Mycobacterium tuberculosis (Mtb) infection is initiated in the distal airways, but the bacteria ultimately disseminate to the lung interstitium. Although various cell types, including alveolar ...macrophages (AM), neutrophils, and permissive monocytes, are known to be infected with Mtb, the initially infected cells as well as those that mediate dissemination from the alveoli to the lung interstitium are unknown. In this study, using a murine infection model, we reveal that early, productive Mtb infection occurs almost exclusively within airway-resident AM. Thereafter Mtb-infected, but not uninfected, AM localize to the lung interstitium through mechanisms requiring an intact Mtb ESX-1 secretion system. Relocalization of infected AM precedes Mtb uptake by recruited monocyte-derived macrophages and neutrophils. This dissemination process is driven by non-hematopoietic host MyD88/interleukin-1 receptor inflammasome signaling. Thus, interleukin-1-mediated crosstalk between Mtb-infected AM and non-hematopoietic cells promotes pulmonary Mtb infection by enabling infected cells to disseminate from the alveoli to the lung interstitium.
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•Early M. tuberculosis infection primarily targets lung alveolar macrophages (AM)•Mtb-infected AM selectively relocalize from the airways to the lung interstitium•AM interstitial localization precedes Mtb dissemination to other immune cell types•Relocalization of infected AM is dependent on Mtb ESX-1 and host IL-1R signaling
Using a mouse model, Cohen et al. demonstrate that early Mycobacterium tuberculosis infection predominantly targets alveolar macrophages (AM). Infected AM relocalize from the alveolar space to lung interstitium, preceding bacterial dissemination into migratory myeloid populations. Relocalization requires IL-1R and bacterial ESX-1, highlighting the host-pathogen interplay required to establish infection.
Pulmonary granulomas are widely considered the epicenters of the immune response to
Mycobacterium tuberculosis
(Mtb), the causative agent of tuberculosis (TB). Recent animal studies have revealed ...factors that either promote or restrict TB immunity within granulomas. These models, however, typically ignore the impact of preexisting immunity on cellular organization and function, an important consideration because most TB probably occurs through reinfection of previously exposed individuals. Human postmortem research from the pre-antibiotic era showed that infections in Mtb-naïve individuals (primary TB) versus those with prior Mtb exposure (postprimary TB) have distinct pathologic features. We review recent animal findings in TB granuloma biology, which largely reflect primary TB. We also discuss our current understanding of postprimary TB lesions, about which much less is known. Many knowledge gaps remain, particularly regarding how preexisting immunity shapes granuloma structure and local immune responses at Mtb infection sites.
Recently developed approaches for highly multiplexed imaging have revealed complex patterns of cellular positioning and cell-cell interactions with important roles in both cellular- and tissue-level ...physiology. However, tools to quantitatively study cellular patterning and tissue architecture are currently lacking. Here, we develop a spatial analysis toolbox, the histo-cytometric multidimensional analysis pipeline (CytoMAP), which incorporates data clustering, positional correlation, dimensionality reduction, and 2D/3D region reconstruction to identify localized cellular networks and reveal features of tissue organization. We apply CytoMAP to study the microanatomy of innate immune subsets in murine lymph nodes (LNs) and reveal mutually exclusive segregation of migratory dendritic cells (DCs), regionalized compartmentalization of SIRPα− dermal DCs, and preferential association of resident DCs with select LN vasculature. The findings provide insights into the organization of myeloid cells in LNs and demonstrate that CytoMAP is a comprehensive analytics toolbox for revealing features of tissue organization in imaging datasets.
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•CytoMAP is a user-friendly, comprehensive platform for spatial analysis of tissues•Allows quantification of cellular positioning and global tissue structure•Enables exploration of cellular and tissue microenvironment heterogeneity•CytoMAP reveals fundamental features of myeloid cell organization in lymph nodes
Stoltzfus et al. present CytoMAP, a spatial analytics platform that incorporates diverse statistical and visualization modules for analysis of cellular positioning, cell-cell interactions, global tissue structure, and heterogeneity of tissue microenvironments. Exploration of myeloid cell localization in lymph nodes reveals fundamental positional relationships between dendritic cell subsets and local vasculature.
Abstract T cells producing IFN-γ have long been considered a stalwart for immune protection against Mycobacterium tuberculosis (Mtb), but their relative importance to pulmonary immunity has been ...challenged by murine studies that achieved protection by adoptively transferred Mtb-specific IFN-γ−/− T cells. Using IFN-γ−/− T cell chimeric mice and adoptive transfer of IFN-γ−/− T cells into TCRβ−/−δ−/− mice, we demonstrate that control of lung Mtb burden is in fact dependent on T cell–derived IFN-γ, and, furthermore, mice selectively deficient in T cell–derived IFN-γ develop exacerbated disease compared with T cell–deficient control animals, despite equivalent lung bacterial burdens. Deficiency in T cell–derived IFN-γ skews infected and bystander monocyte-derived macrophages to an alternative M2 phenotype and promotes neutrophil and eosinophil influx. Our studies support an important role for T cell–derived IFN-γ in pulmonary immunity against tuberculosis.
Tuberculosis (TB) is a heterogeneous disease manifesting in a subset of individuals infected with aerosolized Mycobacterium tuberculosis (Mtb). Unlike human TB, murine infection results in uniformly ...high lung bacterial burdens and poorly organized granulomas. To develop a TB model that more closely resembles human disease, we infected mice with an ultra-low dose (ULD) of between 1–3 founding bacteria, reflecting a physiologic inoculum. ULD-infected mice exhibited highly heterogeneous bacterial burdens, well-circumscribed granulomas that shared features with human granulomas, and prolonged Mtb containment with unilateral pulmonary infection in some mice. We identified blood RNA signatures in mice infected with an ULD or a conventional Mtb dose (50–100 CFU) that correlated with lung bacterial burdens and predicted Mtb infection outcomes across species, including risk of progression to active TB in humans. Overall, these findings highlight the potential of the murine TB model and show that ULD infection recapitulates key features of human TB.
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•Ultra-low dose (ULD) Mtb infection of mice results in heterogeneous bacterial burdens•ULD-infected mice often maintain unilateral pulmonary infection•ULD-infected mice develop single, well-organized, pulmonary granuloma•Blood RNA signatures reflecting murine Mtb burdens can predict human TB risk
Conventional murine Mtb infection fails to recapitulate several human TB features. Plumlee et al. show that ultra-low dose infection results in heterogeneous outcomes, including single, well-organized granulomas and unilateral lung containment. Blood transcriptional signatures derived to correlate with pulmonary bacterial burdens in Mtb-infected mice can predict human TB risk.
CD4 T cell effector function is required for optimal containment of Mycobacterium tuberculosis (Mtb) infection. IFNɣ produced by CD4 T cells is a key cytokine that contributes to protection. However, ...lung-infiltrating CD4 T cells have a limited ability to produce IFNɣ, and IFNɣ plays a lesser protective role within the lung than at sites of Mtb dissemination. In a murine infection model, we observed that IFNɣ production by Mtb-specific CD4 T cells is rapidly extinguished within the granuloma but not within unaffected lung regions, suggesting localized immunosuppression. We identified a signature of TGFβ signaling within granuloma-infiltrating T cells in both mice and rhesus macaques. Selective blockade of TGFβ signaling in T cells resulted in an accumulation of terminally differentiated effector CD4 T cells, improved IFNɣ production within granulomas, and reduced bacterial burdens. These findings uncover a spatially localized immunosuppressive mechanism associated with Mtb infection and provide potential targets for host-directed therapy.
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•Despite antigen sensing, CD4 T cells produce minimal IFNɣ in pulmonary Mtb granulomas•Granuloma-localized TGFβ sensing by CD4 T cells rapidly limits their effector function•TGFβ also limits the expansion and survival of terminally differentiated CD4 T cells•Ablation of T cell TGFβ sensing improves responses and lowers bacterial burdens
CD4 T cell function at the site of infection is crucial to control Mycobacterium tuberculosis. Gern, Adams et al. show that TGFβ inhibits CD4 T cell function specifically within the pulmonary granuloma. Ablation of T cell TGFβ sensing reprograms responses and lowers mycobacterial burdens, suggesting avenues for host-directed therapy.
Growing evidence suggests the outcome of
infection is established rapidly after exposure, but how the current tuberculosis vaccine, bacillus Calmette-Guérin (BCG), impacts early immunity is poorly ...understood. In this study, we found that murine BCG immunization promotes a dramatic shift in infected cell types. Although alveolar macrophages are the major infected cell for the first 2 weeks in unimmunized animals, BCG promotes the accelerated recruitment and infection of lung-infiltrating phagocytes. Interestingly, this shift is dependent on CD4 T cells, yet does not require intrinsic recognition of Ag presented by infected alveolar macrophages.
-specific T cells are first activated in lung regions devoid of infected cells, and these events precede vaccine-induced reduction of the bacterial burden, which occurs only after the colocalization of T cells and infected cells. Understanding how BCG alters early immune responses to
provides new avenues to improve upon the immunity it confers.
To investigate how host and pathogen diversity govern immunity against Mycobacterium tuberculosis (Mtb), we performed a large-scale screen of vaccine-mediated protection against aerosol Mtb infection ...using three inbred mouse strains C57BL/6 (B6), C3HeB/FeJ (C3H), Balb/c x 129/SvJ (C129F1) and three Mtb strains (H37Rv, CDC1551, SA161) representing two lineages and distinct virulence properties. We compared three protective modalities, all of which involve inoculation with live mycobacteria: Bacillus Calmette-Guérin (BCG), the only approved TB vaccine, delivered either subcutaneously or intravenously, and concomitant Mtb infection (CoMtb), a model of pre-existing immunity in which a low-level Mtb infection is established in the cervical lymph node following intradermal inoculation. We examined lung bacterial burdens at early (Day 28) and late (Day 98) time points after aerosol Mtb challenge and histopathology at Day 98. We observed substantial heterogeneity in the reduction of bacterial load afforded by these modalities at Day 28 across the combinations and noted a strong positive correlation between bacterial burden in unvaccinated mice and the degree of protection afforded by vaccination. Although we observed variation in the degree of reduction in bacterial burdens across the nine mouse/bacterium strain combinations, virtually all protective modalities performed similarly for a given strain-strain combination. We also noted dramatic variation in histopathology changes driven by both host and bacterial genetic backgrounds. Vaccination improved pathology scores for all infections except CDC1551. However, the most dramatic impact of vaccination on lesion development occurred for the C3H-SA161 combination, where vaccination entirely abrogated the development of the large necrotic lesions that arise in unvaccinated mice. In conclusion, we find that substantial TB heterogeneity can be recapitulated by introducing variability in both host and bacterial genetics, resulting in changes in vaccine-mediated protection as measured both by bacterial burden as well as histopathology. These differences can be harnessed in future studies to identify immune correlates of vaccine efficacy.
In vitro trends of cefazolin and ceftriaxone susceptibilities from pediatric clinical isolates of methicillin-susceptible Staphylococcus aureus (MSSA) between 2011 and 2016 were analyzed for ...surveillance.
Our laboratory continues to use agar disk diffusion for staphylococcal susceptibilities applying Clinical Laboratory Standard Institute's 2012 breakpoints.
A total of 3992 MSSA clinical isolates in the last 6 years were analyzed for their in vitro cefazolin and ceftriaxone susceptibilities. While all MSSA isolates exhibited cefazolin susceptibilities within the "susceptible" zone range, there have been a proportion of isolates with ceftriaxone susceptibilities falling in "intermediate" zones, ranging from 2.6% in 2011 to 8.3% in 2016.
Cefazolin continues to be the recommended agent for MSSA treatment at our institution, reflected by the finding that only 2% (6/321) of patients who received ceftriaxone as definitive therapy for MSSA bacteremia during the study period. We have confirmed the cefoxitin-predicted MSSA susceptibility to cefazolin, but have found concerning drifts in ceftriaxone susceptibilities by continued in vitro monitoring over the last 6 years.
A polymorphism causing deficiencies in Toll-interacting protein (TOLLIP), an inhibitory adaptor protein affecting endosomal trafficking, is associated with increased tuberculosis (TB) risk. It is, ...however, unclear how TOLLIP affects TB pathogenesis. Here we show that TB severity is increased in Tollip
mice, characterized by macrophage- and T cell-driven inflammation, foam cell formation and lipid accumulation. Tollip
alveolar macrophages (AM) specifically accumulated lipid and underwent necrosis. Transcriptional and protein analyses of Mycobacterium tuberculosis (Mtb)-infected, Tollip
AM revealed increased EIF2 signalling and downstream upregulation of the integrated stress response (ISR). These phenotypes were linked, as incubation of the Mtb lipid mycolic acid with Mtb-infected Tollip
AM activated the ISR and increased Mtb replication. Correspondingly, the ISR inhibitor, ISRIB, reduced Mtb numbers in AM and improved Mtb control, overcoming the inflammatory phenotype. In conclusion, targeting the ISR offers a promising target for host-directed anti-TB therapy towards improved Mtb control and reduced immunopathology.
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