the requirement for MyD88 in BCG-triggered migration was not restricted to the migrating DC itself and that hematopoietic expression of MyD88 was needed in part for full-fledged migration. Our observations thus identify a population of DCs that contribute towards the priming of CD4+ T cells to BCG infection by transporting bacilli into the DLN in an IL-1R-MyD88-dependent manner and reveal both DC-intrinsic and -extrinsic requirements for MyD88 in DC migration.

Mannose-binding lectin regulates host resistance and pathology during experimental infection with Trypanosoma cruzi.

Mannose-binding lectin (MBL) is a humoral pattern-recognition molecule important for host defense. Although recent genetic studies suggest an involvement of MBL/MASP2-associated pathways in Chagas' disease, it is currently unknown whether MBL plays a role in host resistance to the intracellular protozoan Trypanosoma cruzi, the causative agent of Chagas' disease. In this study we employed MBL(-/-) mice to assess the role of MBL in resistance to experimental infection with T. cruzi. T. cruzi infection enhanced tissue expression of MBL both at the mRNA and protein level. Similarly, symptomatic acute Chagas' disease patients displayed increased serum concentrations of MBL compared to patients with indeterminate, asymptomatic forms of the disease. Furthermore, increased parasite loads in the blood and/or tissue were observed in MBL(-/-) mice compared to WT controls. This was associated with reduced systemic levels of IL-12/23p40 in MBL(-/-) mice. Importantly, MBL(-/-) mice infected with a cardiotropic strain of T. cruzi displayed increased myocarditis and cardiac fibrosis compared to WT controls. The latter was accompanied by elevated hydroxyproline content and mRNA levels of collagen-1 and -6 in the heart. These observations point to a previously unappreciated role for MBL in regulating host resistance and cardiac inflammation during infection with a major human pathogen.

IL-10 limits parasite burden and protects against fatal myocarditis in a mouse model of Trypanosoma cruzi infection.

Chagas' disease is a zoonosis prevalent in Latin America that is caused by the protozoan Trypanosoma cruzi. The immunopathogenesis of cardiomyopathy, the main clinical problem in Chagas' disease, has been extensively studied but is still poorly understood. In this study, we systematically compared clinical, microbiologic, pathologic, immunologic, and molecular parameters in two mouse models with opposite susceptibility to acute myocarditis caused by the myotropic Colombiana strain of T. cruzi: C3H/HeSnJ (100% mortality, uncontrolled parasitism) and C57BL/6J (<10% mortality, controlled parasitism). T. cruzi induced differential polarization of immunoregulatory cytokine mRNA expression in the hearts of C57BL/6J versus C3H/HeSnJ mice; however, most differences were small. The difference in IL-10 expression was exceptional (C57BL/6J 8.7-fold greater than C3H/HeSnJ). Consistent with this, hearts from infected C57BL/6J mice, but not C3H/HeSnJ mice, had a high frequency of total IL-10-producing CD8(+) T cells and both CD4(+) and CD8(+) subsets of IFN-γ(+)IL-10(+) double-producing T cells. Furthermore, T. cruzi infection of IL-10(-/-) C57BL/6J mice phenocopied fatal infection in wild-type C3H/HeSnJ mice with complete loss of parasite control. Adoptive transfer experiments indicated that T cells were a source of protective IL-10. Thus, in this system, IL-10 production by T cells promotes T. cruzi control and protection from fatal acute myocarditis.

Intravital imaging reveals limited antigen presentation and T cell effector function in mycobacterial granulomas.

Cell-mediated adaptive immunity is critical for host defense, but little is known about T cell behavior during delivery of effector function. Here we investigate relationships among antigen presentation, T cell motility, and local production of effector cytokines by CD4+ T cells within hepatic granulomas triggered by Bacille Calmette-Guérin or Mycobacterium tuberculosis. At steady-state, only small fractions of mycobacteria-specific T cells showed antigen-induced migration arrest within granulomas, resulting in low-level, polarized secretion of cytokines. However, exogenous antigen elicited rapid arrest and robust cytokine production by the vast majority of effector T cells. These findings suggest that limited antigen presentation and/or recognition within granulomas evoke a muted T cell response drawing on only a fraction of the host's potential effector capacity. Our results provide new insights into the regulation of host-protective functions, especially how antigen availability influences T cell dynamics and, in turn, effector T cell function during chronic infection.

The Syk/CARD9-coupled receptor Dectin-1 is not required for host resistance to Mycobacterium tuberculosis in mice.

There is interest in identifying the pattern recognition receptors involved in initiating protective or non-protective host responses to Mycobacterium tuberculosis (Mtb). Here we explored the role of the Syk/CARD9-coupled receptor, Dectin-1, using an aerosol model of Mtb infection in wild-type and Dectin-1 deficient mice. We observed a reduction in pulmonary bacilli burdens in the Dectin-1 deficient animals, but this did not correlate with significant changes in pulmonary pathology, cytokine levels or ability of these animals to survive the infection. Thus Dectin-1 makes a minor contribution to susceptibility to Mtb infections in mice.

Cysteamine, the natural metabolite of pantetheinase, shows specific activity against Plasmodium.

In mice, loss of pantetheinase activity causes susceptibility to infection with Plasmodium chabaudi AS. Treatment of mice with the pantetheinase metabolite cysteamine reduces blood-stage replication of P. chabaudi and significantly increases survival. Similarly, a short exposure of Plasmodium to cysteamine ex vivo is sufficient to suppress parasite infectivity in vivo. This effect of cysteamine is specific and not observed with a related thiol (dimercaptosuccinic acid) or with the pantethine precursor of cysteamine. Also, cysteamine does not protect against infection with the parasite Trypanosoma cruzi or the fungal pathogen Candida albicans, suggesting cysteamine acts directly against the parasite and does not modulate host inflammatory response. Cysteamine exposure also blocks replication of P. falciparum in vitro; moreover, these treated parasites show higher levels of intact hemoglobin. This study highlights the in vivo action of cysteamine against Plasmodium and provides further evidence for the involvement of pantetheinase in host response to this infection.

In situ IL-12/23p40 production during mycobacterial infection is sustained by CD11bhigh dendritic cells localized in tissue sites distinct from those harboring bacilli.

Although IL-12/23p40 is known to play a major role in host resistance to Mycobacterium spp, the cellular source, tissue localization, and regulation of p40 production during mycobacterial infection in vivo has been unclear. In this study, we used IL-12/23p40eYFP (yet40) reporter mice to track expression of the cytokine following Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection. We found that in spleens of these mice, p40 production is initiated by a transient burst from CD11b(low)CD11c(+) dendritic cells (DC) which are later replaced at the onset of granuloma formation by CD11b(high)CD11c(+) DC as the major source of the cytokine. The latter subset was also found to be the key producer of DC-derived p40 in nonlymphoid tissue and in both spleen and liver optimal production of the cytokine was regulated by endogenous TNF-alpha. Although BCG and p40-expressing DC were both observed in splenic white pulp, p40(+) DC rarely colocalized with bacilli. Indeed, in vitro flow cytometry and confocal microscopy indicated that the presence of intracellular bacteria is not required for p40 production by DC and Transwell experiments confirmed that soluble mycobacterial components are sufficient for inducing cytokine expression by these cells. Moreover, when stimulated with LPS, DC directly infected with BCG showed impaired IL-12p40 production in vitro. Together, our findings establish CD11b(high) DC as a major source of IL-12/23p40 during mycobacterial infection in situ and implicate both soluble mycobacterial products and TNF-alpha in stimulating sustained production of p40 by these cells.