Antibiotic treatment modestly reduces protection against Mycobacterium tuberculosis reinfection in macaques.

Concomitant immunity is generally defined as an ongoing infection providing protection against reinfection . Its role in prevention of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is supported by epidemiological evidence in humans as well as experimental evidence in mice and non-human primates (NHPs). Whether the presence of live Mtb, rather than simply persistent antigen, is necessary for concomitant immunity in TB is still unclear. Here, we investigated whether live Mtb plays a measurable role in control of secondary Mtb infection. Using cynomolgus macaques, molecularly barcoded Mtb libraries, positron emission tomography-computed tomography (PET CT) imaging, flow cytometry, and cytokine profiling, we evaluated the effect of antibiotic treatment after primary infection on immunological response and bacterial establishment, dissemination, and burden post-secondary infection. Our data provide evidence that, in this experimental model, treatment with antibiotics after primary infection reduced inflammation in the lung but was not associated with a significant change in bacterial establishment, dissemination, or burden in the lung or lymph nodes. Nonetheless, treatment of the prior infection with antibiotics did result in a modest reduction in protection against reinfection: none of the seven antibiotic-treated animals demonstrated sterilizing immunity against reinfection, while four of the seven non-treated macaques were completely protected against reinfection. These findings support that antibiotic-treated animals were still able to restrict bacterial establishment and dissemination after rechallenge compared to naïve macaques, but not to the full extent of non-antibiotic-treated macaques.

Antibiotic treatment modestly reduces protection against Mycobacterium tuberculosis reinfection in macaques.

Concomitant immunity is generally defined as an ongoing infection providing protection against reinfection1. Its role in prevention of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is supported by epidemiological evidence in humans as well as experimental evidence in mice and non-human primates (NHPs). Whether the presence of live Mtb, rather than simply persistent antigen, is necessary for concomitant immunity in TB is still unclear. Here, we investigated whether live Mtb plays a measurable role in control of secondary Mtb infection. Using cynomolgus macaques, molecularly barcoded Mtb libraries, PET-CT imaging, flow cytometry and cytokine profiling we evaluated the effect of antibiotic treatment after primary infection on immunological response and bacterial establishment, dissemination, and burden post-secondary infection. Our data provide evidence that, in this experimental model, treatment with antibiotics after primary infection reduced inflammation in the lung but was not associated with a significant change in bacterial establishment, dissemination or burden in the lung or lymph nodes. Nonetheless, treatment of the prior infection with antibiotics did result in a modest reduction in protection against reinfection: none of the 7 antibiotic treated animals demonstrated sterilizing immunity against reinfection while 4 of the 7 non-treated macaques were completely protected against reinfection. These findings support that antibiotic-treated animals were still able to restrict bacterial establishment and dissemination after rechallenge compared to naïve macaques, but not to the full extent of non-antibiotic treated macaques.

Development of an Engineered Mycobacterium tuberculosis Strain for a Safe and Effective Tuberculosis Human Challenge Model.

Human challenge experiments could greatly accelerate the development of a tuberculosis (TB) vaccine. Human challenge for tuberculosis requires a strain that can both replicate in the host and be reliably cleared. To accomplish this, we designed Mycobacterium tuberculosis (Mtb) strains featuring up to three orthogonal kill switches, tightly regulated by exogenous tetracyclines and trimethoprim. The resultant strains displayed immunogenicity and antibiotic susceptibility similar to wild-type Mtb under permissive conditions. In the absence of supplementary exogenous compounds, the strains were rapidly killed in axenic culture, mice and nonhuman primates. Notably, the strain that contained three kill switches had an escape rate of less than 10 -10 per genome per generation and displayed no relapse in a SCID mouse model. Collectively, these findings suggest that this engineered Mtb strain could be a safe and effective candidate for a human challenge model.

CD4+ T cells are homeostatic regulators during Mtb reinfection.

Immunological priming - either in the context of prior infection or vaccination - elicits protective responses against subsequent Mycobacterium tuberculosis (Mtb) infection. However, the changes that occur in the lung cellular milieu post-primary Mtb infection and their contributions to protection upon reinfection remain poorly understood. Here, using clinical and microbiological endpoints in a non-human primate reinfection model, we demonstrate that prior Mtb infection elicits a long-lasting protective response against subsequent Mtb exposure and that the depletion of CD4+ T cells prior to Mtb rechallenge significantly abrogates this protection. Leveraging microbiologic, PET-CT, flow cytometric, and single-cell RNA-seq data from primary infection, reinfection, and reinfection-CD4+ T cell depleted granulomas, we identify differential cellular and microbial features of control. The data collectively demonstrate that the presence of CD4+ T cells in the setting of reinfection results in a reduced inflammatory lung milieu characterized by reprogrammed CD8+ T cell activity, reduced neutrophilia, and blunted type-1 immune signaling among myeloid cells, mitigating Mtb disease severity. These results open avenues for developing vaccines and therapeutics that not only target CD4+ and CD8+ T cells, but also modulate innate immune cells to limit Mtb disease.

YAP regulates cell size and growth dynamics via non-cell autonomous mediators.

The Hippo pathway regulates organ size, regeneration, and cell growth by controlling the stability of the transcription factor, YAP (Yorkie in Drosophila). When there is tissue damage, YAP is activated allowing the restoration of homeostatic tissue size. The exact signals by which YAP is activated are still not fully understood, but its activation is known to affect both cell size and cell number. Here we used cultured cells to examine the coordinated regulation of cell size and number under the control of YAP. Our experiments in isogenic HEK293 cells reveal that YAP can affect cell size and number by independent circuits. Some of these effects are cell autonomous, such as proliferation, while others are mediated by secreted signals. In particular CYR61, a known secreted YAP target, is a non-cell autonomous mediator of cell survival, while another unidentified secreted factor controls cell size.

PKC-mediated inhibitory feedback of the cholecystokinin 1 receptor controls the shape of oscillatory Ca²âº signals.

Translation of extracellular hormonal input into cellular responses is often mediated by repetitive increases in cytosolic free Ca(2+) concentration ([Ca(2+) ]c ). Amplitude, duration and frequency of these so-called [Ca(2+) ]c oscillations then carry information about the nature and concentration of the extracellular signalling molecule. At present, there are different hypotheses concerning the induction and control of these oscillations. Here, we investigated the role of agonist-induced receptor phosphorylation in this process using Chinese hamster ovary cells stably expressing a variant of the cholecystokinin 1 receptor (CCK1R) lacking the four consensus sites for protein kinase C (PKC) phosphorylation and deficient in CCK-induced receptor phosphorylation (CCK1R-mt cells). In the presence of cholecystokinin-(26-33)-peptide amide (CCK-8), these cells displayed Ca(2+) oscillations with a much more pronounced bursting dynamics rather than the dominant spiking dynamics observed in Chinese hamster ovary cells stably expressing the wild-type CCK1R. The bursting behaviour returned to predominantly spiking behaviour following removal of extracellular Ca(2+) , suggesting that CCK-8-induced, PKC-mediated CCK1R phosphorylation inhibits Ca(2+) influx across the plasma membrane. To gain mechanistic insight into the underlying mechanism we developed a mathematical model able to reproduce the experimental observations. From the model we conclude that binding of CCK-8 to the CCK1R leads to activation of PKC which subsequently phosphorylates the receptor to inhibit the receptor-mediated influx of Ca(2+) across the plasma membrane. Receptor-specific differences in this feedback mechanism may, at least in part, explain the observation that different agonists evoke [Ca(2+) ]c oscillations with different kinetics in the same cell type.