Macrophage polarization in THP-1 cell line and primary monocytes: A systematic review.

Macrophages derived from human monocytic leukemia THP-1 cell line are often used as the alternative of human primary macrophage. However, the polarization method of THP-1 to macrophages varies between different laboratories, which may unknowingly affect the relevance of research output across research groups. In this regard, a systematic search was developed in Pubmed, BioOne, Scopus, and Science Direct to identify articles focusing on THP-1 polarization into M1 and M2 macrophages. All selected articles were read and discussed by two independent reviewers. The selection process was based on selected keywords on the title, abstract and full-text level. A total of 85 articles were selected and categorized based on the field of studies, method of THP-1 differentiation, and markers or genes expressed upon differentiation. THP-1 derived macrophages were mainly used together with primary monocyte-derived macrophages in cellular inflammation studies, while it was commonly employed alone in cancer research. THP-1 derived macrophages are also of paramount importance in biomaterials studies to prevent unfavorable immune responses in-vivo. We explored various methods of THP-1 differentiation and suggested several common genes encountered to characterize M1 and M2 macrophages differentiated from THP-1. The systematic review highlights the relevance of using THP-1 derived macrophage as a useful alternative to primary macrophage. Although it is not possible to derive a standard method of THP-1 polarization into M1 and M2 macrophages from this review, it may lead researchers to obtain reproducible polarization protocol based on commonly used stimulants and markers of differentiation.

Impact of primary mouse macrophage cell types on Leishmania infection and in vitro drug susceptibility.

Primary mouse macrophages are frequently used to provide an in vitro intracellular model to evaluate antileishmanial drug efficacy. The present study compared the phenotypic characteristics of Swiss, BALB/c, and C57BL/6 mouse bone marrow-derived macrophages and peritoneal exudate cells using different stimulation and adherence protocols upon infection with a Leishmania infantum laboratory strain and two clinical isolates. Evaluation parameters were susceptibility to infection, permissiveness to amastigote multiplication, and impact on drug efficacy. Observed variations in infection of peritoneal exudate cells can mostly be linked to changes in the inflammatory cytokine profiles (IL-6, TNF-α, KC/GRO) rather than to differences in initial production of nitric oxide and reactive oxygen species. Optimization of the cell stimulation and adherence conditions resulted in comparable infection indices among peritoneal exudate cells and the various types of bone marrow-derived macrophages. BALB/c-derived bone marrow-derived macrophages were slightly more permissive to intracellular amastigote replication. Evaluation of antileishmanial drug potency in the various cell systems revealed minimal variation for antimonials and paromomycin, and no differences for miltefosine and amphotericin B. The study results allow to conclude that drug evaluation can be performed in all tested primary macrophages as only marginal differences are observed in terms of susceptibility to infection and impact of drug exposure. Combined with some practical considerations, the use of 24-h starch-stimulated, 48-h adhered, Swiss-derived peritoneal exudate cells can be advocated as an efficient, reliable, relatively quick, and cost-effective tool for routine drug susceptibility testing in vitro whenever the use of primary cells is feasible.

Analysis of inflammatory and lipid metabolic networks across RAW264.7 and thioglycolate-elicited macrophages.

Studies of macrophage biology have been significantly advanced by the availability of cell lines such as RAW264.7 cells. However, it is unclear how these cell lines differ from primary macrophages such as thioglycolate-elicited peritoneal macrophages (TGEMs). We used the inflammatory stimulus Kdo2-lipid A (KLA) to stimulate RAW264.7 and TGEM cells. Temporal changes of lipid and gene expression levels were concomitantly measured and a systems-level analysis was performed on the fold-change data. Here we present a comprehensive comparison between the two cell types. Upon KLA treatment, both RAW264.7 and TGEM cells show a strong inflammatory response. TGEM (primary) cells show a more rapid and intense inflammatory response relative to RAW264.7 cells. DNA levels (fold-change relative to control) are reduced in RAW264.7 cells, correlating with greater downregulation of cell cycle genes. The transcriptional response suggests that the cholesterol de novo synthesis increases considerably in RAW264.7 cells, but 25-hydroxycholesterol increases considerably in TGEM cells. Overall, while RAW264.7 cells behave similarly to TGEM cells in some ways and can be used as a good model for inflammation- and immune function-related kinetic studies, they behave differently than TGEM cells in other aspects of lipid metabolism and phenotypes used as models for various disorders such as atherosclerosis.

Transcriptome Analyses of Chicken Primary Macrophages Infected With Attenuated Salmonella Typhimurium Mutants.

Salmonella enterica is one of the most common foodborne illnesses in the United States and worldwide, with nearly one-third of the cases attributed to contaminated eggs and poultry products. Vaccination has proven to be an effective strategy to reduce Salmonella load in poultry. The Salmonella Typhimurium Δcrp-cya (MeganVac1) strain is the most commonly used vaccine in the United States; however, the mechanisms of virulence attenuation and host response to this vaccine strain are poorly understood. Here, we profiled the invasion and intracellular survival phenotypes of Δcrp-cya and its derivatives (lacking key genes required for intra-macrophage survival) in HD11 macrophages and the transcriptome response in primary chicken macrophages using RNA-seq. Compared to the parent strain UK1, all the mutant strains were highly defective in metabolizing carbon sources related to the TCA cycle and had greater doubling times in macrophage-simulating conditions. Compared to UK1, the majority of the mutants were attenuated for invasion and intra-macrophage survival. Compared to Δcrp-cya, while derivatives lacking phoPQ, ompR-envZ, feoABC and sifA were highly attenuated for invasion and intracellular survival within macrophages, derivatives lacking ssrAB, SPI13, SPI2, mgtRBC, sitABCD, sopF, sseJ and sspH2 showed increased ability to invade and survive within macrophages. Transcriptome analyses of macrophages infected with UK1, Δcrp-cya and its derivatives lacking phoPQ, sifA and sopF demonstrated that, compared to uninfected macrophages, 138, 148, 153, 155 and 142 genes were differentially expressed in these strains, respectively. Similar changes in gene expression were observed in macrophages infected with these strains; the upregulated genes belonged to innate immune response and host defense and the downregulated genes belonged to various metabolic pathways. Together, these data provide novel insights on the relative phenotypes and early response of macrophages to the vaccine strain and its derivatives. The Δcrp-cya derivatives could facilitate development of next-generation vaccines with improved safety.

Induced CAR-Macrophages as a Novel Therapeutic Cell Type for Cancer Immune Cell Therapies.

The Chimeric antigen receptor (CAR)-T cell therapy has made inroads in treating hematological malignancies. Nonetheless, there are still multiple hurdles in CAR-T cell therapy for solid tumors. Primary CAR-expressing macrophage cells (CAR-Ms) and induced pluripotent stem cells (iPSCs)-derived CAR-expressing macrophage cells (CAR-iMacs) have emerged as attractive alternatives in our quest for an efficient and inexpensive approach for tumor immune cell therapy. In this review, we list the current state of development of human CAR-macrophages and provide an overview of the crucial functions of human CAR-macrophages in the field of tumor immune cell therapy.

Primary Macrophage-Based Microrobots: An Effective Tumor Therapy In Vivo by Dual-Targeting Function and Near-Infrared-Triggered Drug Release.

Macrophages (MΦs) have the capability to sense chemotactic cues and to home tumors, therefore presenting a great approach to engineer these cells to deliver therapeutic agents to treat diseases. However, current cell-based drug delivery systems usually use commercial cell lines that may elicit an immune response when injected into a host animal. Furthermore, premature off-target drug release also remains an enormous challenge. Here, we isolated and differentiated MΦs from the spleens of BALB/c mice and developed dual-targeting MΦ-based microrobots, regulated by chemotaxis and an external magnetic field, and had a precise spatiotemporal controlled drug release at the tumor sites in response to the NIR laser irradiation. These microrobots were prepared by coloading citric acid (CA)-coated superparamagnetic nanoparticles (MNPs) and doxorubicin (DOX)-containing thermosensitive nanoliposomes (TSLPs) into the MΦs. CA-MNPs promoted a magnetic targeting function to the microrobots and also permitted photothermal heating in response to the NIR irradiation, triggering drug release from TSLPs. In vitro experiments showed that the microrobots effectively infiltrated tumors in 3D breast cancer tumor spheroids, particularly in the presence of the magnetic field, and effectively induced tumor cell death, further enhanced by the NIR laser irradiation. In vivo experiments confirmed that the application of the magnetic field and NIR laser could markedly inhibit the growth of tumors with a subtherapeutic dose of DOX and a single injection of the microrobots. In summary, the study proposes a strategy for the effective anticancer treatment using the developed microrobots.

Studies Into ß-Glucan Recognition in Fish Suggests a Key Role for the C-Type Lectin Pathway.

Immune-modulatory effects of ß-glucans are generally considered beneficial to fish health. Despite the frequent application of ß-glucans in aquaculture practice, the exact receptors and downstream signalling remains to be described for fish. In mammals, Dectin-1 is a member of the C-type lectin receptor (CLR) family and the best-described receptor for ß-glucans. In fish genomes, no clear homologue of Dectin-1 could be identified so far. Yet, in previous studies we could activate carp macrophages with curdlan, considered a Dectin-1-specific ß-(1,3)-glucan ligand in mammals. It was therefore proposed that immune-modulatory effects of ß-glucan in carp macrophages could be triggered by a member of the CLR family activating the classical CLR signalling pathway, different from Dectin-1. In the current study, we used primary macrophages of common carp to examine immune modulation by ß-glucans using transcriptome analysis of RNA isolated 6 h after stimulation with two different ß-glucan preparations. Pathway analysis of differentially expressed genes (DEGs) showed that both ß-glucans regulate a comparable signalling pathway typical of CLR activation. Carp genome analysis identified 239 genes encoding for proteins with at least one C-type Lectin Domains (CTLD). Narrowing the search for candidate ß-glucan receptors, based on the presence of a conserved glucan-binding motif, identified 13 genes encoding a WxH sugar-binding motif in their CTLD. These genes, however, were not expressed in macrophages. Instead, among the ß-glucan-stimulated DEGs, a total of six CTLD-encoding genes were significantly regulated, all of which were down-regulated in carp macrophages. Several candidates had a protein architecture similar to Dectin-1, therefore potential conservation of synteny of the mammalian Dectin-1 region was investigated by mining the zebrafish genome. Partial conservation of synteny with a region on the zebrafish chromosome 16 highlighted two genes as candidate ß-glucan receptor. Altogether, the regulation of a gene expression profile typical of a signalling pathway associated with CLR activation and, the identification of several candidate ß-glucan receptors, suggest that immune-modulatory effects of ß-glucan in carp macrophages could be a result of signalling mediated by a member of the CLR family.

Culture, Transfection, and Immunocytochemical Analysis of Primary Macrophages.

Macrophages are the primary phagocytes of the body and found in every tissue; often with tissue specific subtypes, e.g., microglia or Kupffer Cells. These cells are essential players in host defense, immune regulation, tissue repair, and homeostasis. Consistent with their diverse functions, macrophages display a remarkable level of plasticity and undergo rapid changes in morphology and activation state in response to environmental cues. Polarization of macrophages towards pro-inflammatory (classically activated or M1) or anti-inflammatory (alternatively activated or M2) activation states is highly dependent on their environment. These activation states result in either tissue remodeling and repair (M2) or enhanced inflammation (M1). As macrophages are dependent upon environmental cues for changes in their activation state, primary cell culture offers the ability to study macrophages under highly controlled conditions in which activation states are easily manipulated with specific growth factors, cytokines, or other signaling molecules and are readily examined through powerful tools such as immunostaining, ELISA, and Ca2+ imaging. Additionally, this approach allows the researcher to manipulate gene expression in these cells to better understanding the underlying principles and mechanisms of macrophage biology. Unfortunately, macrophages are resistant to most forms of transfection and researchers have to use either macrophages isolated from transgenic mice or viral delivery of transgenes which slows the study of these diverse cells. In this chapter we describe methods for isolating, culturing, transfecting, and immunostaining primary macrophages. Particular emphasis is placed on culture conditions and transfection protocol as we found these significantly impacted the success of this protocol. Pairing these methods with functional Ca2+ imaging enables investigation of the effects of silencing or overexpressing specific proteins on the functional properties of primary macrophages.