Formation, Development, and Cross-Species Interactions in Biofilms

Biofilms, which are essential vectors of bacterial survival, protect microbes from antibiotics and host immune attack and are one of the leading causes that maintain drug-resistant chronic infections. In nature, compared with monomicrobial biofilms, polymicrobial biofilms composed of multispecies bacteria predominate, which means that it is significant to explore the interactions between microorganisms from different kingdoms, species, and strains. Cross-microbial interactions exist during biofilm development, either synergistically or antagonistically. Although research into cross-species biofilms remains at an early stage, in this review, the important mechanisms that are involved in biofilm formation are delineated. Then, recent studies that investigated cross-species cooperation or synergy, competition or antagonism in biofilms, and various components that mediate those interactions will be elaborated. To determine approaches that minimize the harmful effects of biofilms, it is important to understand the interactions between microbial species. The knowledge gained from these investigations has the potential to guide studies into microbial sociality in natural settings and to help in the design of new medicines and therapies to treat bacterial infections.

Defective proinsulin handling modulates the MHC I bound peptidome and activates the inflammasome in β-cells

Background: How immune-tolerance is lost to pancreatic β-cell peptides triggering autoimmune type 1 diabetes is enigmatic. We have shown that loss of the proinsulin ER chaperone glucose-regulated protein (GRP) 94 leads to mishandling of proinsulin, ER stress and activation of the inducible proteasome. We hypothesize that inadequate ER proinsulin folding capacity relative to biosynthetic need may lead to an altered β-cell MHC-I bound peptidome and inflammasome activation, sensitizing β-cells to immune attack. Methods: We used INS-1E cells with or without GRP94 knockout (KO), or in the presence or absence of GRP94 inhibitor PU-WS13 (GRP94i, 20μM), or exposed to proinflammatory cytokines interleukin (IL)-1β or IFNγ (15 pg/ml and 10 ng/ml, respectively) for 24 hours. RT1.A (rat MHC I) expression was evaluated using flow cytometry. The total RT1.A-bound peptidome analysis was performed on cell lysates fractionated by reverse phase high performance liquid chromatography (RP-HPLC) followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). NALP1, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), and (pro) IL-1β expression and secretion were investigated by Western blotting. Results: GRP94 KO increased RT1.A expression in β-cells as did cytokine exposure compared to relevant controls. Immunopeptidome analysis showed increased RT1.A-bound peptide repertoire in GRP94 KO/i cells as well as in the cells exposed to cytokines. The GRP94 KO/cytokine exposure groups showed partial overlap in their peptide repertoire. Notably, proinsulin-derived peptides diversity increased among the total RT1.A peptidome in GRP94 KO/i along with cytokines exposure. NALP1 expression was upregulated in GRP94 deficient cells along with decreased (IκBα) content while proIL-1β cellular levels declined, coupled with an increased secretion of mature IL-1β. Our results suggest that limiting β-cell proinsulin chaperoning enhances RT1.A expression, alters the MHC-I peptidome including proinsulin peptides and activates inflammatory pathways, suggesting that stress impeding proinsulin handling may sensitize β-cells to immune-attack.

CHIP: is clonal hematopoiesis a surrogate for aging and other disease?

Somatic mutations are an unavoidable consequence of aging tissues. Even though most mutations are functionally silent, some may affect genes critical to proper tissue self-renewal and differentiation, resulting in the outgrowth of affected cells, also known as clonal expansion. In hematopoietic tissue such clonal dominance is known as clonal hematopoiesis (CH). Sporadic CH is frequent in aging and affects over 10% of individuals beyond the fifth decade of life. It has been associated with an increased risk of hematologic malignancies and cardiovascular disease. In addition to aging, CH has been observed in other hematologic conditions and confers an adaptation of hematopoietic stem cells (HSCs) to various environmental stressors and cell-intrinsic defects. In the presence of extrinsic stressors such as genotoxic therapies, T-cell-mediated immune attack, or inflammation, somatic mutations may result in augmentation of HSC fitness. Such attuned HSCs can evade the environmental insults and outcompete their unadapted counterparts. Similarly, in inherited bone marrow failures, somatic mutations in HSCs frequently lead to the reversion of inherited defects. This may occur via the direct correction of germline mutations or indirect compensatory mechanisms. Occasionally, such adaptation may involve oncogenes or tumor suppressors, resulting in malignant transformation. In this brief article, we focus on the mechanisms of clonal dominance in various clinical and biological contexts.

The High Mobility Group A1 Chromatin Regulator Drives Immune Evasion during MPN Progression By Repressing Genes Involved in Antigen Presentation and Immune Attack

Introduction: Myeloproliferative Neoplasms (MPN) are blood diseases caused by mutations in hematopoietic stem cells (HSCs) which lead to clonal expansion and overproduction of myeloid lineages. Individuals with MPN are at increased risk for transformation to bone marrow fibrosis (myelofibrosis, MF) and acute myeloid leukemia (AML), both of which are associated with poor clinical outcomes. However, targetable mechanisms underlying progression remain elusive. The High Mobility Group A1 (HMGA1) gene encodes chromatin regulators which are enriched in stem cells and aberrantly overexpressed in aggressive tumors (Xian et al Nature Commun 2017;8:15008, Resar et al Cancer Res 2018;78:1890). Transgenic mice misexpressing Hmga1 in lymphoid cells develop lethal leukemia by dysregulating gene networks associated with aberrant proliferation and inflammation (Hillion et al Cancer Res 2008;68:10121, Schuldenfrei et al BMC Genomics, 2011;12:549). We discovered that HMGA1 is overexpressed in MPN with progression and required for leukemic transformation in preclinical models (Resar et al Blood 2018;132 Suppl 1:102). We therefore sought to: 1) test the hypothesis that HMGA1 drives MPN progression by dysregulating gene networks involved in immune evasion, and, 2) identify mediators of immune escape that could be disrupted in therapy. Methods: To elucidate transcriptional networks regulated by HMGA1 during MPN progression to AML, we integrated multi-omics sequencing (seq) analyses, including RNAseq, chromatin immunoprecipitation seq (ChIPseq), and ATACseq in AML cell lines from JAK2 V617Fmutant MPN after leukemic transformation (DAMI, SET-2) + HMGA1 depletion. HMGA1 gene expression was inactivated using CRISPR/Cas9 or short hairpin RNA (shRNA)-mediated gene silencing. Gene set enrichment analysis was used to dissect molecular mechanisms underlying immune invasion by HMGA1. To validate results in human MPN, RNAseq was performed in peripheral blood mononuclear cells (PBMCs) from matched MF patients who transformed to AML. To reconstruct the immune cell composition of primary MPN samples, we applied xCell, a robust computational method that converts gene expression profiles to immune cell types. Transcriptional networks were validated at the level of mRNA and protein via quantitative RT-PCR and flow cytometry. To identify drugs to disrupt HMGA1 immune evasion networks, we applied the Broad Institute Connectivity Map (CMAP) and cytotoxicity assays. Results: Integration of RNAseq, ChIPseq, and ATACseq in MPN AML cells (DAMI, SET-2) revealed that HMGA1 represses genes involved in immune activation (inflammatory response, TNFa signaling, NF-κB networks) and antigen presentation [Interferon gamma (IFNγ) response networks], including genes encoding the major histocompatibility complex (MHC) class I and II antigens. Inhibiting HMGA1 results in up-regulation of MHC class I and II antigen genes, with greatest induction of HLA-DRA (the alpha paralog for HLA Class II antigens). Similarly, HMGA1 depletion increases cell surface expression of HLA-DRA antigens. Strikingly, RNAseq from MPN patients with MF after transformation to AML reveal that HMGA1 is up-regulated in MPN AML concurrent with repression in gene networks of immune activation and antigen presentation. Immune cell transcriptomes are also depleted in MPN cells after leukemic transformation. To determine if immune evasion by HMGA1 could be modulated in therapy, we applied CMAP which identified histone deacetylase inhibitors (HDACi) as drugs targeting the HMGA1 transcriptome. The HDACi, entinostat, is cytotoxic and synergizes with the JAK inhibitor, ruxolitinib, in MPN AML cell lines. Further, entinostat induces expression of MHC class II genes and antigens. Moreover, HMGA1 depletion enhances sensitivity of MPN AML cells to entinostat. Conclusions: We discovered an epigenetic program whereby HMGA1 drives immune evasion during MPN progression by binding to chromatin and enhancing chromatin accessibility to activate transcriptional networks that repress antigen presentation and immune attack. Most importantly, HMGA1 immune evasion networks are dysregulated in human MPN and can be targeted by HDACi therapy. Together, our studies reveal a new paradigm whereby HMGA1 down-regulates MHC antigens during MPN progression, suggesting that targeting HMGA1 networks could activate an immune attack and prevent MPN progression. Figure 1 Figure 1. Disclosures Rampal: Novartis: Consultancy; Pharmaessentia: Consultancy; Kartos: Consultancy; Blueprint: Consultancy; Disc Medicine: Consultancy; BMS/Celgene: Consultancy; Stemline: Consultancy, Research Funding; Constellation: Research Funding; Jazz Pharmaceuticals: Consultancy; Sierra Oncology: Consultancy; Abbvie: Consultancy; CTI: Consultancy; Incyte: Consultancy, Research Funding; Memorial Sloan Kettering: Current Employment.

727 Resistance to enzalutamide and abiraterone drives tumor phenotypic plasticity and resistance to immune-mediated cytotoxicity

BackgroundBackground: Treatment of patients with castration-resistant prostate cancer (CRPC) includes the use of next-generation hormonal therapies such as abiraterone or enzalutamide. Although these agents extend survival, a significant proportion of patients exhibit primary or acquired resistance to treatment. In recent years, immune checkpoint blockade has led to remarkable responses in patients with several tumor types, however, CRPC has remained resistant to immunotherapy. Previous studies have demonstrated that different tumor variants could emerge along the progression of prostate cancer, including tumors undergoing phenotypic plasticity in the context of an epithelial-mesenchymal transition. Our laboratory and others have shown that phenotypic plasticity is a driver of resistance to immunotherapy. Based on this knowledge, we investigated whether changes in tumor phenotype could affect the response of CRPC to immune-based therapies, and ways this can be mitigated.MethodsThe androgen sensitive LNCAP prostate cancer cell line was used to derive LNCAP cells resistant to enzalutamide (LNCAP-EnzaR) or abiraterone (LNCAP-AbiR). Resistant cell lines and parental LNCAP cells were comparatively evaluated for features of EMT and neuroendocrine phenotype via RT-PCR, ELISA, western blot, immunofluorescence, and RNAseq. Changes in the susceptibility to NK-cell mediated cytotoxicity were evaluated with NK cells isolated from peripheral blood from healthy donors. LNCAP-EnzaR cells were also grown in vivo in NSG MHC-deficient mice, and tumors were characterized for phenotypic markers and potential therapeutic targets.ResultsAcquisition of resistance to both enzalutamide and abiraterone was associated with a significant increase in mesenchymal tumor features, including high levels of vimentin and fibronectin, and the loss of epithelial features and cell-to-cell attachments. LNCAP-Enza-R and LNCAP-AbiR cells showed a significant reduction (up to 90%) in susceptibility to NK-cell mediated cytotoxicity and antibody-dependent cell cytotoxicity (ADCC), compared with parental cells. These results prompted us to investigate approaches to improve immune-mediated lysis, including inhibition of estrogen receptor 1 (ESR1), which was identified as highly upregulated in LNCAP-EnzaR cells via RNAseq analysis. In a xenograft model of LNCAP-EnzaR cells, we corroborated the maintenance of tumor phenotypic plasticity and the expression of actionable targets.ConclusionsOur data indicates that acquisition of resistance to androgen receptor inhibition is associated with marked reduction of susceptibility to immune attack, and the acquisition of tumor phenotypic plasticity. Future studies will investigate approaches that revert tumor plasticity, including blockade of ESR1, TGF-beta or IL-8, for potential improvement of tumor susceptibility to immune attack in CRPC.Ethics ApprovalPBMCs were obtained from healthy donors at the NIH Clinical Center Blood Bank (NCT00001846). All animal studies were approved and conducted in accordance with an IACUC-approved animal protocol (LTIB-57) with the approval the NIH/NCI Institutional Animal Care and Use Committee.

The Link between Cancer, Autoimmunity and Diabetes

In cancer, corrupted neoplastic cell successfully evades the immune attack using two main strategies: avoiding the immune recognition and instigating an immunosuppressive tumor microenvironment. Cancer is able to induce immune tolerance and present itself to immune system as normal tissue. There is a defect in immune tolerance: the tolerance is wrongly induced toward cancer. In autoimmune diseases, immune system attacks normal tissue because it fails to recognize its own constituent parts as self. There is a defect in immune tolerance: the tolerance is lost toward normal tissues. In both diseases wrong information in the communication between tissue (neoplastic) cells and immune system is crucial: in cancer, neoplastic cells avoid immune attack, whilst in autoimmune diseases, immune system attacks normal cells. Diabetes mellitus type I and II are associated with an increased risk of developing different types of cancer. Pathogenesis of diabetes mellitus type I and pathogenesis of cancer overlap in genetic, environmental and immunologic factors. Diabetes mellitus type II and obesity can both induce oncogenesis.

Unraveling How Tumor-Derived Galectins Contribute to Anti-Cancer Immunity Failure

Current data indicates that anti-tumor T cell-mediated immunity correlates with a better prognosis in cancer patients. However, it has widely been demonstrated that tumor cells negatively manage immune attack by activating several immune-suppressive mechanisms. It is, therefore, essential to fully understand how lymphocytes are activated in a tumor microenvironment and, above all, how to prevent these cells from becoming dysfunctional. Tumors produce galectins-1, -3, -7, -8, and -9 as one of the major molecular mechanisms to evade immune control of tumor development. These galectins impact different steps in the establishment of the anti-tumor immune responses. Here, we carry out a critical dissection on the mechanisms through which tumor-derived galectins can influence the production and the functionality of anti-tumor T lymphocytes. This knowledge may help us design more effective immunotherapies to treat human cancers.

Targeting Toll-Like Receptor (TLR) Pathways in Inflammatory Arthritis: Two Better Than One?

Inflammatory arthritis is a cluster of diseases caused by unregulated activity of the immune system. The lost homeostasis is followed by the immune attack of one’s self, what damages healthy cells and tissues and leads to chronic inflammation of various tissues and organs (e.g., joints, lungs, heart, eyes). Different medications to control the excessive immune response are in use, however, drug resistances, flare-reactions and adverse effects to the current therapies are common in the affected patients. Thus, it is essential to broaden the spectrum of alternative treatments and to develop disease-modifying drugs. In the last 20 years, the involvement of the innate immune receptors TLRs in inflammatory arthritis has been widely investigated and targeting either the receptor itself or the proteins in the downstream signalling cascades has emerged as a promising therapeutic strategy. Yet, concerns about the use of pharmacological agents that inhibit TLR activity and may leave the host unprotected against invading pathogens and toxicity issues amid inhibition of downstream kinases crucial in various cellular functions have arisen. This review summarizes the existing knowledge on the role of TLRs in inflammatory arthritis; in addition, the likely druggable related targets and the developed inhibitors, and discusses the pros and cons of their potential clinical use.