Tuberculosis is a chronic disease that displays several features commonly associated with biofilm-associated infections: immune system evasion, antibiotic treatment failures, and recurrence of ...infection. However, although Mycobacterium tuberculosis (Mtb) can form cellulose-containing biofilms in vitro, it remains unclear whether biofilms are formed during infection in vivo. Here, we demonstrate the formation of Mtb biofilms in animal models of infection and in patients, and that biofilm formation can contribute to drug tolerance. First, we show that cellulose is also a structural component of the extracellular matrix of in vitro biofilms of fast and slow-growing nontuberculous mycobacteria. Then, we use cellulose as a biomarker to detect Mtb biofilms in the lungs of experimentally infected mice and non-human primates, as well as in lung tissue sections obtained from patients with tuberculosis. Mtb strains defective in biofilm formation are attenuated for survival in mice, suggesting that biofilms protect bacilli from the host immune system. Furthermore, the administration of nebulized cellulase enhances the antimycobacterial activity of isoniazid and rifampicin in infected mice, supporting a role for biofilms in phenotypic drug tolerance. Our findings thus indicate that Mtb biofilms are relevant to human tuberculosis.
MicroRNAs (miRNAs) are short, noncoding RNA molecules that regulate the expression of a number of genes involved in cancer; therefore, they offer great diagnostic and therapeutic targets. We have ...developed doxorubicin-resistant and -sensitive metastatic human breast cancer cell lines (MDA-MB-231) to study the chemoresistant mechanisms regulated by miRNAs. We found that doxorubicin localized exclusively to the cytoplasm and was unable to reach the nuclei of resistant tumor cells because of the increased nuclear expression of MDR1/P-glycoprotein (P-gp). An miRNA array between doxorubicin-sensitive and -resistant breast cancer cells showed that reduced expression of miR-298 in doxorubicin-resistant human breast cancer cells was associated with increased expression of P-gp. In a transient transfection experiment, miR-298 directly bound to the MDR1 3′ untranslated region and regulated the expression of firefly luciferase reporter in a dose-dependent manner. Overexpression of miR-298 down-regulated P-gp expression, increasing nuclear accumulation of doxorubicin and cytotoxicity in doxorubicin-resistant breast cancer cells. Furthermore, down-regulation of miR-298 increased P-gp expression and induced doxorubicin resistance in sensitive breast cancer cells. In summary, these results suggest that miR-298 directly modulates P-gp expression and is associated with the chemoresistant mechanisms of metastatic human breast cancer. Therefore, miR-298 has diagnostic and therapeutic potential for predicting doxorubicin chemoresistance in human breast cancer.
Vaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease would benefit from validated small animal models. Here, we show that transgenic mice expressing human ...angiotensin-converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2 transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2 transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 6. K18 hACE2 transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.
Emergence of mutant SARS-CoV-2 strains associated with an increased risk of COVID-19-related death necessitates better understanding of the early viral dynamics, host responses and immunopathology. ...Single cell RNAseq (scRNAseq) allows for the study of individual cells, uncovering heterogeneous and variable responses to environment, infection and inflammation. While studies have reported immune profiling using scRNAseq in terminal human COVID-19 patients, performing longitudinal immune cell dynamics in humans is challenging. Macaques are a suitable model of SARS-CoV-2 infection. Our longitudinal scRNAseq of bronchoalveolar lavage (BAL) cell suspensions from young rhesus macaques infected with SARS-CoV-2 (n = 6) demonstrates dynamic changes in transcriptional landscape 3 days post- SARS-CoV-2-infection (3dpi; peak viremia), relative to 14-17dpi (recovery phase) and pre-infection (baseline) showing accumulation of distinct populations of both macrophages and T-lymphocytes expressing strong interferon-driven inflammatory gene signature at 3dpi. Type I interferon response is induced in the plasmacytoid dendritic cells with appearance of a distinct HLADR
CD68
CD163
SIGLEC1
macrophage population exhibiting higher angiotensin-converting enzyme 2 (ACE2) expression. These macrophages are significantly enriched in the lungs of macaques at 3dpi and harbor SARS-CoV-2 while expressing a strong interferon-driven innate anti-viral gene signature. The accumulation of these responses correlated with decline in viremia and recovery.
Tuberculosis (TB) in humans is characterized by formation of immune-rich granulomas in infected tissues, the architecture and composition of which are thought to affect disease outcome. However, our ...understanding of the spatial relationships that control human granulomas is limited. Here, we used multiplexed ion beam imaging by time of flight (MIBI-TOF) to image 37 proteins in tissues from patients with active TB. We constructed a comprehensive atlas that maps 19 cell subsets across 8 spatial microenvironments. This atlas shows an IFN-γ-depleted microenvironment enriched for TGF-β, regulatory T cells and IDO1
PD-L1
myeloid cells. In a further transcriptomic meta-analysis of peripheral blood from patients with TB, immunoregulatory trends mirror those identified by granuloma imaging. Notably, PD-L1 expression is associated with progression to active TB and treatment response. These data indicate that in TB granulomas, there are local spatially coordinated immunoregulatory programs with systemic manifestations that define active TB.
Development of an effective tuberculosis (TB) vaccine has suffered from an incomplete understanding of the correlates of protection against Mycobacterium tuberculosis (Mtb). Intravenous (i.v.) ...vaccination with Bacille Calmette-Guérin (BCG) provides nearly complete protection against TB in rhesus macaques, but the antibody response it elicits remains incompletely defined. Here we show that i.v. BCG drives superior antibody responses in the plasma and the lungs of rhesus macaques compared to traditional intradermal BCG administration. While i.v. BCG broadly expands antibody titers and functions, IgM titers in the plasma and lungs of immunized macaques are among the strongest markers of reduced bacterial burden. IgM was also enriched in macaques that received protective vaccination with an attenuated strain of Mtb. Finally, an Mtb-specific IgM monoclonal antibody reduced Mtb survival in vitro. Collectively, these data highlight the potential importance of IgM responses as a marker and mediator of protection against TB.
Smoking is known to be an added risk factor for tuberculosis (TB), with nearly a quarter of the TB cases attributed to cigarette smokers in the 22 countries with the highest TB burden. Many studies ...have indicated a link between risk of active TB and cigarette smoke. Smoking is also known to significantly decrease TB cure and treatment completion rate and increase mortality rates. Cigarette smoke contains thousands of volatile compounds including carcinogens, toxins, reactive solids, and oxidants in both particulate and gaseous phase. Yet, to date, limited studies have analyzed the impact of cigarette smoke components on
(
), the causative agent of TB. Here we report the impact of cigarette smoke condensate (CSC) on survival, mutation frequency, and gene expression of
. We show that exposure of virulent
to cigarette smoke increases the mutation frequency of the pathogen and strongly induces the expression of the regulon controlled by SigH-a global transcriptional regulator of oxidative stress. SigH has previously been shown to be required for
to respond to oxidative stress, survival, and granuloma formation
. A high-SigH expression phenotype is known to be associated with greater virulence of
. In patients with pulmonary TB who smoke, these changes may therefore play an important, yet unexplored, role in the treatment efficacy by potentially enhancing the virulence of tubercle bacilli.
Tuberculosis (TB), caused by
, remains a leading infectious disease killer worldwide with 1.4 million TB deaths in 2019. While the majority of infected population maintain an active control of the ...bacteria, a subset develops active disease leading to mortality. Effective T cell responses are critical to TB immunity with CD4
and CD8
T cells being key players of defense. These early cellular responses to TB infection have not yet been studied in-depth in either humans or preclinical animal models. Characterizing early T cell responses in a physiologically relevant preclinical model can provide valuable understanding of the factors that control disease development. We studied
-specific T cell responses in the lung compartment of rhesus macaques infected with either a low- or a high-dose of
CDC1551
aerosol. Relative to baseline, significantly higher
-specific CD4
IFN-γ
and TNF-
T cell responses were observed in the BAL of low dose infected macaques as early as week 1 post TB infection. The IFN-γ and TNF-
response was delayed to week 3 post infection in
-specific CD4
and CD8
T cells in the high dose group. The manifestation of earlier T cell responses in the group exposed to the lower
dose suggested a critical role of these cytokines in the antimycobacterial immune cascade, and specifically in the granuloma formation to contain the bacteria. However, a similar increase was not reflected in the CD4
and CD8
IL-17
T cells at week 1 post infection in the low dose group. This could be attributed to either a suppression of the IL-17 response or a lack of induction at this early stage of infection. On the contrary, there was a significantly higher IL-17
response in
-specific CD4
and CD8
T cells at week 3 in the high dose group. The results clearly demonstrate an early differentiation in the immunity following low dose and high dose infection, largely represented by differences in the IFN-γ and TNF-α response by
-specific T cells in the BAL. This early response to antigen expression by the bacteria could be critical for both bacterial growth control and bacterial containment.
The human-specific tropism of Human Immunodeficiency Virus Type 1 (HIV-1) has complicated the development of a macaque model of HIV-1 infection/AIDS that is suitable for preclinical evaluation of ...vaccines and novel treatment strategies. Several innate retroviral restriction factors, such as APOBEC3 family of proteins, TRIM5α, BST2, and SAMHD1, that prevent HIV-1 replication have been identified in macaque cells. Accessory proteins expressed by Simian Immunodeficiency virus (SIV) such as viral infectivity factor (Vif), viral protein X (Vpx), viral protein R (Vpr), and negative factor (Nef) have been shown to play key roles in overcoming these restriction factors in macaque cells. Thus, substituting HIV-1 accessory genes with those from SIV may enable HIV-1 replication in macaques. We and others have constructed macaque-tropic HIV-1 derivatives also called simian-tropic HIV-1 (stHIV-1) or Human-Simian Immunodeficiency Virus (HSIV) carrying SIV
vif
to overcome APOBEC3 family proteins. Additional modifications to HIV-1
gag
in some of the macaque-tropic HIV-1 have also been done to overcome TRIM5α restriction in rhesus and cynomolgus macaques. Although these viruses replicate persistently in macaque species, they do not result in CD4 depletion. Thus, these studies suggest that additional blocks to HIV-1 replication exist in macaques that prevent high-level viral replication. Furthermore, serial animal-to-animal passaging of macaque-tropic HIV-1
in vivo
has not resulted in pathogenic variants that cause AIDS in immunocompetent macaques. In this review, we discuss recent developments made toward developing macaque model of HIV-1 infection.
Mesenchymal stem cells (MSC) derived from bone marrow stem cells (BMSC) and adipose tissue stem cells (ASC) of humans and rhesus macaques were evaluated for their cell cycle properties during ...protracted culture in vitro. Human ASCs (hASC) and rhesus BMSCs (rBMSC) underwent significantly more total population doublings than human BMSCs (hBMSC) and rhesus ASCs (rASC). The cell cycle profile of all MSCs was altered as cultures aged. hMSCs underwent an increase in the frequency of cells in the S phase at P20 and P30. However, rhesus MSCs from both sources developed a distinct polyploid population of cells at P20, which progressed to aneuploidy by P30. Karyotype analysis of MSCs revealed the development of tetraploid or aneuploid karyotypes in the rhesus cells at P20 or P30. Analysis of the transcriptome of the MSCs from early and late passages revealed significant alterations in the patterns of gene expression (8.8% of the genes were differentially expressed in hBMSCs versus hASCs, and 5.5% in rBMSCs versus rASCs). Gene expression changes were much less evident within the same cell type as aging occurred (0.7% in hMSCs and 0.9% in rMSC). Gene ontology analysis showed that functions involved in protein catabolism and regulation of pol II transcription were overrepresented in rASCs, whereas the regulation of I kappa B/nuclear factor-kappaB cascade were overrepresented in hBMSCs. Functional analysis of genes that were differentially expressed in rASCs and hBMSCs revealed that pathways involved in cell cycle, cell cycle checkpoints, protein-ubiquitination, and apoptosis were altered.
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