Summary Toll‐like receptors (TLRs) are increasingly implicated in the pathogenesis of cancer. The present study describes TLR expression and function in healthy and malignant airway epithelial cells. The squamous cell carcinoma cell line Detroit‐562 was compared with the healthy bronchial epithelial cell line NL‐20 and primary human nasal epithelial cells (HNECs). TLR2, TLR3 and TLR5 were present in primary head and neck squamous cell carcinomas (HNSCCs). Consistent with this, Detroit‐562 expressed TLR2, TLR3 and TLR5, whereas NL‐20 expressed mainly TLR3 and HNECs expressed TLR2‐5. In Detroit‐562, Pam3CSK4, poly(I:C) and flagellin, ligands for TLR2, TLR3 and TLR5, respectively, induced an up‐regulation of intercellular adhesion molecule 1 (ICAM‐1), an increase in interleukin (IL)‐6 and IL‐8 secretion and a decrease in cell viability. Additionally, poly(I:C) affected IL‐1β production and the migratory behaviour of Detroit‐562. NL‐20 responded with a slight increase in IL‐8 secretion upon poly(I:C) stimulation. Poly(I:C) induced a small increase in IL‐1β, IL‐6 and IL‐8 production in HNECs, while Pam3CSK4 increased viability. The TLR signalling was transcription‐dependent, but the pathways involved differed among TLRs as well as cells. In Detroit‐562, TLR2 and TLR5 activation was mediated via c‐jun N‐terminal kinase (JNK)‐, p38‐, phosphatidylinositol 3‐kinase (PI3K)‐ and nuclear factor (NF)‐κB‐related pathways, while TLR3 was dependent on NF‐κB. In NL‐20, TLR3 signalled via p38, and in HNECs, NF‐κB, JNK and extracellular signal‐regulated kinase (ERK) appeared to be involved. We found that TLR agonists induced a robust response in HNSCCs, characterized by generation of inflammation and cell death. A similar response was not seen in normal epithelial cells. Thus, the TLR system should be considered an important target in future antitumour immunotherapy.

Nod1, Nod2, and Nalp3 receptors were found to be present in the human nose. The expression of Nod1 and Nalp3 were down-regulated during pollen season among patients with allergic rhinitis. This opens up for new insights and novel therapeutic strategies in inflammatory airway disease.

BackgroundS100A7 is an antimicrobial peptide involved in several inflammatory diseases. The aim of the present study was to explore the expression and regulation of S100A7 in seasonal allergic rhinitis (SAR).MethodsNasal lavage (NAL) fluid was obtained from healthy controls before and after lipopolysaccharide (LPS) provocation, from SAR patients before and after allergen challenge, and from SAR patients having completed allergen-specific immunotherapy (ASIT). Nasal biopsies, nasal epithelial cells and blood were acquired from healthy donors. The airway epithelial cell line FaDu was used for in vitro experiments. Real-time RT-PCR and immunohistochemistry were used to determine S100A7 expression in nasal tissue and cells. Release of S100A7 in NAL and culture supernatants was measured by ELISA. The function of recombinant S100A7 was explored in epithelial cells, neutrophils and peripheral blood mononuclear cells (PBMC).ResultsNasal administration of LPS induced S100A7 release in healthy non-allergic subjects. The level of S100A7 was lower in NAL from SAR patients than from healthy controls, and it was further reduced in the SAR group 6 h post allergen provocation. In contrast, ASIT patients displayed higher levels after completed treatment. S100A7 was expressed in the nasal epithelium and in glands, and it was secreted by cultured epithelial cells. Stimulation with IL-4 and histamine repressed the epithelial S100A7 release. Further, recombinant S100A7 induced activation of neutrophils and PBMC.ConclusionsThe present study shows an epithelial expression and excretion of S100A7 in the nose after microbial stimulation. The levels are diminished in rhinitis patients and in the presence of an allergic cytokine milieu, suggesting that the antimicrobial defense is compromised in patients with SAR.

Background: Bacterial infections can cause a variety of airway diseases. Toll-like receptors (TLRs) directly respond to the presence of microbes and partake in the innate immune defense. TLR4 is activated by lipopolysaccharide (LPS), and has been detected in sinonasal tissue, epithelial cells and various inflammatory cells. Macrophage inflammatory protein 1α (MIP-1α) is a chemokine released during the inflammatory process. The present study investigated the potential role and regulation of MIP-1α in LPS-induced nasal inflammation. Methods: Thirty-two healthy individuals were intranasally challenged with LPS or vehicle. Nasal lavage was performed, followed by a nasal biopsy. Inflammatory cells were counted, MIP-1α levels analyzed and expression of MIP-1α mRNA in biopsies quantified. Neutrophils isolated from peripheral blood were treated with LPS and effects on MIP-1α release, cell survival, and the involved signal pathways, were investigated. Results: LPS challenge caused an increase of MIP-1α in nasal lavage. No corresponding change in mRNA expression was seen in nasal biopsies, suggesting the increase was not due to epithelial synthesis. Neutrophil numbers increased after LPS provocation. Treatment of isolated neutrophils with LPS delayed neutrophil apoptosis and resulted in a time- and concentration-dependent release of MIP-1α, which was reduced by inhibitors of transcription and of nuclear factor (NF)-κB, protein kinase C (PKC) and p38 MAPK pathways. Conclusions: Nasal LPS challenge results in release of MIP-1α. The release most likely originates from recruited neutrophils, via NF-κB-, PKC- and p38 MAPK-dependent pathways. LPS stimulation delayed neutrophil apoptosis. MIP-1α may constitute an important mediator in neutrophilic airway disease.

Breast cancers are divided into different subtypes based on receptor expression status: estrogen receptor (ER), progesterone receptor (PR) and Her2. The luminal A subtype (ER+PR+Her2-) is most often associated with a good prognosis while the triple-negative (TN) cancers (ER-PR-Her2-) have a poor prognosis. ER+ cancer cells depend on estrogen for their growth and can be treated with drugs to block its effects. While most human breast cancers express ER alpha, some tumors are hormone independent despite the presence of ER and others become ER negative over time. Myeloid cells are known to affect tumor development, and infiltration of tumor-associated macrophages is associated with a worse prognosis. Theoretically, however, macrophages can be both beneficial and bad for the patient. Proinflammatory M1 macrophages (CD68+ CD163-) can eliminate cancer cells and promote antitumor immune responses while anti-inflammatory M2 macrophages (CD163+CD68+) or anti-inflammatory myeloid cells (CD68-CD163+) can promote tumor progression by inhibiting proinflammatory immune responses and induce wound-healing processes. This includes angiogenesis and matrix degradation, both of which promote metastatic spread and possibly even EMT. Macrophages have also been proposed to downregulate ER in breast cancer cells through an unknown mechanism. Due to the recent reports suggesting that macrophages can induce EMT and downregulate ER, we evaluated the effect of human primary macrophages on ER expression in breast cancer cells. Immunohistochemistry analyses of breast cancer tumor xenografts, established with co-transplantation of primary human myeloid cells together with ER+ breast cancer cells in NSG mice, showed association of monocytes with downregulation of ER-receptor expression. Using primary human monocyte-derived macrophage cultures In vitro, we show that the different subtypes of macrophages have unique cytokine secretion profiles with M1 macrophages secreting significantly higher levels of TNF alpha compared to M2 macrophages. ER alpha positive MCF-7 and ER alpha negative MDA-MB-231 breast cancer cells were cultured in macrophage condition media, and both Western blot and qPCR analysis showed downregulation of ER was caused by M1 macrophage-derived TNF alpha that inhibits FoxO3A transcription of ER alpha. We propose that proinflammatory macrophages (M1), despite being tumoricidal, may have direct effects on endocrine resistance in breast cancer patients. Our findings suggest that different macrophage subtypes have various and unique impacts on breast cancer progression, and that proinflammatory macrophages, despite being tumoricidal, may have unwanted and direct effects on endocrine resistance mechanisms in breast cancer patients. Citation Format: Frida Björk Gunnarsdottir, Catharina Hagerling, Caroline Bergenfelz, Camilla M. Rydberg, Meliha Mehmeti, Roni Allaoui, Sofie Mohlin, Sven Påhlman, Daniel Bexell, Karin Leandersson. Inflammatory macrophage derived TNF alpha induces downregulation of estrogen receptor alpha in breast cancer cells by inactivation of Foxo3A [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A39.