AIRE is expressed in breast cancer TANs and TAMs to regulate the extrinsic apoptotic pathway and inflammation.

The autoimmune regulator (AIRE) is a transcriptional regulator expressed in the thymus and is necessary for maintaining immunological self-tolerance. Extrathymic AIRE expression is rare, and a role for AIRE in tumor-associated innate immune cells has not yet been established. In this study, we show that AIRE is expressed in human pro-tumor neutrophils. In breast cancer, AIRE was primarily located to tumor-associated neutrophils (TANs), and to a lesser extent to tumor-associated macrophages (TAMs) and tumor cells. Expression of AIRE in TAN/TAMs, but not in cancer cells, was associated with an adverse prognosis. We show that the functional role for AIRE in neutrophils and macrophages is to regulate expression of immune mediators and the extrinsic apoptotic pathway involving the Fas/TNFR death receptors and cathepsin G. Here, we propose that the role for AIRE in TAN/TAMs in breast tumors is to regulate cell death and inflammation, thus promoting tumor progression.

Serum immuno-oncology markers carry independent prognostic information in patients with newly diagnosed metastatic breast cancer, from a prospective observational study.

BACKGROUND: Metastatic breast cancer (MBC) is a challenging disease, and despite new therapies, prognosis is still poor for a majority of patients. There is a clinical need for improved prognostication where immuno-oncology markers can provide important information. The aim of this study was to evaluate serum immuno-oncology markers in MBC patients and their respective relevance for prediction of survival. PATIENTS AND METHODS: We investigated a broad panel of 92 immuno-oncology proteins in serum from 136 MBC patients included in a prospective observational study (NCT01322893) with long-term follow-up. Serum samples were collected before start of systemic therapy and analyzed using multiplex proximity extension assay (Olink Target 96 Immuno-Oncology panel). Multiple machine learning techniques were used to identify serum markers with highest importance for prediction of overall and progression-free survival (OS and PFS), and associations to survival were further evaluated using Cox regression analyses. False discovery rate was then used to adjust for multiple comparisons. RESULTS: Using random forest and random survival forest analyses, we identified the top nine and ten variables of highest predictive importance for OS and PFS, respectively. Cox regression analyses revealed significant associations (P < 0.005) of higher serum levels of IL-8, IL-10 and CAIX with worse OS in multivariable analyses, adjusted for established clinical prognostic factors including circulating tumor cells (CTCs). Similarly, high serum levels of IL-8, IL-10, ADA and CASP8 significantly associated with worse PFS. Interestingly, high serum levels of FasL significantly associated with improved OS and PFS. In addition, CSF-1, IL-6, MUC16, TFNSFR4 and CD244 showed suggestive evidence (P < 0.05) for an association to survival in multivariable analyses. After correction for multiple comparisons, IL-8 still showed strong evidence for correlation to survival. CONCLUSION: To conclude, we found six serum immuno-oncology markers that were significantly associated with OS and/or PFS in MBC patients, independently of other established prognostic factors including CTCs. Furthermore, an additional five serum immuno-oncology markers provided suggestive evidence for an independent association to survival. These findings highlight the relevance of immuno-oncology serum markers in MBC patients and support their usefulness for improved prognostication. Trial registration Clinical Trials (NCT01322893), registered March 25, 2011.

Gene expression in metastatic breast cancer-patterns in primary tumors and metastatic tissue with prognostic potential.

Background: Metastatic breast cancer (MBC) is the main cause of breast cancer-related death. The outcome of MBC varies, and there is a lack of biomarkers to aid in prognostication. The primary aim of this study was to evaluate the prognostic value of gene expression (GEX) signatures in the primary tumor (PT) and distant metastasis (DM) for progression-free survival (PFS) and overall survival (OS). The secondary aim was to describe GEX changes through MBC evolution and to identify MBC subtypes. Methods: RNA was extracted from the PT, lymph node metastasis (LNM), and DM from MBC patients in a prospective observational study (n = 142; CTC-MBC NCT01322893) and was subjected to GEX analysis retrospectively using the NanoString Breast Cancer 360™ panel. 31 continuous GEX variables in DMs and PTs were analyzed for PFS and OS by Cox regression analysis and Kaplan-Meier estimates. Multivariable Cox regressions were adjusted for number of DM sites and CTCs, visceral metastasis, ECOG status, age at MBC diagnosis and, in additional analyses, PAM50 subtype. Differential GEX analyses and Euclidean distances were used to describe subgroup differences and visualize within-patient heterogeneity. Results: Compared to DM GEX, GEX of the PT was at least equally useful for predicting MBC outcome. The strongest marker for a favorable PFS, both when expressed in the PT and the DM was AR, even after adjustment for prognostic markers including PAM50. GEX signatures related to hormone responsiveness, including ESR1, FOXA1, PGR, and AR were favorable prognostic markers, and the p53 signature was unfavorable for PFS when expressed in PT or DM. The previously published PAM50MET signature was prognostic for both PFS and OS. We established five distinct DM GEX profiles where two associated with liver and bone metastases, respectively. Finally, we identified four DM GEX profiles able to identify MBCs with poor OS in this cohort. Conclusion: GEX of both DM and PT are useful in MBC prognostication. GEX of AR adds prognostic information for MBC. Our descriptive analyses illuminate the biological differences between MBCs in relation to outcome and metastatic site.

Peripheral Blood Mononuclear Cell Populations Correlate with Outcome in Patients with Metastatic Breast Cancer.

Local tumor-associated immune cells hold prognostic and predictive value in various forms of malignancy. The role of systemic, circulating leukocytes is, however, not well-characterized. In this prospective and explorative study, we aim to delineate the clinical relevance of a broad panel of circulating immune cells in 32 patients with newly diagnosed metastatic breast cancer (MBC) before the start of systemic treatment. Freshly isolated peripheral blood mononuclear cells (PBMCs) were analyzed by flow cytometry and evaluated for potential associations to clinicopathological variables and patient outcome. We show that the levels of specific circulating leukocyte populations are associated with clinical parameters such as hormone receptor status, histological subtype, number of circulating tumor cells (CTCs) and metastatic burden. Importantly, high levels of CD8+ cytotoxic T lymphocytes (CTLs) are significantly linked to improved overall survival (OS). In patients with estrogen receptor (ER)-positive primary tumors, high levels of circulating CTLs and non-classical (CD14+CD16++) monocytes were associated with improved OS, whereas in patients with ER-negative tumors low levels of circulating natural killer (NK) cells potentially associate with improved OS. We propose that the levels of specific circulating immune cell populations, such as CD8+ CTLs, may be used to predict clinical outcomes in MBC patients. Thus, larger studies are warranted to validate these findings.

HAMLET a human milk protein-lipid complex induces a pro-inflammatory phenotype of myeloid cells.

HAMLET is a protein-lipid complex with a specific and broad bactericidal and tumoricidal activity, that lacks cytotoxic activity against healthy cells. In this study, we show that HAMLET also has general immune-stimulatory effects on primary human monocyte-derived dendritic cells and macrophages (Mo-DC and Mo-M) and murine RAW264.7 macrophages. HAMLET, but not its components alpha-lactalbumin or oleic acid, induces mature CD14low/- CD83+ Mo-DC and M1-like CD14+ CD86++ Mo-M surface phenotypes. Concomitantly, inflammatory mediators, including IL-2, IL-6, IL-10, IL-12 and MIP-1α, were released in the supernatant of HAMLET-stimulated cells, indicating a mainly pro-inflammatory phenotype. The HAMLET-induced phenotype was mediated by calcium, NFκB and p38 MAPK signaling in Mo-DCs and calcium, NFκB and ERK signaling in Mo-M as inhibitors of these pathways almost completely blocked the induction of mature Mo-DCs and M1-like Mo-M. Compared to unstimulated Mo-DCs, HAMLET-stimulated Mo-DCs were more potent in inducing T cell proliferation and HAMLET-stimulated macrophages were more efficient in phagocytosis of Streptococcus pneumoniae in vitro. This indicates a functionally activated phenotype of HAMLET-stimulated DCs and macrophages. Combined, we propose that HAMLET has a two-fold anti-bacterial activity; one inducing direct cytotoxic activity, the other indirectly mediating elimination of bacteria by activation of immune cells of the myeloid lineage.

Human G-MDSCs are neutrophils at distinct maturation stages promoting tumor growth in breast cancer.

Myeloid-derived suppressor cells (MDSCs) are known to contribute to immune evasion in cancer. However, the function of the human granulocytic (G)-MDSC subset during tumor progression is largely unknown, and there are no established markers for their identification in human tumor specimens. Using gene expression profiling, mass cytometry, and tumor microarrays, we here demonstrate that human G-MDSCs occur as neutrophils at distinct maturation stages, with a disease-specific profile. G-MDSCs derived from patients with metastatic breast cancer and malignant melanoma display a unique immature neutrophil profile, that is more similar to healthy donor neutrophils than to G-MDSCs from sepsis patients. Finally, we show that primary G-MDSCs from metastatic breast cancer patients co-transplanted with breast cancer cells, promote tumor growth, and affect vessel formation, leading to myeloid immune cell exclusion. Our findings reveal a role for human G-MDSC in tumor progression and have clinical implications also for targeted immunotherapy.

Niche- and Gender-Dependent Immune Reactions in Relation to the Microbiota Profile in Pediatric Patients with Otitis Media with Effusion.

Otitis media with effusion (OME) is a common inflammatory disease that primarily affects children. OME is defined as a chronic low-grade inflammation of the middle ear (ME), without any signs of infection and with effusion persisting in the ME for more than 3 months. The precise pathogenesis is, however, not fully understood. Here, we comprehensively characterized and compared the host immune responses (inflammatory cells and mediators) and the overall microbial community composition (microbiota) present in matched middle ear effusion (MEE) samples, external ear canal (EEC) lavages, and nasopharynx (NPH) samples from children with OME. Female patients had significantly increased percentages of T lymphocytes and higher levels of a wide array of inflammatory mediators in their MEE compared to that of male patients, which were unrelated to microbiota composition. The relative abundances of identified microorganisms were strongly associated with their niche of origin. Furthermore, specific inflammatory mediators were highly correlated with certain bacterial species. Interestingly, some organisms displayed a niche-driven inflammation pattern in which presence of Haemophilus spp. and Corynebacterium propinquum in MEE was accompanied by proinflammatory mediators, whereas their presence in NPH was accompanied by anti-inflammatory mediators. For Turicella and Alloiococcus, we found exactly the opposite results, i.e., an anti-inflammatory profile when present in MEE, whereas their presence in the the NPH was accompanied by a proinflammatory profile. Together, our results indicate that immune responses in children with OME are highly niche- and microbiota-driven, but gender-based differences were also observed, providing novel insight into potential pathogenic mechanisms behind OME.

The Generation and Identity of Human Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are cells of myeloid lineage with a potent immunosuppressive capacity. They are present in cancer patients as well as in patients with severe inflammatory conditions and infections. MDSCs exist as two main subtypes, the granulocytic (G-MDSCs) and the monocytic (Mo-MDSCs) type, as defined by their surface phenotype and functions. While the functions of MDSCs have been investigated in depth, the origin of human MDSCs is less characterized and even controversial. In this review, we recapitulate theories on how MDSCs are generated in mice, and whether this knowledge is translatable into human MDSC biology, as well as on problems of defining MDSCs by their immature cell surface phenotype in relation to the plasticity of myeloid cells. Finally, the challenge of pharmacological targeting of MDSCs in the future is envisioned.

Inflammatory macrophage derived TNFα downregulates estrogen receptor α via FOXO3a inactivation in human breast cancer cells.

Patients with estrogen receptor α positive (ERα+) breast cancer can respond to endocrine therapy, but treatment resistance is common and associated with downregulation of ERα expression in the dormant residual cells. Here we show, using long-term NSG xenograft models of human breast cancer and primary human monocytes, in vitro primary cell cultures and tumors from breast cancer patients, that macrophage derived tumor necrosis factor alpha (TNFα) downregulates ERα in breast cancer cells via inactivation of the transcription factor Forkhead box O transcription factor 3a (FOXO3a). Moreover, presence of tumor associated macrophages in the primary tumor of breast cancer patients, was associated with ERα negativity, and with worse prognosis in patients with ERα+ tumors. We propose that pro-inflammatory macrophages, despite being tumoricidal, may have direct effects on tumor progression and endocrine resistance in breast cancer patients. Our findings suggest that TNFα antagonists should be evaluated for treatment of ERα+ breast cancer.

Clinical relevance of systemic monocytic-MDSCs in patients with metastatic breast cancer.

The overall aim of this prospective study was to delineate the role of monocytic myeloid-derived suppressor cells (Mo-MDSCs) in patients with metastatic breast cancer (MBC). MDSCs are a heterogeneous group of immunosuppressive cells often enriched in different malignancies which hold prognostic and predictive value for clinical outcomes. Here, we assessed the clinical significance of Mo-MDSCs in 54 patients with de novo or distant recurrent MBC. We show that high levels of Mo-MDSCs significantly correlated with de novo MBC (metastatic disease at initial diagnosis), estrogen receptor (ER) negativity, and liver- and bone metastasis. A trend towards an association between high levels of Mo-MDSCs and survival (P = 0.053) was also found in patients with distant recurrent ER-positive MBC. We therefore propose that an increased population of Mo-MDSCs may be related to the metastatic or immunoregulatory switch associated with transition to a more systemic disease. Our data imply that high levels of systemic Mo-MDSCs represent patients with more aggressive disease and worse outcome.

The serine protease HtrA plays a key role in heat-induced dispersal of pneumococcal biofilms.

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx by forming multicellular biofilms. Due to the high level of asymptomatic carriage, transition to infections, such as otitis media, pneumonia, sepsis, and meningitis, occurs often enough that the pneumococcus remains a major cause of disease and death globally. Virus infection and virus-induced responses, such as increased temperature (fever), trigger release of virulent bacteria from colonizing biofilms. The exact mechanisms involved in pneumococcal egress during biofilm dispersal remain unknown, although we hypothesize that disruption of the biofilm matrix encasing the bacteria is necessary. Here, we utilized established in vitro biofilm dispersal models to investigate the involvement of proteases in bacterial egress from pneumococcal biofilms. We demonstrate the importance of protease activity, both through increased bacterial release following addition of proteases and reduced heat-induced biofilm dispersal in the presence of protease inhibitors. We identify a key role for the surface-exposed serine protease HtrA, but not PrtA, in heat-induced biofilm dispersal. Bacterial release from htrA-negative biofilms was significantly reduced compared to wild-type isogenic strains but was restored and increased above wild-type levels following addition of recombinant HtrA. Understanding the specific mechanisms involved in bacterial egress may provide novel targets for future strategies aimed to specifically interfere with disease progression without disturbing nasopharyngeal biofilm colonization.

Impact of systemic therapy on circulating leukocyte populations in patients with metastatic breast cancer.

Tumors affect the immune system, locally and systemically. The frequencies of specific circulating immune cell populations correlate with disease progression as well as prognosis of the patients. Although largely neglected, conventional antitumoral therapies often possess immunomodulatory properties and affect the levels of specific immune cell populations. Most information, however, derive from animal or in vitro studies. As this could impact prognosis as well as response to therapy, further studies of the effects of treatment on circulating immune cells in patients are warranted. In this pilot study, we evaluated a wide panel of circulating immune cells over time (up to six months) in ten patients with metastatic breast cancer receiving standard antitumoral regimens. Overall, endocrine therapy tends to enrich for natural killer (NK) and natural killer T (NKT) cells in the circulation, whereas both chemotherapy and endocrine therapy reduce the levels of circulating monocytic myeloid-derived suppressor cells (Mo-MDSCs). This indicates that the systemic immunosuppressive profile observed in patients tends to revert over the course of systemic therapy and holds promise for future combination treatment with standard antitumoral agents and immunotherapy.

Wnt5a is a TLR2/4-ligand that induces tolerance in human myeloid cells.

Innate immune responses are rapid, dynamic and highly regulated to avoid overt reactions. This regulation is executed by innate immune tolerance mechanisms that remain obscure. Wnt5a is a signalling protein mainly involved in developmental processes and cancer. The effect of Wnt5a on inflammatory myeloid cells is controversial. Here, we combine primary cell cultures, in vitro binding studies, mass spectrometry and Drosophila protein modelling to show that Wnt5a is a direct ligand of toll-like receptor (TLR) 2 and 4. The binding promotes a MyD88-non-canonical nuclear factor of kappa B (NFκB) and AP-1 signalling cascade, with contradictory profiles in mouse (pro-inflammatory) and human (anti-inflammatory) myeloid immune cells. These data reveal that the true nature of Wnt5a in inflammatory cells, is to regulate TLR signals, and in human myeloid cells it acts as an endogenous, tolerance-associated molecular pattern (TAMP), inducing IL-10 and innate immune tolerance.

Growing and Characterizing Biofilms Formed by Streptococcus pneumoniae.

It is estimated that over 80% of bacterial infections are associated with biofilm formation. Biofilms are organized bacterial communities formed on abiotic surfaces, such as implanted or inserted medical devices, or on biological surfaces, such as epithelial linings and mucosal surfaces. Biofilm growth is advantageous for the bacterial organism as it protects the bacteria from antimicrobial host factors and allows the bacteria to reside in the host without causing excessive inflammation. Like many other opportunistic pathogens of the respiratory tract, Streptococcus pneumoniae forms biofilms during asymptomatic carriage, which promotes, among other things, persistence in the niche, intraspecies and interspecies communication, and spread of bacterial DNA. Changes within the colonizing environment resulting from host assaults, such as virus infection, can induce biofilm dispersion where bacteria leave the biofilm and disseminate to other sites with ensuing infection. In this chapter, we present methodology to form complex biofilms in the nasopharynx of mice and to evaluate the biofilm structure and function in this environment. Furthermore, we present methods that recapitulate this biofilm phenotype in vitro by incorporating crucial factors associated with the host environment and describe how these models can be used to study biofilm function, transformation, and dispersion.

Streptococcus pneumoniae Otitis Media Pathogenesis and How It Informs Our Understanding of Vaccine Strategies.

PURPOSE OF REVIEW: This study aimed to review the literature regarding the mechanisms of transition from asymptomatic colonization to induction of otitis media and how the insight into the pathogenesis of otitis media has the potential to help design future otitis media-directed vaccines. RECENT FINDINGS: Respiratory viruses have long been shown to predispose individuals to bacterial respiratory infections, such as otitis media. Recent information suggests that Streptococcus pneumoniae, which colonize the nasopharynx asymptomatically, can sense potentially "threatening" changes in the nasopharyngeal environment caused by virus infection by upregulating specific sets of genes involved in biofilm release, dissemination from the nasopharynx to other sites, and protection against the host immune system. Furthermore, an understanding of the transcriptional and proteomic changes occurring in bacteria during transition to infection has led to identification of novel vaccine targets that are disease-specific and will not affect asymptomatic colonization. This approach will avoid major changes in the delicate balance of microorganisms in the respiratory tract microbiome due to elimination of S. pneumoniae. SUMMARY: Our recent findings are reviewed in the context of the current literature on the epidemiology and pathogenesis of otitis media. We also discuss how other otopathogens, such as Haemophilus influenzae and Moraxella catarrhalis, as well as the normal respiratory microbiome, can modulate the ability of pneumococci to cause infection. Furthermore, the unsatisfactory protection offered by the pneumococcal conjugate vaccines is highlighted and we review potential future strategies emerging to confer a more specific protection against otitis media.

On the origin of myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) have a strong immunosuppressive character that allows them to regulate immune responses and hinder overt inflammatory responses. In cancer, this leads to tumor immune evasion and disease progression. MDSCs come in at least two forms: monocytic (Mo-MDSCs) and granulocytic (G-MDSCs). The classical definition of MDSCs as immature myeloid cells blocked from differentiating has been challenged by recent studies suggesting that Mo-MDSCs and G-MDSCs may represent monocytes and granulocytes that have acquired immunosuppressive properties. The molecular mechanism behind their generation and their true origins are now widely debated. In this review we discuss the different proposed mechanisms of the generation of both types of MDSCs, with a special focus on human MDSCs in cancer.

In Vitro and In Vivo Biofilm Formation by Pathogenic Streptococci.

This manuscript presents novel approaches to grow and evaluate Streptococcal biofilm formation using the human respiratory pathogen Streptococcus pneumoniae (the pneumococcus) as the main model organism on biological surfaces in vitro and in vivo. Most biofilm models are based on growth on abiotic surfaces, which is relevant for many pathogens whose growth on surfaces or medical devices is a major cause of disease transmission and infections, especially in hospital environments. However, most infections with commensal organisms require biofilm formation on biological surfaces in the host at the site of colonization or infection. In vitro model systems incorporating biological components from the host and taking into account the host environment of the infectious site are not well described.In a series of publications, we have shown that S. pneumoniae form complex biofilms in the nasopharynx of mice and have devised methodology to evaluate the biofilm structure and function in this environment. We have also been able to recapitulate this biofilm phenotype in vitro by incorporating crucial factors associated with the host environment. Although the protocols presented in this manuscript are focused on S. pneumoniae, the same methodology can and has been used for other Streptococcal species that form biofilms on mucosal surfaces.

Cancer-associated fibroblast-secreted CXCL16 attracts monocytes to promote stroma activation in triple-negative breast cancers.

Triple-negative (TN) breast cancers (ER-PR-HER2-) are highly metastatic and associated with poor prognosis. Within this subtype, invasive, stroma-rich tumours with infiltration of inflammatory cells are even more aggressive. The effect of myeloid cells on reactive stroma formation in TN breast cancer is largely unknown. Here, we show that primary human monocytes have a survival advantage, proliferate in vivo and develop into immunosuppressive myeloid cells expressing the myeloid-derived suppressor cell marker S100A9 only in a TN breast cancer environment. This results in activation of cancer-associated fibroblasts and expression of CXCL16, which we show to be a monocyte chemoattractant. We propose that this migratory feedback loop amplifies the formation of a reactive stroma, contributing to the aggressive phenotype of TN breast tumours. These insights could help select more suitable therapies targeting the stromal component of these tumours, and could aid prediction of drug resistance.

S100A9 expressed in ER(-)PgR(-) breast cancers induces inflammatory cytokines and is associated with an impaired overall survival.

BACKGROUND: Breast cancer is the most common cancer form among women today. Depending on hormone receptor status, breast cancers are divided into different subtypes with vastly varying prognosis. S100A9 is a calcium-binding protein that is associated with inflammation and expressed not only in myeloid cells but also in some tumours. The role for S100A9 in the malignant cells is not well characterised; however, previous studies have shown that the protein could have important immune-modulating properties. METHODS: Using a human breast cancer cohort consisting of 144 tumour samples and in vitro analysis of human breast cancer cell lines, we investigated the expression and function of S100A9 in human breast cancer. RESULTS: We show that S100A9 expression in breast cancer correlated with the ER(-)PgR(-) breast tumour subtype (P<0.001) and with Ki67 (P=0.024) and was expressed both in the malignant cells and in the tumour-infiltrating anti-inflammatory CD163(+) myeloid cells (P<0.001). Stromal expression of S100A9 also correlated to nodal stage, tumour size and Her2 positivity. Within the ER(-)PgR(-) subgroup, all Her2(+) and EGFR(+) tumours expressed S100A9 in the cytoplasm. Both cytoplasmic staining in the malignant cells as well as stromal S100A9 expression in myeloid cells correlated with a decreased overall survival in breast cancer patients. Furthermore, rS100A9 homodimers induced expression of pro-inflammatory cytokines (IL-6, IL-8 and IL-1ß) in a TLR4- and EGFR-dependent manner in human breast cancer cells in vitro. CONCLUSION: We suggest that S100A9 could be viewed as a novel therapeutic target for patients with ER(-)PgR(-) breast cancers.