Abstract
Mycobacterium tuberculosis (Mtb) infection is initiated in the distal airways, a site patrolled by alveolar macrophages (AM), and infection outcomes are governed by early immune events that ...remain poorly understood. We show that Mtb replicates almost exclusively within airway-resident AM during the first week after aerosol exposure. Although AM are typically thought to reside within the alveolar lumen, confocal imaging demonstrated that Mtb-infected AM establish a novel niche within the lung interstitium, where they undergo proliferation and physically associate with recruited monocyte-derived cells (MC). Localization of AM to the interstitium precedes subsequent Mtb uptake by MC and neutrophils and is driven by non-hematopoietic MyD88/IL-1R inflammasome signaling and the Mtb ESX-1 secretion system. The interstitial localization of infected AM occurs in the absence of recruited monocytes and neutrophils, suggesting that this pulmonary niche may result from AM-intrinsic egress from the alveolar space rather than inflammation-driven alveolar consolidation. Comparisons of the transcriptomes of infected AM localized to the airway or interstitium by RNA-sequencing revealed unique transcriptional profiles in the two populations, suggesting that these two AM subsets may be functionally distinct. For example, Mtb-infected AM in the interstitium dramatically upregulated interferon-response genes, lending support to the idea that these cells are in unique lung environments. Thus, crosstalk between Mtb-infected AM and non-hematopoietic cells establishes pulmonary Mtb infection by promoting the translocation of infected cells from the alveoli to lung interstitium, facilitating dissemination to other myeloid subsets.
Growing evidence suggests the outcome of
Mycobacterium tuberculosis
(Mtb) infection is established rapidly after exposure, but how the current tuberculosis vaccine, BCG, impacts early immunity is ...poorly understood. Here we found that murine BCG immunization promotes a dramatic shift in infected cell types. While alveolar macrophages (AM) are the major infected cell for the first two 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 antigen presented by infected AM. Mtb-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 co-localization of T cells and infected cells. Understanding how BCG alters early immune responses to Mtb provides new avenues to improve upon the immunity it confers.
Keywords Mycobacterium tuberculosis; adaptive immunity; IFN?; granuloma; quantitative imaging; TGFbeta; immune suppression; lung inflammation; immune cell trafficking; T cell function Highlights * ...Despite antigen sensing, CD4 T cells produce minimal IFN? in pulmonary Mtb granulomas * Granuloma-localized TGFbeta sensing by CD4 T cells rapidly limits their effector function * TGFbeta also limits the expansion and survival of terminally differentiated CD4 T cells * Ablation of T cell TGFbeta sensing improves responses and lowers bacterial burdens Summary CD4 T cell effector function is required for optimal containment of Mycobacterium tuberculosis (Mtb) infection. IFN? 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 IFN?, and IFN? plays a lesser protective role within the lung than at sites of Mtb dissemination. In a murine infection model, we observed that IFN? 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 TGFbeta signaling within granuloma-infiltrating T cells in both mice and rhesus macaques. Selective blockade of TGFbeta signaling in T cells resulted in an accumulation of terminally differentiated effector CD4 T cells, improved IFN? 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. Author Affiliation: (1) Center for Global Infectious Disease Research, Seattle Children's Research Institute, Seattle, WA 98109, USA (2) Department of Pediatrics, University of Washington, Seattle, WA 98195, USA (3) Department of Immunology, University of Washington, Seattle, WA 98109, USA (4) Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR 97006, USA * Corresponding author Article History: Received 6 February 2020; Revised 2 December 2020; Accepted 22 January 2021 (miscellaneous) Published: March 11, 2021 (footnote)5 These authors contributed equally (footnote)6 Lead contact Byline: Benjamin H. Gern (1,2,5), Kristin N. Adams (1,5), Courtney R. Plumlee (1), Caleb R. Stoltzfus (3), Laila Shehata (3), Albanus O. Moguche (3), Kathleen Busman-Sahay (4), Scott G. Hansen (4), Michael K. Axthelm (4), Louis J. Picker (4), Jacob D. Estes (4), Kevin B. Urdahl kevin.urdahl@seattlechildrens.org (1,2,3,6,*), Michael Y. Gerner gernermy@uw.edu (3,**)
Abstract
Tuberculosis (TB) is a highly heterogeneous disease that develops in a subset of individuals infected with aerosolized Mycobacterium tuberculosis (Mtb). To improve upon the standard ...experimental mouse model, we infected mice with an ultra-low Mtb dose (ULD), consisting of 1-3 founding bacteria, which reflects the physiologic inoculum of humans. These mice exhibited a broad range of outcomes, with bacterial burdens ranging from <10 to ~106 CFUs in individual lungs. Furthermore, they exhibited well-circumscribed granulomas that share features with human granulomas. To monitor outcomes in live mice, we identified a blood RNA signature that correlated with lung bacterial burdens. Remarkably, this mouse-derived signature predicted Mtb infection outcomes across species, including risk of progression to active TB in humans. Given these improvements in the mouse model, we wondered whether ULD infection would provide a better platform for assessing vaccine-induced immunity. We now report that BCG-immunized mice, compared to unimmunized controls, exhibited a lower percentage of infected mice at late timepoints after ULD Mtb challenge. Interestingly, this difference was not observed at earlier timepoints. Taken together, these results suggest that some BCG-immunized mice, upon subsequent Mtb aerosol challenge, are capable of eradicating their initial infection, a finding that has not previously been observed in the TB mouse model. We are currently developing methods to verify initial infection prior to potential clearance and are also investigating immune correlates of protection.
Abstract
Tuberculosis (TB) is a highly heterogeneous human disease that develops in a subset of individuals who inhale Mycobacterium tuberculosis (Mtb). Using advanced machine-learning algorithms, we ...discovered a blood transcriptional signature that identifies Mtb-exposed individuals in the process of progressing to active TB up to 18 months before they exhibit clinical symptoms. This signature may help prevent TB disease by identifying individuals who could benefit from early intervention. However, it fails to identify ~30% of progressors and does not reveal insights into the diverse outcome-governing pathways within the infected lung that underlie TB progression. To develop a tractable system that enables blood-based signatures to be linked to mechanistic pathways within the lung, we pioneered a novel murine TB model that recapitulates key aspects of human Mtb infection. We find that mice infected with a physiologic, ultra-low dose (i.e., 1–3 CFUs) of aerosolized Mtb exhibit a broad range of outcomes, with bacterial burdens ranging from less than 10 to ~106 CFUs within individual lungs. Mice that contain Mtb exhibit well-circumscribed granulomatous structures that share many features with human Mtb granulomas. In addition, we have identified a blood transcriptional signature that distinguishes “controller” and “progressor” mice. Remarkably, this mouse-derived signature is equally as effective as our previously identified human-derived signature at predicting TB risk in humans, confirming the model’s relevance to human disease. This finding enables us to use the tools of the tractable mouse system to address questions central to TB pathogenesis in a clinically relevant model.
Abstract
Background
CD4 T-cell production of IFNɣ is essential to prevent dissemination of pulmonary Mtb, though is less effective at controlling infection within the lung. Because T cells are most ...effective in the context of direct interactions with Mtb-infected cells, we sought to determine the location of CD4 T-cell antigen sensing and IFNɣ production within granulomas.
Methods
We used a murine ultra-low-dose aerosol infection, developed by our lab, where most mice are infected by a single bacillus, resulting in a solitary granuloma that is more similar to human granulomas. We examined the lungs of wild-type mice 35 days later for patterns of T-cell activation with quantitative confocal imaging. This analysis was next performed following adoptive co-transfer of Th1 polarized Mtb-specific cells and control cells of irrelevant specificity (OVA). To determine the effect of TGFβ on IFNɣ production, we examined mice that lack the TGFβR on T cells (TGFβR.KO). Finally, we examined whether this effect was Th1 cell-intrinsic with an adoptive transfer of Th1-polarized Mtb-specific cells with (Tg.WT) and without (Tg.KO) the TGFβR.
Results
Despite many CD4 T cells localizing to the granuloma and undergoing T-cell receptor (TCR) activation (pS6), IFNɣ production was not significantly increased in the granuloma compared with a distal lung site (Figure 1). This pattern of localization and activation was found to be similar in the Mtb-specific (but not control) Th1 cells (Figure 2). In contrast, CD4 T cells from TGFβR.KO mice produced more IFNɣ within the granuloma (Figure 3). Lastly, Tg.KO cells transferred into wild-type mice produced increased IFNɣ compared with Tg.WT, mirroring the findings in TGFβR.KO mice (Figure 4).
Conclusion
CD4 T cell production of IFNɣ is decreased within the granuloma, where it can be most effective, despite evidence of ongoing TCR stimulation in Mtb-specific cells. We have shown that by alleviating the effects of TGFβ signaling, even terminally-differentiated Mtb-specific Th1 cells can produce more IFNɣ within the granuloma. While this modest increase suggests that there are additional mechanisms at play which warrant further exploration, these findings have the potential to guide immunotherapeutic development, especially given that TGFβ inhibitors are already in phase III clinical trials for other purposes.
Disclosures
All Authors: No reported Disclosures.
Asthma is a descriptive label for an obstructive, inflammatory disease in the lower airways manifesting with symptoms including breathlessness, cough, difficulty in breathing and wheezing. From a ...clinician's point of view, asthma symptoms can commence at any age although most asthma patients - regardless of their age of onset - seem to have had some form of airway problems during childhood. Asthma inception and related pathophysiologic processes are therefore very likely to occur early in life, further evidenced by recent lung physiologic and mechanistic research. Herein, we present state-of-the-art updates on the role of genetics and epigenetics, early viral and bacterial infections, immune response and pathophysiology as well as lifestyle and environmental exposures in asthma across the life-course. We conclude early environmental insults in genetically vulnerable individuals to induce an abnormal, pre-asthmatic airway response as key events in asthma inception and highlight disease heterogeneity - across ages - and the potential shortness of treating all patients with asthma using the same treatments. Although there are no interventions that, at present, can modify long-term outcomes, a precision-medicine approach should be implemented to optimize treatment and tailor follow-up for all patients with asthma.Asthma is a descriptive label for an obstructive, inflammatory disease in the lower airways manifesting with symptoms including breathlessness, cough, difficulty in breathing and wheezing. From a clinician's point of view, asthma symptoms can commence at any age although most asthma patients - regardless of their age of onset - seem to have had some form of airway problems during childhood. Asthma inception and related pathophysiologic processes are therefore very likely to occur early in life, further evidenced by recent lung physiologic and mechanistic research. Herein, we present state-of-the-art updates on the role of genetics and epigenetics, early viral and bacterial infections, immune response and pathophysiology as well as lifestyle and environmental exposures in asthma across the life-course. We conclude early environmental insults in genetically vulnerable individuals to induce an abnormal, pre-asthmatic airway response as key events in asthma inception and highlight disease heterogeneity - across ages - and the potential shortness of treating all patients with asthma using the same treatments. Although there are no interventions that, at present, can modify long-term outcomes, a precision-medicine approach should be implemented to optimize treatment and tailor follow-up for all patients with asthma.
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