Remodeling the bladder tumor immune microenvironment by mycobacterial species with changes in their cell envelope composition.

Intravesical BCG instillation after bladder tumor resection is the standard treatment for non-muscle invasive bladder cancer; however, it is not always effective and frequently has undesirable side effects. Therefore, new strategies that improve the clinical management of patients are urgently needed. This study aimed to comprehensively evaluate the bladder tumor immune microenvironment profile after intravesical treatment with a panel of mycobacteria with variation in their cell envelope composition and its impact on survival using an orthotopic murine model to identify more effective and safer therapeutic strategies. tumor-bearing mice were intravesically treated with a panel of BCG and M. brumae cultured under different conditions. Untreated tumor-bearing mice and healthy mice were also included as controls. After mycobacterial treatments, the infiltrating immune cell populations in the bladder were analysed by flow cytometry. We provide evidence that mycobacterial treatment triggered a strong immune infiltration into the bladder, with BCG inducing higher global absolute infiltration than M. brumae. The induced global immune microenvironment was strikingly different between the two mycobacterial species, affecting both innate and adaptive immunity. Compared with M. brumae, BCG treated mice exhibited a more robust infiltration of CD4+ and CD8+ T-cells skewed toward an effector memory phenotype, with higher frequencies of NKT cells, neutrophils/gMDSCs and monocytes, especially the inflammatory subset, and higher CD4+ TEM/CD4+ Treg and CD8+ TEM/CD4+ Treg ratios. Conversely, M. brumae treatment triggered higher proportions of total activated immune cells and activated CD4+ and CD8+ TEM cells and lower ratios of CD4+ TEM cells/CD4+ Tregs, CD8+ TEM cells/CD4+ Tregs and inflammatory/reparative monocytes. Notably, the mycobacterial cell envelope composition in M. brumae had a strong impact on the immune microenvironment, shaping the B and myeloid cell compartment and T-cell maturation profile and thus improving survival. Overall, we demonstrate that the bladder immune microenvironment induced by mycobacterial treatment is species specific and shaped by mycobacterial cell envelope composition. Therefore, the global bladder immune microenvironment can be remodelled, improving the quality of infiltrating immune cells, the balance between inflammatory and regulatory/suppressive responses and increasing survival.

Modulation of the autophagic pathway inhibits HIV-1 infection in human lymphoid tissue cultured ex vivo.

A complex link exists between HIV-1 and autophagy, and discordant results have been reported in different in vitro models regarding the way HIV and autophagy modulate each other. Despite this, there is very limited knowledge about the interplay between HIV and autophagy in vivo in lymphoid tissue, due in part by the lack of cell models that recapitulate the in vivo setting. Here, we evaluate the interrelationship between HIV and autophagy using human ex vivo lymphoid tissue cultures as an HIV infection model. Our results showed that human lymphoid aggregated cultures (HLACs) from tonsillar tissue displayed fully functional autophagic activity. In this system, HIV infection resulted in an increase in autophagy. Notably, we observed that both, autophagy-enhancing (rapamycin) or blocking drugs (3-methyladenine, chloroquine and bafilomycin), were able to decrease HIV-DNA levels and HIV replication. Therefore, efficient HIV-1 replication requires a fine-tuned level of autophagy, so modifications of this balance will have a negative impact on its replication. Therefore, targeting the autophagic pathway could be a new therapeutic approach to be explored to treat HIV-1 infection. Ex vivo cultures of human lymphoid tissue are a suitable model to obtain further insights into HIV and its intricate relationship with autophagy.

Mycobacterial surface characters remodeled by growth conditions drive different tumor-infiltrating cells and systemic IFN-γ/IL-17 release in bladder cancer treatment.

The mechanism of action of intravesical Mycobacterium bovis BCG immunotherapy treatment for bladder cancer is not completely known, leading to misinterpretation of BCG-unresponsive patients, who have scarce further therapeutic options. BCG is grown under diverse culture conditions worldwide, which can impact the antitumor effect of BCG strains and could be a key parameter of treatment success. Here, BCG and the nonpathogenic Mycobacterium brumae were grown in four culture media currently used by research laboratories and BCG manufacturers: Sauton-A60, -G15 and -G60 and Middlebrook 7H10, and used as therapies in the orthotopic murine BC model. Our data reveal that each mycobacterium requires specific culture conditions to induce an effective antitumor response. since higher survival rates of tumor-bearing mice were achieved using M. brumae-A60 and BCG-G15 than the rest of the treatments. M. brumae-A60 was the most efficacious among all tested treatments in terms of mouse survival, cytotoxic activity of splenocytes against tumor cells, higher systemic production of IL-17 and IFN-É£, and bladder infiltration of selected immune cells such as ILCs and CD4TEM. BCG-G15 triggered an antitumor activity based on a massive infiltration of immune cells, mainly CD3+ (CD4+ and CD8+) T cells, together with high systemic IFN-É£ release. Finally, a reduced variety of lipids was strikingly observed in the outermost layer of M. brumae-A60 and BCG-G15 compared to the rest of the cultures, suggesting an influence on the antitumor immune response triggered. These findings contribute to understand how mycobacteria create an adequate niche to help the host subvert immunosuppressive tumor actions.

SARS-CoV-2 Infection Modulates ACE2 Function and Subsequent Inflammatory Responses in Swabs and Plasma of COVID-19 Patients.

Angiotensin converting enzyme 2 (ACE2) is a host ectopeptidase and the receptor for the SARS-CoV-2 virus, albeit virus-ACE2 interaction goes far beyond viral entry into target cells. Controversial data exists linking viral infection to changes in ACE2 expression and function, which might influence the subsequent induction of an inflammatory response. Here, we tested the significance of soluble ACE2 enzymatic activity longitudinally in nasopharyngeal swabs and plasma samples of SARS-CoV-2 infected patients, along with the induction of inflammatory cytokines. Release of soluble functional ACE2 increases upon SARS-CoV-2 infection in swabs and plasma of infected patients, albeit rapidly decreasing during infection course in parallel with ACE2 gene expression. Similarly, SARS-CoV-2 infection also induced the expression of inflammatory cytokines. These changes positively correlated with the viral load. Overall, our results demonstrate the existence of mechanisms by which SARS-CoV-2 modulates ACE2 expression and function, intracellular viral sensing and subsequent inflammatory response, offering new insights into ACE2 dynamics in the human upper respiratory tract and pointing towards soluble ACE2 levels as a putative early biomarker of infection severity.

Kidney cancer PDOXs reveal patient-specific pro-malignant effects of antiangiogenics and its molecular traits.

An open debate in antiangiogenic therapies is about their consequence on tumor invasiveness and metastasis, which is undoubtedly relevant for patients currently treated with antiangiogenics, such as renal cell carcinoma patients. To address, this we developed an extensive series of 27 patient biopsy-derived orthotopic xenograft models (Ren-PDOX) that represent inter-patient heterogeneity. In specific tumors, antiangiogenics produced increased invasiveness and metastatic dissemination, while in others aggressiveness remained unchanged. Mechanistically, species-discriminative RNA sequencing identified a tumor cell-specific differential expression profile associated with tumor progression and aggressivity in TCGA RCC patients. Gene filtering using an invasion-annotated patient series pinpointed two candidate genes, of which ALDH1A3 differentiated the pro-invasive subtype of Ren-PDOXs. Validation in an independent series of 15 antiangiogenic-treated patients confirmed that pre-treatment ALDH1A3 can significantly discriminate patients with pro-aggressive response upon treatment. Overall, results confirm that effects of antiangiogenic drugs on tumor invasion and metastasis are heterogeneous and may profoundly affect the natural progression of tumors and promote malignancy. Furthermore, we identify a specific molecular biomarker that could be used to select patients that better benefit from treatment.

Analysis of Runx1 Using Induced Gene Ablation Reveals Its Essential Role in Pre-liver HSC Development and Limitations of an In Vivo Approach.

Hematopoietic stem cells (HSCs) develop in the embryonic aorta-gonad-mesonephros (AGM) region and subsequently relocate to fetal liver. Runx1 transcription factor is essential for HSC development, but is largely dispensable for adult HSCs. Here, we studied tamoxifen-inducible Runx1 inactivation in vivo. Induction at pre-liver stages (up to embryonic day 10.5) reduced erythromyeloid progenitor numbers, but surprisingly did not block the appearance of Runx1-null HSCs in liver. By contrast, ex vivo analysis showed an absolute Runx1 dependency of HSC development in the AGM region. We found that, contrary to current beliefs, significant Cre-inducing tamoxifen activity persists in mouse blood for at least 72 hr after injection. This deferred recombination can hit healthy HSCs, which escaped early Runx1 ablation and result in appearance of Runx1-null HSCs in liver. Such extended recombination activity in vivo is a potential source of misinterpretation, particularly in analysis of dynamic developmental processes during embryogenesis.

Tracing the origin of the HSC hierarchy reveals an SCF-dependent, IL-3-independent CD43(-) embryonic precursor.

Definitive hematopoietic stem cells (HSCs) develop in the aorta gonad mesonephros (AGM) region in a stepwise manner. Type I pre-HSCs express CD41 but lack CD45 expression, which is subsequently upregulated in type II pre-HSCs prior to their maturation into definitive HSCs. Here, using ex vivo modeling of HSC development, we identify precursors of definitive HSCs in the trunk of the embryonic day 9.5 (E9.5) mouse embryo. These precursors, termed here pro-HSCs, are less mature than type I and II pre-HSCs. Although pro-HSCs are CD41(+), they lack the CD43 marker, which is gradually upregulated in the developing HSC lineage. We show that stem cell factor (SCF), but not interleukin-3 (IL-3), is a major effector of HSC maturation during E9-E10. This study extends further the previously established hierarchical organization of the developing HSC lineage and presents it as a differentially regulated four-step process and identifies additional targets that could facilitate the generation of transplantable HSCs from pluripotent cells for clinical needs.

Hierarchical organization and early hematopoietic specification of the developing HSC lineage in the AGM region.

The aorta-gonad-mesonephros region plays an important role in hematopoietic stem cell (HSC) development during mouse embryogenesis. The vascular endothelial cadherin⁺ CD45⁺ (VE-cad⁺CD45⁺) population contains the major type of immature pre-HSCs capable of developing into long-term repopulating definitive HSCs. In this study, we developed a new coaggregation culture system, which supports maturation of a novel population of CD45-negative (VE-cad⁺CD45⁻CD41⁺) pre-HSCs into definitive HSCs. The appearance of these pre-HSCs precedes development of the VE-cad⁺CD45⁺ pre-HSCs (termed here type I and type II pre-HSCs, respectively), thus establishing a hierarchical directionality in the developing HSC lineage. By labeling the luminal surface of the dorsal aorta, we show that both type I and type II pre-HSCs are distributed broadly within the endothelial and subendothelial aortic layers, in contrast to mature definitive HSCs which localize to the aortic endothelial layer. In agreement with expression of CD41 in pre-HSCs, in vivo CD41-Cre-mediated genetic tagging occurs in embryonic pre-HSCs and persists in all lymphomyeloid lineages of the adult animal.

ZNRD1 (zinc ribbon domain-containing 1) is a host cellular factor that influences HIV-1 replication and disease progression.

BACKGROUND: Human immunodeficiency virus (HIV) takes advantage of multiple host proteins to support its own replication. The gene ZNRD1 (zinc ribbon domain-containing 1) has been identified as encoding a potential host factor that influenced disease progression in HIV-positive individuals in a genomewide association study and also significantly affected HIV replication in a large-scale in vitro short interfering RNA (siRNA) screen. Genes and polymorphisms identified by large-scale analysis need to be followed up by means of functional assays and resequencing efforts to more precisely map causal genes. METHODS: Genotyping and ZNRD1 gene resequencing for 208 HIV-positive subjects (119 who experienced long-term nonprogression [LTNP] and 89 who experienced normal disease progression) was done by either TaqMan genotyping assays or direct sequencing. Genetic association analysis was performed with the SNPassoc package and Haploview software. siRNA and short hairpin RNA (shRNA) specifically targeting ZNRD1 were used to transiently or stably down-regulate ZNRD1 expression in both lymphoid and nonlymphoid cells. Cells were infected with X4 and R5 HIV strains, and efficiency of infection was assessed by reporter gene assay or p24 assay. RESULTS: Genetic association analysis found a strong statistically significant correlation with the LTNP phenotype (single-nucleotide polymorphism rs1048412; P = .0004), independently of HLA-A10 influence. siRNA-based functional analysis showed that ZNRD1 down-regulation by siRNA or shRNA impaired HIV-1 replication at the transcription level in both lymphoid and nonlymphoid cells. CONCLUSION: Genetic association analysis unequivocally identified ZNRD1 as an independent marker of LTNP to AIDS. Moreover, in vitro experiments pointed to viral transcription as the inhibited step. Thus, our data strongly suggest that ZNRD1 is a host cellular factor that influences HIV-1 replication and disease progression in HIV-positive individuals.

HIV endocytosis after dendritic cell to T cell viral transfer leads to productive virus infection.

Contacts between HIV-producing T cells and primary CD4+ T cells may induce the uptake of HIV by target cells that are endocytosed into trypsin-resistant compartments. We have now compared the mechanism of virus transmission from T cell-to-T cell versus infected dendritic cells (DCs)-to-T cell. In cocultures of HIV-1-infected DCs with primary CD4+ T cells, virus transmission to the target cells was resistant to trypsin treatment and could only be prevented by the anti-SUgp120 antibody IgGb12 but not by TAK-779, C34 or AZT. Importantly, upon stimulation of purified HIV-1-loaded CD4+ T cells with PHA/IL-2, cells became productively infected as measured by intracellular CAp24 staining and antigen determination in the cell supernatant. These results suggest that the viral endocytic transfer may represent a escape mechanism in the presence of drugs targeting HIV-1 entry or the host immune system.

Cell adhesion through alphaV-containing integrins is required for efficient HIV-1 infection in macrophages.

Monocytes and macrophages are an important reservoir of human immunodeficiency virus (HIV) and may represent the largest reservoir of this virus in tissues. Differentiation of monocytes into macrophages leads to cell attachment and susceptibility to infection and replication of HIV. Among other cell-surface molecules, integrins are overexpressed during monocyte-macrophage differentiation and may play a role in the replication cycle of envelope viruses including HIV. Here, we show that inhibition of alphaV integrin in monocyte-derived macrophages, by RNA interference or their inhibition by a selective small heterocyclic RGD-mimetic nonpeptide compound, inhibited the replication of HIV in the absence of cytotoxicity. Interference or inhibition of alphaV integrins triggered a signal transduction pathway, leading to down-regulation of nuclear factor-kappaB-dependent HIV-1 transcription. Such inhibition was mediated by a MAP-kinase signaling cascade, probably involving ERK1/2, p38-mitogen-activated protein kinases, and HSP27. In conclusion, our results reveal a significant role of integrin alphaV-mediated adhesion in HIV-1 infection of macrophages.

A clathrin-dynamin-dependent endocytic pathway for the uptake of HIV-1 by direct T cell-T cell transmission.

Cellular contacts between HIV-1-infected cells and target primary T CD4+ lymphocytes trigger the formation of a structure known as the virological synapse. As a consequence, viral production in HIV-1-infected cells is polarized towards the virological synapse and nascent viral particles are directly transferred to target T CD4+ lymphocytes. In this study, we performed short time cocultures of target primary T CD4+ lymphocytes with effector T cells infected by either HIV-1 NL4-3 or BaL. Using flow cytometry and immuno-confocal analyses, we investigated the transfer of HIV-1 virion antigens. We found that after 3 h of coculture, unstimulated T CD4+ lymphocytes captured complete HIV-1 virions from infected T cells during cell-cell contacts. Virus transfer occurred through a dynamin-dependent pathway and could be inhibited by chlorpromazine, an inhibitor of clathrin-dependent endocytosis. Transferred HIV-1 virions were located in compartments close to the surface of the target cell in a polarized manner. These compartments were positive for clathrin and the early endosomal marker EEA1 but were negative for caveolin-1. Furthermore, the great majority of internalized HIV-1 particles did not colocalize with Lamp1, a well-known marker for the lysosomal-degradative pathway. Similar results were observed when stimulated primary T CD4+ lymphocytes were the target cells. Our results suggest a mechanism of cell to cell HIV-1 transfer through a clathrin- and dynamin-dependent early endocytic pathway where internalized HIV-1 particles would not reach Lamp1 positive compartments, suggesting that during HIV-1 transfer by cell-cell contacts, virions can be taken up by endocytosis but not be degraded in lysosomes.

HIV-1 resistance to the anti-HIV activity of a shRNA targeting a dual-coding region.

We generated a lymphoid cell line (Sup-T1-Rev/Env) that stably expresses a 19-bp short hairpin RNA (shRNA) targeting a conserved region of HIV-1 encoding for the Envelope and Rev proteins, which potently inhibited viral replication. However, continuous passage of HIV-1 in Sup-T1-Rev/Env generated virus mutants able to overcome the RNAi restriction. Sequence analysis of the emerging viruses showed that mutations were located at positions 5 and 17 of the target sequence. Both mutations are silent in the Env frame, but the mutation 5 generated an amino acid change (V47M) in the Rev reading frame. We have analyzed the impact of these two mutations on the RNAi mechanism, showing a more crucial role of the mutation 17 in the resistance to RNAi. We show that even targeting a conserved region of the HIV-1 genome involved in the biosynthesis of two essential genes, env and rev, the virus could evolve to escape by single point mutations in the target sequence, without a significant fitness cost.

Inhibition of HIV-1 replication by RNA interference of p53 expression.

p53 expression and activation have been associated to faster human immunodeficiency virus (HIV) disease progression, most probably by inducing CD4+ T cell death but also through its cooperative effect in the control of viral gene transcription by viral regulatory proteins. Here, we show that RNA interference of p53 in HIV-1 reporter (HeLa P4-R5 MAGI) and lymphoid (SupT1) cell lines blocked HIV-1 and Tat-induced transcription from the HIV-1 promoter and HIV-1 replication in acutely infected cells, suggesting a cooperative role of p53 in HIV-1 transcription. Contrary to SupT1 cells, which encode several mutations on the p53 DNA binding domain, death of HIV-1-induced syncytia was reduced in cocultures of HeLa P4-R5 MAGI with persistently infected HIV-1 cells. To our knowledge, this is the first demonstration of the effect of the loss of function of p53 in HIV-1 replication, which is independent on its classical DNA binding activity. Our results suggest two independent roles for p53 in HIV-1 infection: cooperation in HIV long-terminal repeat transcription and virus-induced cell death.