Bone-marrow-derived blood monocytes were for many years thought to be the precursor of all tissue macrophages. However, relatively recent studies have shown that most tissue macrophages originate from two early waves of cells from the yolk sac and that it is only the blood monocytes and one population of intestinal macrophages that originate from the adult bone marrow [1
]. Single cell analysis of different macrophage subpopulations has also shown large differences in phenotype and therefore likely indicates tissue-specific functions [8
]. Two different subpopulations of human blood monocytes have been identified, the CD14high
population that constitute approximately 80%, and a minor CD14 low and CD16 + population that constitutes approximately 20% of the circulating monocytes [9
]. These two population show large similarities but may have partly different functions in the amount of cytokines produced and levels of MHC class II expressed [9
]. In addition to the circulating monocytes, there seems to be a relatively large pool of immature monocytes, more numerous than the circulating pool, residing in the spleen as a reservoir of monocytes ready to exit the spleen and accumulate in injured tissue [10
During inflammatory conditions, monocytes can enter tissues and become tissue macrophages and their phenotype likely depends on the tissue environment [11
]. A similar situation has recently been observed for mast cells. The majority of the tissue mast cells seem to originate from an early wave of cells from the yolk sac and bone-marrow-derived mast cell precursors primarily enter tissue during inflammatory conditions such as parasite-infected intestinal regions or inflamed lungs [16
]. Following the clearance of the infection, the majority of these cells disappear, most likely by apoptosis. Most of the tissue macrophages and mast cells seem therefore to have the capacity to proliferate and thereby to restore homeostatic levels of cells if they have been consumed during an inflammatory reaction. What is then the primary function of blood monocytes when they apparently take minor part in homeostatic maintenance of the majority of tissue macrophage populations?
In order to look deeper into this issue, we here present a quantitative analysis of the transcriptome of human CD14 positive blood monocytes and how they respond to bacterial lipopolysaccharides (LPS). CD14 acts as a coreceptor to Toll-like receptor 4 (TLR-4) that together with myeloid differentiation factor 2 (MD-2) is the key sensor of Escherichia coli
]. Most previous studies of monocytes and macrophages have been single cell analysis with lineage tracing as the primary aim, which provides very limited quantitative information. For studies aiming to clarify the biological function and relevance of cells or molecules, high-resolution quantitative information is essential. By performing transcriptome analysis of purified human blood monocytes, we can here show that human peripheral blood monocytes act as very potent and rapid activators of inflammation by quick upregulation of a highly selective set of inflammatory cytokines, such as the classical IL-1α, IL-1β, IL-6 and TNF-α, and a number of inflammatory chemokines in response to LPS. One of the most extreme upregulations was seen for IL-6, which was upregulated more than 58,000 times within four hours of in vitro culture in the presence of Escherichia coli
LPS. A low basal expression level of IL-8 was detected, along with very strong upregulation of this chemokine already after four hours of LPS stimulation, when it became the most highly expressed gene in these monocytes. Only a few additional genes were upregulated, among them the super oxide dismutase (SOD2) that was increased by 28 times in expression levels. The complement factor B was also upregulated by more than 2500 times and coagulation factor 3, the tissue factor, by more than 7000 times, already at four hours of in vitro culture in the presence of LPS. This shows that monocytes act as extremely potent and rapid activators of an inflammatory response by producing massive amounts of a selective set of inflammatory cytokines and chemokines and a few additional proteins of importance for their role as inflammatory initiators.
In humans, monocytes constitute between 2 and 10% of the white blood cells and are thereby a relatively abundant immune cell of the peripheral blood. Recently, they have been found to only contribute to a very minor extent to the majority of tissue macrophage populations, so the question is, what are their major functions?
Evidence clearly shows that they can migrate to inflamed tissue, enter the tissues and become tissue macrophages of a type determined by the tissue they enter, through cell-to-cell contacts and the cytokine environment of the tissue [11
]. There, they support the local macrophage population together with incoming neutrophils by phagocytosis and possibly also additional recruitment by the production of cytokines and chemokines. However, is this their primary function? The very rapid and extremely potent upregulation of a very selective panel of inflammatory cytokines and chemokines strongly supports the hypothesis that they predominantly function as sensitive detectors for the presence of bacteria in the circulation and act as potent inducers of an inflammatory response. The very restrictive set of inflammatory cytokines essentially involving only the traditional inflammatory cytokines IL-1α, IL-1β, IL-6 and TNF-α strongly supports this conclusion. What is then the role of the set of chemokines produced upon this rapid response? One of the major upregulated chemokines is IL-8, a potent chemoattractant of neutrophils, one of the key cells for combating a bacterial infection. IL-8 is also an activator of both phagocytosis, neutrophil extracellular trap (NET) formation and later also angiogenesis, and is thereby an important player in the inflammatory response. In these activated monocytes, IL-8 actually becomes the dominating transcript only four hours after activation with 47,095 reads and is thereby the most highly expressed transcript of the activated monocyte (Table S8
and Figure 3
). A few additional chemokines including Ccl2, Ccl3, CclL3, Ccl4, Ccl20, Cxcl1, Cxcl2 and Cxcl3 were also very strongly upregulated already at four hours after activation (Table S8
and Figure 3
and Figure 4
). With a slight delay, peaking at 24 to 48 h, there is also Cxcl5 (Table S8
and Figure 4
). Ccl4, also named MIP-1β, the second most highly expressed chemokine with 20 000 reads at four hours after activation, is a potent chemoattractant for NK-cells, monocytes and a number of other inflammatory cells [20
]. The third most highly expressed chemokine, Ccl3, also named MIP-1α, with 8000 reads, is also a strong chemoattractant of neutrophils but also acts on monocytes and macrophages (Table S8
and Figure 3
]. Ccl2 shows a modest increase in expression by four hours with 1267 reads, but then increases dramatically by 24 h to 25,206 reads, being the second highest expressed chemokine by 24 h (Table S8
and Figure 3
). Ccl2 primarily attracts monocytes and basophils, indicating that activated monocytes can further enhance the response by attracting more of their own kind. This delay in response may have functional implications. If infection does not clear within 12 h, monocytes at the site of infection may need to recruit more monocytes to keep the inflammation going. This timing thereby indicates a nicely orchestrated response.
A potent upregulation of G-CSF already by four hours after LPS stimulation was also observed, indicating that monocytes do not only contribute to the inflammatory response through the classical inflammatory cytokines. In addition, they recruit neutrophils and other inflammatory cells by production of a panel of chemokines, which may contribute to triggering the bone marrow to produce more neutrophils and to activate them to be ready for phagocytosis of bacteria in the area of inflammation by producing significant amounts of G-CSF.
The rapid and potent induction of the IL-1 receptor antagonist, IL1RN and of TNIP3, the inhibitor of NFkB signaling, after activation by IL-1, TLR-4 and TNF-α, also indicates that the monocytes modulate the response to these potent inflammatory cytokines by producing receptor antagonists and potent inhibitors of the NFkB-triggered inflammatory response.
We also observed a marked upregulation of coagulation factor 3 (F3) (Table S6
). F3, also named tissue factor, is an important initial trigger of coagulation, indicating that coagulation may be part of the bacterial defense by trapping the bacteria in the area of entry by forming a local blood clot similar to the formation of extracellular traps by neutrophils.
A marked upregulation of one microRNA was also observed upon LPS stimulation of the MIR155HG, indicating its involvement in regulating the massive upregulation of cytokines and chemokines during the response to LPS. This microRNA seems to have a very complex role during inflammation, initially to suppress negative regulators of inflammation and later to enhance NFkB activation [21
The response to IFN-γ was remarkably different from that to LPS. Only a relatively modest upregulation of a few cytokines and chemokines was observed, primarily IL-27, CXCL9, CXCL10 and CXCL11 (Table S8
). The most pronounced response was for CXCL11, which went from 2.5 to 6084 reads after 24 h and CXCL9, which went from 0.2 to 1494 reads by 4 h incubation in the presence of IFN-γ (Table S8
). We found, instead of a massive increase in inflammatory cytokines, an upregulation of components connected to antigen presentation such as the MHC class II alpha and beta chains, the invariant chain, the TAP peptide transporter and also the B7 molecules. The B7 molecules are essential for the triggering of a T cell response by binding to CD28. Interestingly, for both the response to IFN-γ and LPS, we see a marked shift in the expression of the two B7 molecules. Both IFN-γ and LPS stimulation results in a marked reduction in the expression of B7:2 and an upregulation of B7:1 (Table S6
). At least in some studies, B7:1 seems to be a more potent activator of T cells in stoichiometric terms, indicating that the monocytes after inflammatory signaling can become better antigen presenters to naïve T cells [23
The non-adhesive coating of the culturing flasks has resulted in a major improvement in the culturing of blood monocytes. We observed that the absolute majority of the cells stayed non-adherent even after 24 and 48 h in culture, better mimicking the in vivo conditions compared to previous culture flasks. However, even if the cells were non-adherent, some changes in the transcriptome occurred due to culture, primarily after 24 and 48 h and not at 4 h, as for the very rapid activation of cytokines and chemokines by LPS. We did not observe any major increase in any of the inflammatory cytokines and chemokines but instead in molecules involved in lipid metabolism such as APOE and OLR1. The mechanism and importance of these changes in transcriptome are not known but need to be kept in mind using in vitro cultured monocytes in studies of their in vivo function.
One obvious question is also how well transcriptome data match with protein expression. Large combined studies of transcriptome and proteome have shown good correlation between the two, as exemplified by the study by Meissner of LPS-activated macrophages on the secretome of these cells [24
]. However, there are exceptions. In the human lung, there are mast cells that express high levels of tryptase and carboxypeptidase A3 (CPA3) mRNA but where the CPA3 protein most likely is degraded in the lysosomal compartment and therefore is not granule-stored and cannot be detected upon histochemical analysis [25
]. We therefore expect the massive increase in cytokine and chemokine transcripts to also result in a similar increase in secreted protein. However, there could be some discrepancies between the two, due to processing and transport.
Two studies of the effect of LPS stimulation in vivo in mice have recently also shown strong effects on both mRNA levels in inflammatory cytokines and chemokines and on protein levels in the serum of IL-6, TNF-α, IL-1β and IL-8 when using levels of LPS comparable or higher than what we use in vitro [26
In summary, these data indicate that human monocytes act as a highly sensitive and very potent mobile sensor of infection. Putting the cells in culture along with the presence of bacterial LPS trigger the cells to a massive inflammatory response and the production of massive amounts of a selective panel of cytokines and chemokines, a cytokine storm. The cytokines produced trigger upregulation of an acute phase response by the liver, primarily by IL-6, to increase levels of C-reactive protein (CRP) and other complement components and serum amyloids. The TNF-α upregulates adhesion molecules on the blood vessel endothelial cell surface to increase influx of inflammatory cells, including the monocytes themselves but primarily neutrophils, and the chemokines guide the inflammatory cells into the area of infection and enhance phagocytic activity. All of these findings point in the direction that the monocyte primarily acts as a sensitive sensor and a potent amplifier of the inflammatory response to various pathogens. The response to IFN-γ was quite different, with an upregulation of a few other chemokines, primarily CXCL9, 10 and 11, almost no upregulation of cytokines, except for a minor upregulation of IL-27, and also an upregulation of proteins connected to antigen presentation such as the MHC Class II genes, the invariant chain, the TAP transporter and the B7:1 molecule. This shows that monocytes adapt the response to the type of inflammatory challenge, one highly relevant and extremely potent cytokine and chemokine response to a bacterial challenge and a completely different response to viruses, with upregulation of a small set of chemokines and of the components involved in antigen presentation.
4. Materials and Methods
4.1. Purification of Monocytes from Human Peripheral Blood
Peripheral blood monocytes were isolated from whole blood, obtained as buffy coats, from five healthy donors at the University Hospital in Uppsala, Sweden. These five donors were of different age and sex, three men of age 51, 43 and 28, and two women of age 47 and 61. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll–Paque Plus (GE Healthcare, Uppsala, Sweden) and standard density gradient centrifugation. PBMCs were further washed with PBS containing 2 mM of EDTA, and incubated with anti-CD14-coated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Positive selection of CD14+ cells was performed through magnetic cell separation. Subsequently, CD14 cells were stained with anti-human CD14 PE antibody (clone: 61D3, Invitrogen, Carlsbad, CA, USA) and the purity was verified (average of 95%) by flow cytometry.
Four million of these cells were immediately frozen and stored at −80 °C for preparation of total RNA. The remaining cells were transferred into six different culture flasks with approximately 2.5 million cells per flask. We used Cellstar culture flasks with a cell-repellent surface, developed for minimal activating properties, with white filter screw cap sterile 50 mL (25 cm2) (Greiner Bio-One GmbH, Kremsmünster, Austria, product number 690985). Three culture flasks were used to culture cells without any immunostimulant, only in the presence of culture medium, RPMI-1640 with 10% fetal bovine serum (FBS). Three flasks were used to culture the cells with 1 ug/mL of Escherichia coli LPS (Sigma-Aldrich, Saint Louis, Missouri, USA, L4516- from E. coli O127:B8), or 200 ng/mL of recombinant human IFN-γ (Bio-Rad, Hercules, CA, USA, cat. PHP050). Cells from these cultures were harvested at three time points, 4, 24 and 48 h of in vitro culture.
4.2. Ampliseq Analysis of the Total Transcriptome
Total RNA was prepared from the CD14+ monocytes, both the freshly isolated and the different in vitro cultures from each donor, using the RNeasy Plus mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s recommendations. The RNA was eluted with 30 μL of DEPC-treated water, and the concentration of RNA was determined by using a Nanodrop ND-1000 (Nano Drop Technologies, Wilmington, DE, USA). Later, the integrity of the RNA was confirmed by visualization on 1.2% agarose gel using ethidium bromide staining.
The transcriptome of freshly isolated monocytes and the different cultures were analyzed for their total transcriptome by the Thermo Fisher chip-based Ampliseq transcriptomic platform at the SciLife lab in Uppsala, Sweden (Ion-Torrent next-generation sequencing system). The sequence results were delivered in the form of Excel files with normalized expression levels for an easy comparison between samples.