Group A Streptococcus induces lysosomal dysfunction in THP-1 macrophages.

The human-specific bacterial pathogen group A Streptococcus (GAS) is a significant cause of morbidity and mortality. Macrophages are important to control GAS infection, but previous data indicate that GAS can persist in macrophages. In this study, we detail the molecular mechanisms by which GAS survives in THP-1 macrophages. Our fluorescence microscopy studies demonstrate that GAS is readily phagocytosed by macrophages, but persists within phagolysosomes. These phagolysosomes are not acidified, which is in agreement with our findings that GAS cannot survive in low pH environments. We find that the secreted pore-forming toxin Streptolysin O (SLO) perforates the phagolysosomal membrane, allowing leakage of not only protons but also large proteins including the lysosomal protease cathepsin B. Additionally, GAS recruits CD63/LAMP-3, which may contribute to lysosomal permeabilization, especially in the absence of SLO. Thus, although GAS does not inhibit fusion of the lysosome with the phagosome, it has multiple mechanisms to prevent proper phagolysosome function, allowing for persistence of the bacteria within the macrophage. This has important implications for not only the initial response but also the overall functionality of the macrophages, which may lead to the resulting pathologies in GAS infection. Our data suggest that therapies aimed at improving macrophage function may positively impact patient outcomes in GAS infection.

Virus specificity and nucleoporin requirements for MX2 activity are affected by GTPase function and capsid-CypA interactions.

Human myxovirus resistance 2 (MX2/MXB) is an interferon-induced GTPase that inhibits human immunodeficiency virus-1 (HIV-1) infection by preventing nuclear import of the viral preintegration complex. The HIV-1 capsid (CA) is the major viral determinant for sensitivity to MX2, and complex interactions between MX2, CA, nucleoporins (Nups), cyclophilin A (CypA), and other cellular proteins influence the outcome of viral infection. To explore the interactions between MX2, the viral CA, and CypA, we utilized a CRISPR-Cas9/AAV approach to generate CypA knock-out cell lines as well as cells that express CypA from its endogenous locus, but with specific point mutations that would abrogate CA binding but should not affect enzymatic activity or cellular function. We found that infection of CypA knock-out and point mutant cell lines with wild-type HIV-1 and CA mutants recapitulated the phenotypes observed upon cyclosporine A (CsA) addition, indicating that effects of CsA treatment are the direct result of blocking CA-CypA interactions and are therefore independent from potential interactions between CypA and MX2 or other cellular proteins. Notably, abrogation of GTP hydrolysis by MX2 conferred enhanced antiviral activity when CA-CypA interactions were abolished, and this effect was not mediated by the CA-binding residues in the GTPase domain, or by phosphorylation of MX2 at position T151. We additionally found that elimination of GTPase activity also altered the Nup requirements for MX2 activity. Our data demonstrate that the antiviral activity of MX2 is affected by CypA-CA interactions in a virus-specific and GTPase activity-dependent manner. These findings further highlight the importance of the GTPase domain of MX2 in regulation of substrate specificity and interaction with nucleocytoplasmic trafficking pathways.

Virus specificity and nucleoporin requirements for MX2 activity are affected by GTPase function and capsid-CypA interactions.

Human myxovirus resistance 2 (MX2/MXB) is an interferon-induced GTPase that inhibits human immunodeficiency virus-1 (HIV-1) infection by preventing nuclear import of the viral preintegration complex. The HIV-1 capsid (CA) is the major viral determinant for sensitivity to MX2, and complex interactions between MX2, CA, nucleoporins (Nups), cyclophilin A (CypA), and other cellular proteins influence the outcome of viral infection. To explore the interactions between MX2, the viral CA, and CypA, we utilized a CRISPR-Cas9/AAV approach to generate CypA knock-out cell lines as well as cells that express CypA from its endogenous locus, but with specific point mutations that would abrogate CA binding but should not affect enzymatic activity or cellular function. We found that infection of CypA knock-out and point mutant cell lines with wild-type HIV-1 and CA mutants recapitulated the phenotypes observed upon cyclosporine A (CsA) addition, indicating that effects of CsA treatment are the direct result of blocking CA-CypA interactions and are therefore independent from potential interactions between CypA and MX2 or other cellular proteins. Notably, abrogation of GTP hydrolysis by MX2 conferred enhanced antiviral activity when CA-CypA interactions were abolished, and this effect was not mediated by the CA-binding residues in the GTPase domain, or by phosphorylation of MX2 at position T151. We additionally found that elimination of GTPase activity also altered the Nup requirements for MX2 activity. Our data demonstrate that the antiviral activity of MX2 is affected by CypA-CA interactions in a virus-specific and GTPase activity-dependent manner. These findings further highlight the importance of the GTPase domain of MX2 in regulation of substrate specificity and interaction with nucleocytoplasmic trafficking pathways.

Insights into the role of HIV-1 Vpu in modulation of NF-ĸB signaling pathways.

HIV-1 inhibits the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) to prevent the induction of a proinflammatory state but also activates the NF-κB pathway to promote viral transcription. Thus, optimal regulation of this pathway is important for the viral life cycle. In recent work, Pickering et al. (3) demonstrate that HIV-1 viral protein U has contrasting effects on the two distinct paralogs of ß-transducin repeat-containing protein (ß-TrCP1 and ß-TrCP2) and that this interaction has important implications for the regulation of both the canonical and non-canonical NF-κB pathways. Additionally, the authors identified the viral requirements for the dysregulation of ß-TrCP. In this commentary, we discuss how these findings further our understanding of how the NF-κB pathway functions during viral infection.

Genetic Differences between 129S Substrains Affect Antiretroviral Immune Responses.

Inbred mouse lines vary in their ability to mount protective antiretroviral immune responses, and even closely related strains can exhibit opposing phenotypes upon retroviral infection. Here, we found that 129S mice inherit a previously unknown mechanism for the production of anti-murine leukemia virus (MLV) antibodies and control of infection. The resistant phenotype in 129S1 mice is controlled by two dominant loci that are independent from known MLV resistance genes. We also show that production of anti-MLV antibodies in 129S7 mice, but not 129S1 mice, is independent of interferon gamma signaling. Thus, our data indicate that 129S mice inherit an unknown mechanism for control of MLV infection and demonstrate that there is genetic variability in 129S substrains that affects their ability to mount antiviral immune responses. IMPORTANCE Understanding the genetic basis for production of protective antiviral immune responses is crucial for the development of novel vaccines and adjuvants. Additionally, characterizing the genetic and phenotypic variability in inbred mice has implications for the selection of strains for targeted mutagenesis, choice of controls, and for broader understanding of the requirements for protective immunity.

Spatial distribution of B cells and lymphocyte clusters as a predictor of triple-negative breast cancer outcome.

While tumor infiltration by CD8+ T cells is now widely accepted to predict outcomes, the clinical significance of intratumoral B cells is less clear. We hypothesized that spatial distribution rather than density of B cells within tumors may provide prognostic significance. We developed statistical techniques (fractal dimension differences and a box-counting method 'occupancy') to analyze the spatial distribution of tumor-infiltrating lymphocytes (TILs) in human triple-negative breast cancer (TNBC). Our results indicate that B cells in good outcome tumors (no recurrence within 5 years) are spatially dispersed, while B cells in poor outcome tumors (recurrence within 3 years) are more confined. While most TILs are located within the stroma, increased numbers of spatially dispersed lymphocytes within cancer cell islands are associated with a good prognosis. B cells and T cells often form lymphocyte clusters (LCs) identified via density-based clustering. LCs consist either of T cells only or heterotypic mixtures of B and T cells. Pure B cell LCs were negligible in number. Compared to tertiary lymphoid structures (TLS), LCs have fewer lymphocytes at lower densities. Both types of LCs are more abundant and more spatially dispersed in good outcomes compared to poor outcome tumors. Heterotypic LCs in good outcome tumors are smaller and more numerous compared to poor outcome. Heterotypic LCs are also closer to cancer islands in a good outcome, with LC size decreasing as they get closer to cancer cell islands. These results illuminate the significance of the spatial distribution of B cells and LCs within tumors.

Physics approaches to the spatial distribution of immune cells in tumors.

The goal of immunotherapy is to mobilize the immune system to kill cancer cells. Immunotherapy is more effective and, in general, the prognosis is better, when more immune cells infiltrate the tumor. We explore the question of whether the spatial distribution rather than just the density of immune cells in the tumor is important in forecasting whether cancer recurs. After reviewing previous work on this issue, we introduce a novel application of maximum entropy to quantify the spatial distribution of discrete point-like objects. We apply our approach to B and T cells in images of tumor tissue taken from triple negative breast cancer patients. We find that the immune cells are more spatially dispersed in good clinical outcome (no recurrence of cancer within at least 5 years of diagnosis) compared to poor clinical outcome (recurrence within 3 years of diagnosis). Our results highlight the importance of spatial distribution of immune cells within tumors with regard to clinical outcome, and raise new questions on their role in cancer recurrence.