Cutting Edge: Cytosolic Receptor AIM2 Is Induced by Peroxisome Proliferator-activated Receptor γ following Mycobacterium tuberculosis Infection of Human Macrophages but Does Not Contribute to IL-1ß Release.

AIM2 (absent in melanoma 2), an inflammasome component, mediates IL-1ß release in murine macrophages and cell lines. AIM2 and IL-1ß contribute to murine control of Mycobacterium tuberculosis (M.tb) infection, but AIM2's impact in human macrophages, the primary niche for M.tb, remains unclear. We show that M.tb, Mycobacterium bovis bacillus Calmette-Guérin (BCG), and M. smegmatis induce AIM2 expression in primary human macrophages. M.tb-induced AIM2 expression is peroxisome proliferator-activated receptor γ (PPARγ)-dependent and M.tb ESX-1-independent, whereas BCG- and M. smegmatis-induced AIM2 expression is PPARγ-independent. PPARγ and NLRP3, but not AIM2, are important for IL-1ß release in response to M.tb, and NLRP3 colocalizes with M.tb. This is in contrast to the role for AIM2 in inflammasome activation in mice and peritoneal macrophages. Altogether, we show that mycobacteria induce AIM2 expression in primary human macrophages, but AIM2 does not contribute to IL-1ß release during M.tb infection, providing further evidence that AIM2 expression and function are regulated in a cell- and/or species-specific manner.

Considerations and Approaches for Cancer Immunotherapy in the Aging Host.

Advances in cancer immunotherapy are improving treatment successes in many distinct cancer types. Nonetheless, most tumors fail to respond. Age is the biggest risk for most cancers, and the median population age is rising worldwide. Advancing age is associated with manifold alterations in immune cell types, abundance, and functions, rather than simple declines in these metrics, the consequences of which remain incompletely defined. Our understanding of the effects of host age on immunotherapy mechanisms, efficacy, and adverse events remains incomplete. A deeper understanding of age effects in all these areas is required. Most cancer immunotherapy preclinical studies examine young subjects and fail to assess age contributions, a remarkable deficit given the known importance of age effects on immune cells and factors mediating cancer immune surveillance and immunotherapy efficacy. Notably, some cancer immunotherapies are more effective in aged versus young hosts, while others fail despite efficacy in the young. Here, we review our current understanding of age effects on immunity and associated nonimmune cells, the tumor microenvironment, cancer immunotherapy, and related adverse effects. We highlight important knowledge gaps and suggest areas for deeper enquiries, including in cancer immune surveillance, treatment response, adverse event outcomes, and their mitigation.

Pharmacological tumor PDL1 depletion with chlorambucil treats ovarian cancer and melanoma: improves antitumor immunity and renders anti-PDL1-resistant tumors anti-PDL1-sensitive through NK cell effects.

BACKGROUND: Tumor intracellular programmed cell death ligand-1 (PDL1) mediates pathologic signals that regulate clinical treatment responses distinctly from surface-expressed PDL1 targeted by αPDL1 immune checkpoint blockade antibodies. METHODS: We performed a drug screen for tumor cell PDL1 depleting drugs that identified Food and Drug Administration (FDA)-approved chlorambucil and also 9-[2-(phosphonomethoxy)ethyl] guanine. We used in vitro and in vivo assays to evaluate treatment and signaling effects of pharmacological tumor PDL1 depletion focused on chlorambucil as FDA approved, alone or plus αPDL1. RESULTS: PDL1-expressing mouse and human ovarian cancer lines and mouse melanoma were more sensitive to chlorambucil-mediated proliferation inhibition in vitro versus corresponding genetically PDL1-depleted lines. Orthotopic peritoneal PDL1-expressing ID8agg ovarian cancer and subcutaneous B16 melanoma tumors were more chlorambucil-sensitive in vivo versus corresponding genetically PDL1-depleted tumors. Chlorambucil enhanced αPDL1 efficacy in tumors otherwise αPDL1-refractory, and improved antitumor immunity and treatment efficacy in a natural killer cell-dependent manner alone and plus αPDL1. Chlorambucil-mediated PDL1 depletion was relatively tumor-cell selective in vivo, and treatment efficacy was preserved in PDL1KO hosts, demonstrating tumor PDL1-specific treatment effects. Chlorambucil induced PDL1-dependent immunogenic tumor cell death which could help explain immune contributions. Chlorambucil-mediated PDL1 reduction mechanisms were tumor cell-type-specific and involved transcriptional or post-translational mechanisms, including promoting PDL1 ubiquitination through the GSK3ß/ß-TRCP pathway. Chlorambucil-mediated tumor cell PDL1 depletion also phenocopied genetic PDL1 depletion in reducing tumor cell mTORC1 activation and tumor initiating cell content, and in augmenting autophagy, suggesting additional treatment potential. CONCLUSIONS: Pharmacological tumor PDL1 depletion with chlorambucil targets tumor-intrinsic PDL1 signaling that mediates treatment resistance, especially in αPDL1-resistant tumors, generates PDL1-dependent tumor immunogenicity and inhibits tumor growth in immune-dependent and independent manners. It could improve treatment efficacy of selected agents in otherwise treatment-refractory, including αPDL1-refractory cancers, and is rapidly clinically translatable.

Alternative splicing of the SUMO1/2/3 transcripts affects cellular SUMOylation and produces functionally distinct SUMO protein isoforms.

Substantial increases in the conjugation of the main human SUMO paralogs, SUMO1, SUMO2, and SUMO3, are observed upon exposure to different cellular stressors, and such increases are considered important to facilitate cell survival to stress. Despite their critical cellular role, little is known about how the levels of the SUMO modifiers are regulated in the cell, particularly as it relates to the changes observed upon stress. Here we characterize the contribution of alternative splicing towards regulating the expression of the main human SUMO paralogs under normalcy and three different stress conditions, heat-shock, cold-shock, and Influenza A Virus infection. Our data reveal that the normally spliced transcript variants are the predominant mature mRNAs produced from the SUMO genes and that the transcript coding for SUMO2 is by far the most abundant of all. We also provide evidence that alternatively spliced transcripts coding for protein isoforms of the prototypical SUMO proteins, which we refer to as the SUMO alphas, are also produced, and that their abundance and nuclear export are affected by stress in a stress- and cell-specific manner. Additionally, we provide evidence that the SUMO alphas are actively synthesized in the cell as their coding mRNAs are found associated with translating ribosomes. Finally, we provide evidence that the SUMO alphas are functionally different from their prototypical counterparts, with SUMO1α and SUMO2α being non-conjugatable to protein targets, SUMO3α being conjugatable but targeting a seemingly different subset of protein from those targeted by SUMO3, and all three SUMO alphas displaying different cellular distributions from those of the prototypical SUMOs. Thus, alternative splicing appears to be an important contributor to the regulation of the expression of the SUMO proteins and the cellular functions of the SUMOylation system.

Genomic and phenotypic analyses suggest moderate fitness differences among Zika virus lineages.

RNA viruses have short generation times and high mutation rates, allowing them to undergo rapid molecular evolution during epidemics. However, the extent of RNA virus phenotypic evolution within epidemics and the resulting effects on fitness and virulence remain mostly unknown. Here, we screened the 2015-2016 Zika epidemic in the Americas for lineage-specific fitness differences. We engineered a library of recombinant viruses representing twelve major Zika virus lineages and used them to measure replicative fitness within disease-relevant human primary cells and live mosquitoes. We found that two of these lineages conferred significant in vitro replicative fitness changes among human primary cells, but we did not find fitness changes in Aedes aegypti mosquitoes. Additionally, we found evidence for elevated levels of positive selection among five amino acid sites that define major Zika virus lineages. While our work suggests that Zika virus may have acquired several phenotypic changes during a short time scale, these changes were relatively moderate and do not appear to have enhanced transmission during the epidemic.

Pharmacologic Tumor PDL1 Depletion with Cefepime or Ceftazidime Promotes DNA Damage and Sensitivity to DNA-Damaging Agents.

The interaction between tumor surface-expressed PDL1 and immune cell PD1 for the evasion of antitumor immunity is well established and is targeted by FDA-approved anti-PDL1 and anti-PD1 antibodies. Nonetheless, recent studies highlight the immunopathogenicity of tumor-intrinsic PDL1 signals that can contribute to the resistance to targeted small molecules, cytotoxic chemotherapy, and αPD1 immunotherapy. As genetic PDL1 depletion is not currently clinically tractable, we screened FDA-approved drugs to identify those that significantly deplete tumor PDL1. Among the candidates, we identified the ß-lactam cephalosporin antibiotic cefepime as a tumor PDL1-depleting drug (PDD) that increases tumor DNA damage and sensitivity to DNA-damaging agents in vitro in distinct aggressive mouse and human cancer lines, including glioblastoma multiforme, ovarian cancer, bladder cancer, and melanoma. Cefepime reduced tumor PDL1 post-translationally through ubiquitination, improved DNA-damaging-agent treatment efficacy in vivo in immune-deficient and -proficient mice, activated immunogenic tumor STING signals, and phenocopied specific genetic PDL1 depletion effects. The ß-lactam ring and its antibiotic properties did not appear contributory to PDL1 depletion or to these treatment effects, and the related cephalosporin ceftazidime produced similar effects. Our findings highlight the rapidly translated potential for PDDs to inhibit tumor-intrinsic PDL1 signals and improve DNA-damaging agents and immunotherapy efficacy.

Tumor Intrinsic PD-L1 Promotes DNA Repair in Distinct Cancers and Suppresses PARP Inhibitor-Induced Synthetic Lethality.

BRCA1-mediated homologous recombination is an important DNA repair mechanism that is the target of FDA-approved PARP inhibitors, yet details of BRCA1-mediated functions remain to be fully elucidated. Similarly, immune checkpoint molecules are targets of FDA-approved cancer immunotherapies, but the biological and mechanistic consequences of their application are incompletely understood. We show here that the immune checkpoint molecule PD-L1 regulates homologous recombination in cancer cells by promoting BRCA1 nuclear foci formation and DNA end resection. Genetic depletion of tumor PD-L1 reduced homologous recombination, increased nonhomologous end joining, and elicited synthetic lethality to PARP inhibitors olaparib and talazoparib in vitro in some, but not all, BRCA1 wild-type tumor cells. In vivo, genetic depletion of tumor PD-L1 rendered olaparib-resistant tumors sensitive to olaparib. In contrast, anti-PD-L1 immune checkpoint blockade neither enhanced olaparib synthetic lethality nor improved its efficacy in vitro or in wild-type mice. Tumor PD-L1 did not alter expression of BRCA1 or its cofactor BARD1 but instead coimmunoprecipitated with BARD1 and increased BRCA1 nuclear accumulation. Tumor PD-L1 depletion enhanced tumor CCL5 expression and TANK-binding kinase 1 activation in vitro, similar to known immune-potentiating effects of PARP inhibitors. Collectively, these data define immune-dependent and immune-independent effects of PARP inhibitor treatment and genetic tumor PD-L1 depletion. Moreover, they implicate a tumor cell-intrinsic, immune checkpoint-independent function of PD-L1 in cancer cell BRCA1-mediated DNA damage repair with translational potential, including as a treatment response biomarker. SIGNIFICANCE: PD-L1 upregulates BRCA1-mediated homologous recombination, and PD-L1-deficient tumors exhibit BRCAness by manifesting synthetic lethality in response to PARP inhibitors, revealing an exploitable therapeutic vulnerability and a candidate treatment response biomarker. See related commentary by Hanks, p. 2069.

Reversing Post-Infectious Epigenetic-Mediated Immune Suppression.

The immune response must balance the pro-inflammatory, cell-mediated cytotoxicity with the anti-inflammatory and wound repair response. Epigenetic mechanisms mediate this balance and limit host immunity from inducing exuberant collateral damage to host tissue after severe and chronic infections. However, following treatment for these infections, including sepsis, pneumonia, hepatitis B, hepatitis C, HIV, tuberculosis (TB) or schistosomiasis, detrimental epigenetic scars persist, and result in long-lasting immune suppression. This is hypothesized to be one of the contributing mechanisms explaining why survivors of infection have increased all-cause mortality and increased rates of unrelated secondary infections. The mechanisms that induce epigenetic-mediated immune suppression have been demonstrated in-vitro and in animal models. Modulation of the AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR), nuclear factor of activated T cells (NFAT) or nuclear receptor (NR4A) pathways is able to block or reverse the development of detrimental epigenetic scars. Similarly, drugs that directly modify epigenetic enzymes, such as those that inhibit histone deacetylases (HDAC) inhibitors, DNA hypomethylating agents or modifiers of the Nucleosome Remodeling and DNA methylation (NuRD) complex or Polycomb Repressive Complex (PRC) have demonstrated capacity to restore host immunity in the setting of cancer-, LCMV- or murine sepsis-induced epigenetic-mediated immune suppression. A third clinically feasible strategy for reversing detrimental epigenetic scars includes bioengineering approaches to either directly reverse the detrimental epigenetic marks or to modify the epigenetic enzymes or transcription factors that induce detrimental epigenetic scars. Each of these approaches, alone or in combination, have ablated or reversed detrimental epigenetic marks in in-vitro or in animal models; translational studies are now required to evaluate clinical applicability.

SDHD gene mutation in Mexican population with carotid body tumor.

INTRODUCTION: Among the U.S. population, the p81L SDHD (11q23) gene mutation is present in 6-36% of patients with sporadic carotid body tumor (CBT), but in familial cases is high as 80%. That is why the P81L mutation is used as a screening method for carotid body tumor in the U.S. METHODS: We included 25 patients who underwent resection of a CBT from January 2010 to June 2015. After informed consent, a blood sample was taken for genetic testing on real-time polymerase chain reaction, in order to identify p81L mutation in the SDHD gene. The information was analyzed with descriptive statistics, using central tendency and description measures. RESULTS: In our group, 92% were females, a mean age of 55.5 years, and 52% were Shamblin type II. The most common place of residence was Mexico City, 8% of the patients had family history, about 20% of the patients had a contralateral tumor and 16% had antecedent of another kind of tumor, 4 (16%) p81L SDHD gene mutations were detected, all of them were heterozygous. CONCLUSIONS: The p81L mutation in the SDHD gene was found in the Mexican population in higher grade that in the U.S. population, which explain the high incidence of this pathology in our country, but we need more studies about this subject.

INTRODUCCIÓN: La mutación p81L del gen SDHD (11q23) se encuentra presente en el 6-36% de los pacientes con tumores del cuerpo carotídeo (TCC) esporádicos y hasta en el 80% de los que presentan TCC familiares. En los EE.UU. se usa como método de cribado para TCC. MÉTODO: Se incluyeron 25 pacientes consecutivos operados de resección de TCC entre enero de 2010 y junio de 2015. Se les tomó muestra sanguínea venosa que se sometió a reacción en cadena de la polimerasa en tiempo real para identificar la mutación p81L del gen SDHD (11q23). La información se analizó con estadística descriptiva mediante medidas de tendencia central y dispersión. RESULTADOS: Del grupo en estudio, el 92% eran mujeres, la edad promedio era de 55.5 años y el 52% tenían tumor Shamblin tipo II. El lugar de residencia más frecuente fue la Ciudad de México. El 8% presentaban antecedentes familiares, el 20% tumor bilateral y el 16% presentaron un tumor en otra región. Se encontró la mutación p81L del gen SDHD (11q23) en el 16% de los pacientes de forma heterocigota. CONCLUSIONES: La mutación p81L del gen SDHD se encuentra presente en la población mexicana en un grado más elevado que lo reportado en los EE.UU., lo que podría explicar la alta incidencia en nuestro medio.

SDHD gene mutation in Mexican population whit carotid body tumor. / Mutación del gen SDHD en población mexicana con tumor del cuerpo carotídeo

Introduction: Among the U.S. population, the p81L SDHD (11q23) gene mutation is present in 6-36% of patients with sporadic carotid body tumor (CBT), but in familial cases is high as 80%. That is why the P81L mutation is used as a screening method for carotid body tumor in the U.S. Methods: We included 25 patients who underwent resection of a CBT from January 2010 to June 2015. After informed consent, a blood sample was taken for genetic testing on real-time polymerase chain reaction, in order to identify p81L mutation in the SDHD gene. The information was analyzed with descriptive statistics, using central tendency and description measures. Results: In our group, 92% were females, a mean age of 55.5 years, and 52% were Shamblin type II. The most common place of residence was Mexico City, 8% of the patients had family history, about 20% of the patients had a contralateral tumor and 16% had antecedent of another kind of tumor, 4 (16%) p81L SDHD gene mutations were detected, all of them were heterozygous. Conclusions: The p81L mutation in the SDHD gene was found in the Mexican population in higher grade that in the U.S. population, which explain the high incidence of this pathology in our country, but we need more studies about this subject.

Introducción: La mutación p81L del gen SDHD (11q23) se encuentra presente en el 6-36% de los pacientes con tumores del cuerpo carotídeo (TCC) esporádicos y hasta en el 80% de los que presentan TCC familiares. En los EE.UU. se usa como método de cribado para TCC. Método: Se incluyeron 25 pacientes consecutivos operados de resección de TCC entre enero de 2010 y junio de 2015. Se les tomó muestra sanguínea venosa que se sometió a reacción en cadena de la polimerasa en tiempo real para identificar la mutación p81L del gen SDHD (11q23). La información se analizó con estadística descriptiva mediante medidas de tendencia central y dispersión. Resultados: Del grupo en estudio, el 92% eran mujeres, la edad promedio era de 55.5 años y el 52% tenían tumor Shamblin tipo II. El lugar de residencia más frecuente fue la Ciudad de México. El 8% presentaban antecedentes familiares, el 20% tumor bilateral y el 16% presentaron un tumor en otra región. Se encontró la mutación p81L del gen SDHD (11q23) en el 16% de los pacientes de forma heterocigota. Conclusiones: La mutación p81L del gen SDHD se encuentra presente en la población mexicana en un grado más elevado que lo reportado en los EE.UU., lo que podría explicar la alta incidencia en nuestro medio.

Potential therapeutic impact of omega-3 long chain-polyunsaturated fatty acids on inflammation markers in Duchenne muscular dystrophy: A double-blind, controlled randomized trial.

BACKGROUND & AIMS: Duchenne Muscular Dystrophy (DMD) is the most frequent dystrophy in childhood generated by a deficiency in dystrophin. DMD is a neuromuscular disease and its clinical course comprises chronic inflammation and gradual muscle weakness. Supplementation of omega-3 long chain-Polyunsaturated Fatty Acids (ω-3 long chain-PUFA) reduces inflammatory markers in various disorders. The goal of this research was to analyze the influence of ω-3 long chain-PUFA intake on gene expression and blood inflammatory markers in boys with DMD. METHODS: In a placebo-controlled, double. Blind, randomized trial, boys with DMD (n = 36) consumed 2.9 g/day of ω-3 long chain-PUFA or sunflower oil as control, in capsules, for a period of 6 months. Blood was analyzed at baseline and at months 1, 2, 3, and 6 of supplementation for expression of inflammatory markers in leukocytes and serum. RESULTS: There was high adherence to capsule intake (control: 95.3% ± 7.2%, and ω-3 long chain-PUFA: 97.4% ± 3.7% at month 6). Enrichment of EicosaPentaenoic Acid (EPA) and DocosaHexaenoic Acid (DHA) in erythrocytes increased significantly in patients supplemented with ω-3 long chain-PUFA compared with the placebo group during the 6 months of supplementation. Messenger RNA (mRNA) of the Nuclear Factor kappa beta (NF-κB) and its target genes InterLeukin 1 beta (IL-1ß) and IL-6 was downregulated significantly (p < 0.05) in leukocytes from DMD boys supplemented with ω-3 long chain-PUFA for 6 months, compared to the placebo group. Omega-3 long chain-PUFA intake decreased the serum IL-1ß (-59.5%; p = 0.011) and IL-6 (-54.8%; p = 0.041), and increased the serum IL-10 (99.9%, p < 0.005), in relation to those with placebo treatment. CONCLUSION: Supplementation with ω-3 long chain-PUFA 2.9 g/day is well-tolerated, has a beneficial reductive effect on proinflammatory markers, and increases an anti-inflammatory marker, indicating that ω-3 long chain-PUFA could have a potential therapeutic impact on chronic inflammation in DMD. This research is registered at clinicaltrials.gov (NCT018264229).