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In Part II I presented some case reports of IgG4-RD after COVID transfection. A literature view of various journals failed to present many papers discussing IgG4-RA (aortitis), or any research that directly investigated causality, beyond discussing immune cell histology. Part III provides essential background into the patrolling lions of our immune system - the macrophages. These have a significant role in IgG4-RD.
After reading this you will probably think of macrophages in a whole new light.
Discussion
- Macrophages on the prowl
Immunology is an area that gets very complex, very quickly, and you could easily fill a book or three. And on reading the literature you very quickly reach the limits of our understanding, with the authors proposing more research to help confirm their hypothesis or fill a knowledge gap. Our review conclusions frequently mirror this, and new papers add to our understanding each week.
For the basics, let’s go over to Wiki:
Macrophages (/ˈmækroʊfeɪdʒ/; abbreviated Mφ, MΦ or MP) are a type of white blood cell of the innate immune system that engulf and digest pathogens, such as cancer cells, microbes, cellular debris, and foreign substances, which do not have proteins that are specific to healthy body cells on their surface.[1][2] This process is called phagocytosis, which acts to defend the host against infection and injury.[3]
Macrophages are found in essentially all tissues,[4] where they patrol for potential pathogens by amoeboid movement. They take various forms (with various names) throughout the body (e.g., histiocytes, Kupffer cells, alveolar macrophages, microglia, and others), but all are part of the mononuclear phagocyte system. Besides phagocytosis, they play a critical role in nonspecific defense (innate immunity) and also help initiate specific defense mechanisms (adaptive immunity) by recruiting other immune cells such as lymphocytes. For example, they are important as antigen presenters to T cells. In humans, dysfunctional macrophages cause severe diseases such as chronic granulomatous disease that result in frequent infections.
In 2023 Bond et al. published version 2 of an excellent preprint from original research into macrophage priming: “Prior Fc Receptor activation primes macrophages for increased sensitivity to IgG via long term and short term mechanisms“1
They created a light-activated Fc receptor so that they could trigger phagocytosis on demand in a controlled fashion. This enabled them to accurately recreate repeatable conditions and look for changes (i.e. dependent variables).
“Phagocytosis is a type of endocytosis whereby a cell engulfs a particle in an internal compartment- the phagosome. The cell rearranges its membrane to surround and internalise the target particle. Phagocytosis is a major mechanism for detecting and removing potentially pathogenic material. Phagocytes also have lysosomes which are membrane-bound organelles containing hydrolytic enzymes. These fuse with phagosomes and release their cargo into the phagosome, degrading any internalised particles.” Source: https://teachmephysiology.com/immune-system/innate-immune-system/phagocytosis/
Macrophages measure the ‘eat-me’ signal IgG to identify targets for phagocytosis. We wondered if prior encounters with IgG influence macrophage appetite. IgG is recognized by the Fc Receptor.
The researchers used light to activate the macrophage FcR receptor.
CRY2, or cryptochrome circadian regulator 2, is a flavoprotein that is sensitive to blue light.
Oligomerization is where an oligomer is created from a few repeating smaller units. Oligomers can activate a wide range of signalling pathways, including CRY2 to the FcR’s.
To temporally control Fc Receptor activation, we engineered an Fc Receptor that is activated by light-induced oligomerization of Cry2, triggering phagocytosis. Using this tool, we demonstrate that Fc Receptor activation primes macrophages to be more sensitive to IgG in future encounters.
Macrophages that have previously experienced Fc Receptor activation eat more IgG-bound cancer cells.
Increased phagocytosis occurs by two discrete mechanisms – a short- and long-term priming.
ERK is short for a type of protein called “extracellular signal-regulated kinase”. A kinase (KY-nase) is type of enzyme that speeds up chemical reactions in the body by adding phosphates to other molecules, such as sugars or proteins.
ERK is part of the MAPK family (mitogen-activated protein kinase), which partakes in signalling cascades and the transmission of extracellular signals to intracellular targets:
This is just a small part of it. From: “Figure 1. MAPK cascades. MAPKs, which are present in the cytoplasm and can be translocated into the nucleus, catalyse the phosphorylation of dozens of cytosolic proteins and numerous nuclear transcription factors. Adapted from (29). MAPK, mitogen-activated protein kinase; MAP4K, MAPK kinase kinase kinase; MAP3K, MAPK kinase kinase; MAPKK, MAPK kinase; MAPKAPK, mitogen-activated protein kinase-activated protein kinases; MEK, Ras/Raf/MAPK; RSK, ribosomal s6 kinase; MSK, mitogen- and stress-activated protein kinases; MNK, MAP kinase-interacting serine/threonine-protein kinases; cPLA2, cytosolic phospholipase A2; c-FOS, proto-oncogene c-Fos; Elk1, ETS domain-containing protein Elk-1; Ets1, Protein C-ets-1; SP-1, transcription factor Sp1.” Source: ERK/MAPK signalling pathway and tumorigenesis
Priming is where an immune cell is stimulated by an antigen, the functional status of the cell changes and the immune status does not return to basal levels before the secondary infection or stimulus. The second challenge to a primed cell is often synergistic with or additive to the effects of the first.2
A variation on this is “trained immunity”, where the activation status returns to the basal level but epigenetic changes persist, and when next challenged the response is much greater than with the first stimulus:
From: “Fig. 1: Schematic presentation of the behavior of innate immune responses during the different adaptive programs induced in innate immune cells. a, Differentiation. b, Priming. c, Trained immunity (innate immune memory). d, Tolerance.” Source: https://www.nature.com/articles/s41590-020-00845-6
Long term priming requires new protein synthesis and Erk activity. Short term priming does not require new protein synthesis and correlates with an increase in Fc Receptor mobility.
Our work demonstrates that IgG primes macrophages for increased phagocytosis, suggesting that therapeutic antibodies may become more effective after initial priming doses.
Macrophages eat pathogens and infected, cancerous or dying cells via phagocytosis. To select targets for phagocytosis, macrophages measure ‘eat-me’ signals, like IgG antibodies. IgG is recognized by the Fc Receptor (FcR), which is phosphorylated and recruits the kinase Syk, triggering downstream signaling1,2.
Like a lion, they get “eat-me” signals from their prey. Only it’s via their antibody fragment chain receptors (FcR’s).
What affects macrophage appetite? One important parameter is how sensitive a macrophage is to ‘eat-me’ signals. Antibody-dependent phagocytosis requires the coordinated activation of a sufficient number of FcRs10.
Evolution minimised the risks of autoimmunity, by requiring a threshold of stimulation to be reached before it goes after its prey. But it does leave them primed and ready to pounce:
Targets with a subthreshold amount of IgG are not phagocytosed, despite triggering the initial steps in the phagocytosis signaling pathway10. In other macrophage signaling pathways, low levels of activating signal do not elicit any response on their own, but prime macrophages for rapid and intense response to future stimuli11. Whether subthreshold FcR signaling has any effect on macrophage appetite is not clear.
If the lion isn’t hungry enough it may just nibble its prey without killing it. This is a problem with macrophages too, as they can just nibble (or trogocytose) not just pathogens, but cancer cells.
Opsonisation is the process whereby antibodies or complement proteins help your immune system by identifying and marking old cells or pathogens for destruction:
During an immune response or treatment with a therapeutic antibody, macrophages encounter multiple potential targets for phagocytosis sequentially, leading to bursts of FcR activation.
Some encounters with antibody-opsonized cells result in phagocytosis of the entire cell3,5, but many cells do not have sufficient antibodies to trigger phagocytosis. Instead macrophages may trogocytose, or nibble, a target cell or simply ignore it12–15.
Just like an overfed lion, a macrophage can also lose its appetite:
In some circumstances, prior phagocytosis increases macrophage appetite16,17. In contrast, other studies demonstrated that phagocytosing several whole cancer cells reduces macrophage appetite18.
There is no clear, unifying model explaining these differences, which could be dependent on the specific ‘eat-me’ signal presented, the time since phagocytosis, the intensity of the ‘eat-me’ signal, digestion of the internalized particle, or any number of other factors19.
Less is more with macrophages, and this has implications for monoclonal Abs dosing when treating cancer:
To quantitatively control the duration and strength of FcR activation, we developed an optogenetic FcR (optoFcR). We found that prior FcR activation primes macrophages for greater responses to subsequent stimuli.
Counterintuitively, low levels of optoFcR activation induced stronger priming than high levels of optoFcR activation. Macrophage priming is controlled by two independent mechanisms, one short-term (<1 hour) and one long-term response (starting at 4 hours, and lasting up to 3 days).
FcR’s are not static constructs. Unlike what the textbooks might show, they are highly dynamic in number and location. Different classes of macrophage bristle with different receptors, and these vary according to their role (phenotype):
From: “Figure 1 Receptors or molecules of resident and inflammatory macrophages. MΦ express opsonic (FcR and C3bR) or nonopsonic receptors, such as CKRs, MRs, SRs, fMLP and TLRs, as well as express high levels of MHC Ⅱ. However, there are some differences between resident MΦ and inflammatory MΦ. Resident MΦ (left side) do not express high levels of costimulatory molecules such as CD40, CD80 and CD86, and present hyporesponsiveness to TLRs to suppress inflammation. However, inflammatory MΦ (right side) show the opposite trend. The PAMPs lead to inflammation by connecting with hyperresponsive TLRs. CKR: Cytokine receptor; fMLP: Formyl-methionine-leucyl-phenylalanine; MR: Mannose receptor; MΦ: Macrophages; PAMP: Pathogen-associated molecular pattern; SR: Scavenger receptor; TLR: Toll-like receptor.“ Source: https://www.researchgate.net/publication/323917057_Functional_macrophages_and_gastrointestinal_disorders/figures?lo=1
The short term response is associated with an increase in FcR mobility, accelerated initiation of phagocytosis and increased phagocytic cup formation.
Transcription is the process by which a piece of DNA is copied to messenger RNA (mRNA) to make a protein. In this case, it is part of the MAPK signalling:
The long term response requires activation of Erk to drive a transcriptional response. These data suggest that macrophages can integrate signaling from previous encounters with IgG to modify the response to the next target. This study provides insight into how macrophage appetite is regulated, and may suggest strategies to enhance antibody-dependent cellular phagocytosis.
Using blue light-induced CRY2 oligomerization, the researchers found that IgG binding caused the FcR receptors to cluster together, and the clustering was sufficient to induce signalling via FcR phosphorylation.
The supplement includes an awesome movie of the FcR clustering in action, - light ON vs light OFF:
Video S1:
“Representative movie of an optoFcR (visualized with mScarlet; white) expressing bone marrow derived macrophage stimulated with high intensity light. Light was on from 0-15 min and was turned off from 15-55 min. Images were taken every 30s.”
Clusters dissociate when the cells are returned to the dark (Figure 1b). Clustering also results in the recruitment of the downstream effector protein Syk, suggesting that clustering is sufficient to induce FcR phosphorylation (Figure 1c,d; Video S2).
Video S2:
“Representative movie of a RAW macrophages expressing the optoFcR (mScarlet; green) and SYK-neon (magenta) stimulated with high intensity light for the entire duration of the movie. Images were taken every 30s.”
SYK is a spleen-associated tyrosine kinase.
Of note here is that it may also act as a tumour suppressor:
“This gene encodes a member of the family of non-receptor type Tyr protein kinases. This protein is widely expressed in hematopoietic cells and is involved in coupling activated immunoreceptors to downstream signaling events that mediate diverse cellular responses, including proliferation, differentiation, and phagocytosis. It is thought to be a modulator of epithelial cell growth and a potential tumour suppressor in human breast carcinomas. Alternatively spliced transcript variants encoding different isoforms have been found for this gene.”3
From: “Figure 1. Optogenetic Fc Receptor (optoFcR) controls phagocytosis. A) Schematic of optoFcR design, containing a myristoylation signal for membrane localization, the ITAM-containing intracellular domain from the FcR gamma-chain for signaling, iRFP fluorophore, and the homo-oligomerizing peptide - cryptochrome 2 (CRY2olig). B) Representative images of optoFcR in bone marrow derived macrophages (BMDMs). The optoFcR starts dispersed on the membrane (T0m; n=0/22 cells with visible clusters), and clusters after 15 minutes of constant high intensity light (T15m; n=21/22 cells with visible clusters). The optoFcR is declustered after 30 minutes in the dark (T45m; n=0/22 cells with visible clusters). See also supplemental movie 1. C) Representative images of downstream effector protein, SYK, colocalized with optoFcR in Raw macrophages stably expressing both the optoFcR and SYK-mNeonGreen. On the right, a line scan shows the Syk and optoFcR intensity at the position indicated by the yellow arrow in the inset. See also supplemental movie 2. D) Graph shows the Pearson’s correlation coefficient for Syk and optoFcR at the cell cortex. Each data point represents a cell with an ROI drawn around a membrane region. The same ROI was used for both T0 (OFF) and T2 (ON). E) Schematic of experimental design for f-g. F) Representative images show optoFcR (green) expressing macrophages and ICAM conjugated beads visualized by atto390 in the supported lipid bilayer (magenta) before (OFF) and after (ON) 15 min of optoFcR activation with high intensity light. Internalized beads are labeled with a yellow star. See also supplemental movie 3. G) Quantification of phagocytosis in BMDMs after 15 minutes of optoFcR stimulation at low (5 uW/cm2), medium (90 uW/cm2), and high (1390 uW/cm2) intensity light compared to control cells that do not express the optoFcR but receive the high intensity light stimulus. Both medium and high intensity light stimulate phagocytosis. Each data point represents the mean of an independent experiment. Data collected in the same replicate are denoted by symbol shape. In all graphs, bars represent the mean and SEM. * indicates p<0.05, *** indicates p<0.0005 using a paired t-test (d), one way ANOVA with dunnett correction (g). Scale bars are 10 um. See also supplemental figure 1.” Source: http://biorxiv.org/content/10.1101/2023.11.14.567059v2.full-text
Macrophages expressing the optoFcR engulfed three times as many beads as control macrophages when stimulated with the highest intensity light and twice as many beads when stimulated with medium intensity light (Figure 1f,g; Figure S1; Video S3).
Video S3:
“Representative movie of an optoFcR (mScarlet; green) expressing bone marrow derived macrophage phagocytosing ICAM conjugated beads (atto390; magenta) with high intensity light stimulation. Light was on for the entire duration of the movie. Images were taken every 30s.”
Low intensity light did not activate phagocytosis. This dose response is similar to the dose response seen in IgG mediated phagocytosis (Figure S1).
The FcR ITAM (immunoreceptor tyrosine-based activation motif) domain is a conserved sequence of four amino acids that is integral to initiating the signalling pathways and activation of immune cells:
Together, these data demonstrate that clustering of the FcR ITAM domain is sufficient to initiate phagocytosis in macrophages without a specific ‘eat-me’ signal.
We found that prior optoFcR activation increased the amount of eating roughly 2-fold compared to cells that either did not receive prior light stimulation or did not express the optoFcR (Figure 2b). This demonstrates that prior FcR activation primes macrophages to respond to future IgG.
From: “Figure 2. Prior FcR activation specifically enhances macrophage sensitivity to IgG. A) Schematic of experimental design for light-induced priming. Control (mCherry-CAAX) and optoFcR expressing BMDMs were stimulated with light for 15 min. The cells were returned to the dark for either 1 or 12 hrs, then targets opsonized with 1 nM IgG were introduced. After 15 minutes of phagocytosis, beads were washed out. Cells were imaged and the number of targets phagocytosed per cell was counted. B) Quantification of experiment described in (a). OptoFcR expressing cells that received light phagocytosed significantly more than cells that did not receive light, or cells that received light but did not express the optoFcR. C) Schematic of experimental design for priming macrophages with IgG beads. Wildtype BMDMs were given either beads with 0.1 nM or 0 nM IgG for 15 min as a priming stimulus. Those beads were then washed out and the cells were allowed to recover for 1 hr. A second set of different color beads ligated to IgG at the indicated concentration were added and the amount of phagocytosis was determined after 15 min. D) Quantification of phagocytosis for experiment described in (c). Macrophages that were preincubated with 0.1 nM IgG-conjugated beads phagocytosed significantly more than macrophages preincubated with unopsonized beads. E) Phagocytosis of bead targets without an ‘eat-me’ signal. Experiment was performed as described in (a) except the beads did not contain IgG. Phagocytosis is normalized to unstimulated control cells. F) Phagocytosis of bead targets with the efferocytic ‘eat-me’ signal, phosphatidylserine. Phagocytosis is normalized to unstimulated control cells. Experiment was performed as described in (a) except the beads were covered in a 10% phosphatidylserine bilayer to mimic an apoptotic corpse. In all graphs, each point represents the mean of an independent experiment. Data collected in the same replicate are denoted by symbol shape. Bars represent the mean and SEM. * indicates p<0.05, **indicates p<0.005, **** indicates p<0.0001 by two way anova with Sidak corrections (b), multiple T-tests with Holm-Sidak corrections (d), and unpaired t-test (e,f). See also supplemental figure 2.” Source: http://biorxiv.org/content/10.1101/2023.11.14.567059v2.full-text
They found that priming was particular to FcR signalling. PS (phosphatidylserine) coated, efferocytic ‘eat-me’ coated beads did not change outcomes. In other words, in this study, they confirmed that only antibodies may prime a macrophage:
We incubated PS-coated beads with macrophages at 1 and 12 hours post light stimulation and measured the amount of eating. There was no change in the amount of eating at either timepoint (Figure 2f).
These data suggest that prior FcR activation primes macrophages to specifically react to IgG. This also suggests that the molecular regulators of priming are unique to FcR signaling, rather than in one of the pathways shared by efferocytosis and antibody-dependent phagocytosis.
Less is more:
In all cases, we found that low doses of light, and thus less FcR activity, led to the highest macrophage priming (Figure S2).
The amount of light that best primed macrophages was not sufficient to activate phagocytosis on its own (Figure 1h). This suggests that a sub-threshold level of FcR activation, not sufficient to activate phagocytosis, primes macrophages for future encounters with IgG.
They also verified experimentally that their optoFcR system was priming macrophages just like native IgG does.
Next, they found that priming only altered macrophage sensitivity to low levels of IgG, not the overall capacity for phagocytosis. The lion’s appetite was whetted beforehand, but it couldn’t eat any more of the kill than usual:
To do this, we added beads with various concentrations of IgG to BMDMs and calculated the phagocytic index (Figure S2). Primed macrophages show enhanced eating of beads with low concentration of IgG.
The total capacity for phagocytosis is not significantly changed in primed macrophages. This indicates that priming primarily alters macrophage sensitivity to low levels of IgG, rather than overall capacity for phagocytosis.
These findings were especially useful for clinical applications: as well as lower concentrations being more effective, priming leads to improved anti-cancer cell phagocytosis:
We next sought to determine if primed macrophages can increase whole cell eating of opsonized cancer cell targets. In addition to phagocytosis, macrophages often trogocytose or nibble target cells, stripping the cancer cells of target antigen without killing them15.
Prior clinical studies have shown that the anti-CD20 therapeutic antibody (rituximab) is most effective when administered more frequently at a low dose, which minimizes antigen shaving28.
The next step was to see if these findings extended to mechanisms of cancer cell phagocytosis too.
Raji lymphoblast-like B cells were isolated from the jaw of a Burkitt's lymphoma patient in 1963:
Given our results, we hypothesized that prior FcR activation might also enhance phagocytosis of cancer cells. We first measured phagocytosis of Raji B cells incubated with increasing concentrations of anti-CD20 antibody to find an antibody concentration where we could detect a change in macrophage sensitivity (Figure S3).
To measure both the amount of whole cell phagocytosis and trogocytosis, we incubated IgG opsonized Raji cell targets with primed or unprimed optoFcR-expressing BMDMs and analyzed the cells with timelapse microscopy (Figure 3a-b; Video S4-6).
Video S4:
“Representative movie of a primed optoFcR (mScarlet; green) expressing macrophage phagocytosing a Raji cell target (CellTrace Far Red; magenta). The macrophage was stimulated with low intensity light for 15 minutes 12 hrs prior to the experiment. Images were taken every 3 min.”
Video S5:
You can slow these down in the settings. A playback speed of x 0.25 works well.
To do this, pause the playback and press the 3 dots bottom right:
“Representative movie of a primed optoFcR (mScarlet; green) expressing macrophage (green) trogocytosing a Raji cell target (CellTrace Far Red; magenta). The macrophage was stimulated with low intensity light 12 hrs prior to the experiment. Images were taken every 3 min.”
OK, I know, I couldn't afford a real lion.
Video S6:
“Representative movie of an optoFcR (mScarlet; green) expressing macrophage trogocytosing a Raji cell target (CellTrace Far Red; magenta). The macrophage was not stimulated with light prior to the experiment. Images were taken every 3 min.”
We found that the number of Raji cells phagocytosed, and the percent of phagocytic macrophages increased in primed macrophages (Figure 3c-d). The percentage of primed macrophages that trogocytosed was not significantly increased compared to unprimed macrophages (Figure 3e). This suggests that primed macrophages are better at phagocytosing antibody-opsonized cancer cells.
From: “Figure 3. Macrophages are primed for increased cancer cell engulfment. A) Schematic of experimental design for cancer cell targets. optoFcR cells were exposed to light for 15 minutes, then returned to the dark. Raji B cells opsonized with 5 ng/ml IgG were added after 12 hours, and the cancer cell-macrophage interactions were captured by timelapse microscopy. B) Representative images of optoFcR expressing macrophages completing phagocytosis and trogocytosis. Raji B cells were visualized using cell trace far red (magenta) and macrophages were visualized using optoFcR-mSc (green). Yellow arrows point to phagocytosis of a whole cancer cell and white arrows point to trogocytosis of cancer cell fragments. See also supplemental movies 4-C) Percent of BMDMs that engulfed a whole target cell increased with prior FcR stimulation. D) The number of whole raji cells engulfed was greater in primed macrophages. E) Percent of BMDMs that trogocytosed a raji target was not significantly different with priming. Each data point represents the mean of an independent experiment, denoted by symbol shape, and bars represent the mean and SEM. **indicates p<0.005 using an unpaired t-test. See also supplemental figure 3.” Source: http://biorxiv.org/content/10.1101/2023.11.14.567059v2.full-text
They found that macrophage priming occurs through a short-term (peaking around 1 hour after FcR activation) and a long-term mechanism (begins at 4 hours after activation) and persists for at least 24 hours:
While 12 hours post-stimulation is likely enough time for changes in transcription or translation to affect macrophage phenotypes, 1 hour is likely too short for this mechanism.
From: “Figure 4.FcR mediated priming occurs via a short- and long-term mechanism. A) Phagocytosis of 1nM IgG beads added at the indicated time post 15 min low intensity light stimulation. Blue line denotes macrophages expressing the optoFcR and red denotes macrophages expressing a control mCh-CAAX. Enhanced phagocytosis occurs in two discrete peaks and lasts for at least 72 hrs. Points denote the mean of 4 independent replicates. Phagocytosis is normalized to unstimulated control cells. B) Macrophages were treated with Actonomycin D (AD, 10nM) or Cycloheximide (CHX, 10ug/ml) to block transcription or translation respectively starting 7 hours before phagocytosis. Macrophages were stimulated with light to activate the optoFcR at 1, 4 or 6 hours before phagocytosis. AD and CHX did not eliminate priming at 1 hour post-stimulation. AD and CHX eliminated the enhanced phagocytosis phenotype at 4 and 6 hours post stimulation. C) Macrophages were treated with Erk inhibitor (PD0325901, 0.5uM) or DMSO control for 16 hrs before measuring phagocytosis. optoFcR macrophages stimulated with light 1 hour before bead addition still showed enhanced phagocytosis. Macrophages stimulated with light 12 hours before bead addition phagocytosed the same number of beads as controls. Phagocytosis is normalized to unstimulated control cells. Each data point represents the mean of an independent experiment. Data collected in the same replicate are denoted by symbol shape. Bars represent the mean and SEM. *indicates p<0.05, **indicates p<0.005, ***indicates p<0.0005, **** indicates p<0.0001 using a two way anova with Sidak corrections (a-c).” Source: http://biorxiv.org/content/10.1101/2023.11.14.567059v2.full-text
The authors demonstrated that long-term priming likely requires de novo mRNA and protein synthesis:
We evaluated priming in macrophages treated with cycloheximide (CHX) and actinomycin D (AD) to inhibit translation and transcription respectively.
Treatment with either CHX or AD significantly reduced phagocytosis in primed macrophages compared to DMSO treated control macrophages at 4 and 6 hours post light stimulation (Figure 4b).
This suggests that de novo mRNA and protein synthesis is required for a long-term memory response. Blocking new protein synthesis did not significantly reduce priming at 1 hour post stimulation, suggesting that short-term memory is not reliant on new protein production (Figure 4b).
Overall this suggests that there are two mechanisms for macrophage priming – one that operates on a short timescale and does not require synthesis of new proteins, and one that operates on a long time scale and requires changes in gene expression.
The use of an ERK (MAPK pathway) blocker also demonstrated that long-term priming requires de novo protein synthesis via ERK signalling:
Erk, a nuclear kinase, functions downstream of the FcR and regulates the macrophage dose dependent response to LPS as well as many other immune signaling pathways11.
To determine if Erk contributes to macrophage priming, we used PD0325901 to block Erk activity. We then stimulated the optoFcR for 15 minutes, waited 1 or 12 hours, and measured phagocytosis of IgG-coated beads.
Inhibiting Erk signaling blocked long-term memory with no effect on short-term memory (Figure 4c).
These results indicate that long-term priming requires a transcriptional response mediated by Erk activation.
Next, they investigated the mechanism behind short-term priming:
We quantified the kinetics of engulfment using live cell imaging, breaking the process of phagocytosis into three steps: target binding, initiation of phagocytosis, and completion21 (Figure 5a,b; Video S7).
Video S7:
“Representative movie of an optoFcR expressing macrophage (caax-mCherry and optoFcR-mScarlet; green) phagocytosing a 1nM IgG conjugated bead (atto647; magenta). Movie was generated from a maximum projection of 7 z-stacks, 1.5um apart. Images were taken every 20s.“
Primed macrophages were more likely to initiate phagocytosis of bound beads, and the time between bead binding and initiation was less (Figure 5c,d).
In contrast, after initiation the chance of successfully completing phagocytosis and the speed of phagocytosis were the same in primed and unprimed macrophages (Figure 5e,f).
Overall, the percent of bead contacts that result in successful phagocytic events is significantly increased in primed macrophages (Figure 5g).
These data indicate that prior sub-threshold FcR activation primes macrophages for faster target recognition and more frequent signal initiation, implicating early phagocytic machinery.
Primed macrophages had a bead phagocytosis success rate of 55% vs 31% for the unprimed. Without priming, macrophages had about half the initiation rate (A and G):
From: “Figure 5. Initiation of phagocytosis is faster and more likely in primed macrophages. A) optoFcR macrophages received a 15 min light stimulus 1 hour before bead addition (primed) and were compared to optoFcR macrophages that did not receive light stimulation (unprimed). Timelapse imaging was used to quantify the kinetics of phagocytosis. Schematic shows each step in phagocytosis: binding (target contact with cell), initiation (formation of the phagocytic cup), and completion (cup closure and bead internalization). Below, the schematic shows percent of macrophage-bead contacts that progress from one stage to the next for primed and unprimed macrophages. B) Representative images of each stage. Scale bar is 10 um. See also supplemental movie 7 C) Percent of bead contacts that initiate phagocytosis is higher in primed macrophages. D) Time from binding to initiation in optoFcR primed macrophages is decreased compared with unprimed macrophages. E) Probability of completing phagocytosis after initiation is comparable in primed and unprimed macrophages. F) The speed of cup closure is comparable in primed and unprimed macrophages. G) The overall success rate of phagocytosis is higher in primed macrophages compared to unprimed macrophages. Each filled data point represents the mean of an independent experiment, denoted by symbol shape, and corresponding outlined data points represent individual bead times. Bars represent the mean and SEM. *indicates p<0.05, **indicates p<0.005, ***indicates p<0.0005 using an unpaired t-test on the means of each replicate. See also supplemental figure 4.” Source: http://biorxiv.org/content/10.1101/2023.11.14.567059v2.full-text
To conclude their investigations, they managed to optically track individual FcR molecules to demonstrate that priming is associated with enhanced mobility:
Clustering of IgG, and subsequently FcR, increases the frequency and speed of initiating phagocytosis but not the speed of cup closure, similar to the phenotype we observed in our phagocytosis kinetics analysis21.
FcR cluster formation and subsequent activation is dependent on the lateral mobility of FcRs, which is constrained by a heterogeneous F-actin ‘fence’ and other mechanisms29–32.
Increased FcR mobility correlates with increased binding of IgG-coated targets and phagocytosis29,31.
We hypothesized that primed macrophages may have higher FcR mobility, which could explain the increased speed and frequency of initiating phagocytosis.
To determine if optoFcR priming increases receptor mobility, we tracked single FcR molecules on optoFcR primed and unprimed cells (Figure 6a).
On average, the FcR molecules on primed macrophages had a higher mean jump distance (MJD; average distance traveled between frames) than on unprimed macrophages (Figure 6b).
Graphing the MJD of individual tracks, primed macrophages had a multi-modal distribution of track MJDs, suggesting there may be a more mobile population in the primed macrophages (Figure 6c).
From: “Priming increases FcR mobility. A) optoFcR macrophages were primed with a 15 minute light stimulus or were not stimulated. One hour later, single FcR molecules were labeled with QDots and tracked for 60 seconds. Tracks from a representative primed and unprimed image are shown. Scale bar is 1 um. B) Quantification of the per image mean jump distance (MJD) of all tracks. FcRs from primed macrophages had increased MJD compared to unprimed macrophages. C) Histogram of MJD from all acquired tracks from primed and unprimed macrophages. Primed macrophages show a population of FcRs that have a greater MJD than unprimed macrophages. Histogram was smoothed in prism using a second order polynomial with 6 neighbors on each size. D) Examples of ‘free’ and ‘confined’ tracks. E) Quantification of motion type analysis shows the proportion of tracks categorized as ‘free’ (circles) or ‘confined’ (triangles) for individual images. The percent of tracks defined as ‘free’ increases in optoFcR expressing macrophages exposed to light is compared to optoFcR macrophages with no light exposure. F) Quantification of the per image mean diffusion coefficients. Tracks from primed macrophages have a higher diffusion coefficient compared to unprimed macrophages. Each filled data point represents the mean from a single image. Images were acquired in three separate experiments, denoted by symbol shape (b and e). Bars represent the mean and SEM. *indicates p<0.05, **indicates p<0.005 using an unpaired t-test (b and f) or a one-way anova (e).” Source: http://biorxiv.org/content/10.1101/2023.11.14.567059v2.full-text
To conclude the paper they discuss possible mechanisms to explain these observations.
A phosphatase is an enzyme enzymes that catalyze the hydrolysis and removal of phosphate groups from other molecules.
“CD45 (lymphocyte common antigen) is a receptor-linked protein tyrosine phosphatase that is expressed on all leucocytes, and which plays a crucial role in the function of these cells.”4
CD45 may have stimulatory or inhibitory effects. The authors of this important study from 2021 only use the word “cancer” in the abstract, but their work obviously has implications for cancer patients:
… CD45, the predominant transmembrane tyrosine phosphatase in leukocytes, is required for the efficient induction of T cell receptor signaling and activation. We recently reported that the CD45-intracellular signals in peripheral blood mononuclear cells (PBMCs) of triple negative breast cancer (TNBC) patients are inhibited.
We also reported that C24D, an immune modulating therapeutic peptide, binds to CD45 on immune-suppressed cells and resets the functionality of the immune system via the CD45 signaling pathway.
Various studies have demonstrated that also viruses can interfere with the functions of CD45 and that patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are immune-suppressed.
Given the similarity between the role of CD45 in viral immune suppression and our findings on TNBC, we hypothesized that the C24D peptide may have a similar “immune-resetting” effect on PBMCs from COVID-19 patients as it did on PBMCs from TNBC patients.
We tested this hypothesis by comparing the CD45/TCR intracellular signaling in PBMCs from ten COVID-19 patients vs. PBMCs from ten healthy volunteers.
“Downregulation of CD45 Signaling in COVID-19 Patients Is Reversed by C24D, a Novel CD45 Targeting Peptide” (2021)
The FcR has no inherent kinase activity, so why does clustering promote receptor activation? ITAM phosphorylation is controlled by the opposing actions of Src family kinases, which favor activation, and transmembrane phosphatases like CD45 that deactivate the receptor1.
Prior work has shown that particle binding drives CD45 exclusion from the phagocytic synapse because the bulky extracellular domain is sterically excluded from the tight membrane-membrane interface35–38.
To illustrate this, the graphical abstract from a study by Bakalar et al:
This allows Src family kinases to dominate, tipping the kinase-phosphatase balance in favor of FcR activation39.
Our prior work has shown that FcR clustering enhances ITAM phosphorylation independent of avidity effects that would increase receptor binding to the target21.
Ordered lipid-dense rafts may house clusters of B cell receptors (BCRs), and prevent phosphatases like CD45 from deactivating the FcR:5
This may be mediated by the formation of lipid ordered domains that segregate kinases and phosphatases, favoring immune receptor activation40,41.
The optoFcR is tethered to the membrane via a myristoylation motif, favoring inclusion in lipid ordered domains, which may promote activation by Src family kinases and segregation from CD45 without the need for particle binding. Alternatively, the geometry of the clustered intracellular FcR domains may promote activation through another mechanism.
More on the important protein SYK:
“Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that has long been thought to exclusively mediate signalling by receptors of the adaptive immune response (B cell receptors (BCRs) and Fc receptors (FcRs)). However, recent studies indicate that it also participates in innate immunity and non-immune functions
… SYK participates in innate recognition of fungal and other microbial pathogens, as well as of tissue damage, by C-type lectins. SYK activation by C-type lectins activates the caspase-recruitment domain 9–B cell lymphoma 10–mucosa-associated lymphoid tissue lymphoma translocation protein 1 (CARD9–BCL-10–MALT1) pathway and it is also required for NLR family, pyrin domain-containing 3 (NLRP3) inflammasome activation following fungal infection.”6
Prior work has shown a critical threshold of FcR activation is required for macrophages to commit to phagocytosis10. FcR signaling that is below this threshold activates the initial steps in the phagocytic signaling pathway, including recruitment of the downstream effector kinase Syk10.
Whether this low level of activation has any effect on macrophages was not clear. Our data suggests that this low level of activation may have a purpose in the macrophage, allowing the cell to prepare for future encounters with IgG-bound targets.
Our data show that lower levels of optoFcR stimulation prime phagocytosis better than high levels. Prior work has shown that low levels of TLR activation prime macrophages for a rapid and strong response to future stimuli, without activating an inflammatory response alone11. Our study suggests this may be true for the FcR pathway as well.
The effect of high levels of FcR activation may be different because it is associated with receptor internalization, which can lead to decreased phagocytic capacity18.
Previous studies have shown that phagocytosing many antibody-coated cancer cells causes macrophage ‘hypophagia’ or reduced phagocytosis. This suggests that the signaling consequences of successful phagocytosis and sub-threshold FcR activation may be quite different.
While the adaptive immune system is traditionally thought of as the source of immunological memory, a growing body of evidence shows that the innate immune system also remembers prior infections and threats. This is often called “trained immunity” and occurs via epigenetic reprogramming of myeloid cells to increase or decrease their transcriptional response to reinfection16,43,44.
This is a problem if the trained immunity promotes IgG4 and Spike tolerance. Failed HIV vaccine studies demonstrated recall of IgG4 abs 8 years after boosting:7
Trained immunity can persist for years if myeloid progenitor cells are affected. Most of the work on trained immunity has focused on a memory of pathogenic molecules or inflammatory cytokines.
Our work builds on this, suggesting that FcR activation also elicits a long term molecular memory. In contrast, the short term priming we describe is distinct from prior descriptions of trained immunity since it does not involve changes in gene expression.
Implications for treating cancer:
The FcR is required for the full efficacy of many cancer immunotherapies, including popular immunotherapies like PD-1 and CTLA-4 blockades9,45. Some therapies, like the anti-CD20 antibody Rituximab, heavily rely on antibody-dependent cellular phagocytosis as a mechanism for eliminating cancer cells4.
Interestingly, more frequent low dose treatments of Rituximab are more effective at treating Chronic Lymphocytic Leukemia (CLL) patients than higher dose treatments28.
A key reason for this dosing schedule is to mitigate antigen shaving, or trogocytosis of target antigen. Enhancing phagocytosis without increasing antigen shaving is important but difficult.
Our data shows that primed macrophages are better at phagocytosing whole cancer cells, but equally likely to trogocytose. This suggests that the current dosing regimen of frequent, low doses may already benefit from the effects of macrophage priming.
Additionally, monocytes expressing Chimeric Antigen Receptors that signal through the FcR intracellular signaling domain are an exciting new avenue of cancer research46–49.
How can we engineer hungrier macrophages to attack cancer cells? Our studies reveal that macrophage priming could enhance phagocytosis or other anti-cancer signaling pathways in these macrophages.
Competing interests
The authors A.B., M.Z.W. and M.A.M. have filed a patent relating to this material. The authors have no other competing interests.
Subinfectious viral exposure leads to priming against severe disease
Even if you live like a hypochondriac behind a wall of NPI’s this may be counter-productive. Efficacy is debatable. If they do work then all you may achieve is to disrupt the normal process of gaining immunity to pathogens in our environment through subinfectious exposure to viruses and potentially cross-reactive variants. This can lead to a severe infection later.
There aren’t many studies into this, probably because it runs counter to vaccine dogma.
From this study, note that herd immunity increased rapidly prior to the gene therapy rollout, from the end of 2020. There was never any need for it by then, even if it had been effective and didn’t poison a large part of the population.
From “Repeated Exposure to Subinfectious Doses of SARS-CoV-2 May Promote T Cell Immunity and Protection against Severe COVID-19”8 (2021) by De Angelis et al.:
Abstract
Europe is experiencing a third wave of COVID-19 due to the spread of highly transmissible SARS-CoV-2 variants. A number of positive and negative factors constantly shape the rates of COVID-19 infections, hospitalization, and mortality. Among these factors, the rise in increasingly transmissible variants on one side and the effect of vaccinations on the other side create a picture deeply different from that of the first pandemic wave. Starting from the observation that in several European countries the number of COVID-19 infections in the second and third pandemic wave increased without a proportional rise in disease severity and mortality, we hypothesize the existence of an additional factor influencing SARS-CoV-2 dynamics. This factor consists of an immune defence against severe COVID-19, provided by SARS-CoV-2-specific T cells progressively developing upon natural exposure to low virus doses present in populated environments. As suggested by recent studies, low-dose viral particles entering the respiratory and intestinal tracts may be able to induce T cell memory in the absence of inflammation, potentially resulting in different degrees of immunization. In this scenario, non-pharmaceutical interventions would play a double role, one in the short term by reducing the detrimental spreading of SARS-CoV-2 particles, and one in the long term by allowing the development of a widespread (although heterogeneous and uncontrollable) form of immune protection.
Figure 1. Epidemiological data of the Italian National Institute of Health (Istituto Superiore di Sanità, Rome, Italy) showing the clinical status of COVID-19 patients from January 2020 to April 2021. Colored bars represent the percentage of cases, respectively, with critical clinical conditions (top bars, dark red, clinical manifestations affecting the respiratory tract and/or other organ systems that require hospitalization in intensive care), with severe disease (orange bars, clinical manifestations affecting the respiratory tract and/or other organ systems that require hospitalization, not in intensive care), with mild disease (yellow bars, clinical manifestations affecting the respiratory tract and/or other organ systems that would not normally require hospitalization), with paucisymptomatic disease (blue bars, mild and general symptoms e.g., general malaise, fever, fatigue, etc.), and with asymptomatic disease (green bars, no apparent signs or symptoms of disease). Raw data in the xlsx format are publicly available at the Istituto Superiore di Sanità website www.epicentro.iss.it/coronavirus/sars-cov-2-dashboard (accessed on 23 April 2021).
Figure 2. Modifications occurring in the lung microenvironment upon exposure to high doses (left) or low doses (right) of SARS-CoV-2. High (left) or low (right) numbers of SARS-CoV-2 particles enter pulmonary alveoli and infect airway epithelial cells. In the left panel, massively infected airway epithelial cells undergo cell death releasing damage-associated molecular patterns (DAMPs). DAMPs activate neighboring cells to produce inflammatory cytokines and recruit activated monocytes, macrophages, and neutrophils. In the inflamed lung microenvironment T lymphocytes, after an initial activation, undergo functional exhaustion, while inflammatory monocyte-derived macrophages (inflammatory MACs) substitute alveolar macrophages. In the right panel, low virus doses come in contact with airway epithelial cells and alveolar macrophages. Viral particles are processed and presented to T cells by lung epithelial cells and alveolar macrophages (alveolar MACs), thus generating a controlled immune response. Protective immunity may evolve into efficient long-term immunological memory upon repeated antigenic stimulation.
Keywords:
COVID-19; SARS-CoV-2; protective immunity; T cell responses; facial masking; fomites; environmental exposure; memory T cells
Another study used a model to test the hypothesis that COVID lockdowns and NPIs contributed to a more severe outbreak of RSV in 2021-22, when measures were finally relaxed and children and newborns could start to socialise again.
The authors also appear to unintentionally make a case for natural infection to maintain immunity rather than inferior short-term immunity gained through vaccination, with its inherent risks, - including death.
Key takes from “Natural immune boosting can cause synchrony in susceptibility and outbreaks of respiratory infections with rapidly waning immunity”9 (2023) by Pritchard et al.:
After periods of reduced transmissibility, representing non-pharmaceutical interventions, simulations with more immune boosting exhibit larger rebound outbreaks that occurred sooner.
Respiratory syncytial virus (RSV) infection elicits remarkably brief immunity. Rein-fection is possible within two months [19], and each year 4–10% of adults are reinfected [20]. In 2020, many governments mandated non-pharmaceutical interventions to reduce the spread of SARS-CoV-2, such as school closures, restrictions on gathering, and requirements to wear face coverings [21]. This disrupted RSV transmission, and the typical annual outbreaks were not seen in 2020 [22–24]. Unusually early and large outbreaks of RSV occurred in the following two years, attributed to increased susceptibility in the population [25–27].
From “Figure 4: Respiratory syncytial virus in Scotland. The top plot, and black points in subsequent plots, show recorded respiratory syncytial virus infections in Scotland. Green lines represent simulations with different boosting coefficient values. Grey shaded area represents the period of high stringency index (>50) in Scotland. In each plot, birth rate is 0.087 per year, and mean generation time is 7.5 days.” Source: https://www.medrxiv.org/content/10.1101/2023.11.23.23298952v1.full
This also means that it’s usually a relatively mild illness:
Assuming that, before non-pharmaceutical interventions, all infants were infected in the first two years of life [29] and 4–10% of adults were reinfected annually [20], at least 98% of RSV infections were unrecorded.
Rather than benefiting from oscillations, large peaks in numbers infected may overwhelm healthcare systems, and public health authorities may instead need to direct efforts to ‘flattening the curve’ [37].
Or perhaps reconsider the use of NPI’s in future outbreaks instead of vaccinating. Adequate nutrition too, especially:
We have not included vaccination in our model, but the potential for an outbreak driven by the return to susceptibility suggests a possible strategy for repeat vaccination to be timed with the nadir of the force of infection.
Explicit consideration of the balance between the effects of immune waning and seasonal forcing would be needed to determine the optimal vaccination strategy.
The authors list the simplifying assumptions used in their model. We know how models can tell you anything - garbage in, garbage out (Ferguson, cough). Backtesting against outcomes is needed, but in my opinion, the theory is sound and supported by research into priming the immune system:
Our analysis has many simplifying assumptions. We assumed all-or-nothing immunity with those whose immunity had waned being indistinguishable from those who were immune-näıve. This does not reflect realistic patterns of immunity for RSV, influenza [43], or SARS-CoV-2 [44].
Our model also neglects clinically-important protection against severe disease and death, which persists after mucosal immunity to infection has largely waned [45]. We assumed that the effects of starting and stopping non-pharmaceutical interventions were immediate, but during the covid-19 pandemic some restrictions remained in place for longer than others [21], and levels of engagement with restrictions varied over time [46].
Our model differs from many RSV-specific models that include distinct sequential susceptible, infectious and immune states to represent accumulating effects of re-exposure [47].
We assumed the pathogen was homogeneous, with no variation in strains and no evolution over time. Even for RSV, in which the immunogenic F protein is highly conserved [48], this may not accurately capture all relevant effects, as the dominant RSV group varies between outbreaks [49].
SIRS:“The basic SIR model1 has three groups: susceptible (S), infectious (I) and recovered (R), with a total population size N = S + I + R. It is parametrized by the infectious period 1/γ, the basic reproduction number R0 (the number of secondary cases for each infection in a completely susceptible population) and the contact rate β = γR0.”10
In models with SIRS dynamics, loss of immunity due to waning is equivalent to loss of immunity due to pathogen evolution [50]. Given that exposure to new strains of influenza virus can boost immune response to multiple previous strains [43], it may be plausible that a similar equivalence exists for our model of immune boosting.
Another plus for naturally acquired immunity, and this isn’t helped by vaccine-induced original antigenic sin (OAS):
Alternatively, if a new variant caused infection rather than boosting immunity to a previous variant then different mechanisms of immune loss would lead to different patterns of immunity.
Other studies reached similar conclusions, even with lifelong immunity and a high R number. In other words, repeat exposure at subinfectious levels:
Baker and colleagues’ [52, 53] model of the effects of non-pharmaceutical interventions on transmission produced qualitatively equivalent results to our model, but assumed lifelong immunity and R0 ≈ 7.
I think what they are trying to say here is that whilst lockdowns and other NPIs should work, in reality, they make things worse by underestimating natural immunity and its maintenance:
Each model specification and set of parameters can lead to different predictions for disease dynamics. Underestimating natural immune boosting leads to underestimates of transmissibility and overestimates of the effect on incidence of a reduction in transmission.
More research is called for into natural immunity and priming vs using NPIs and vaccine programs. I would add nutritional and microbiome research to this:
It is therefore vital to establish more robust understanding of natural immune boosting for respiratory-tract infections to inform successful disease control interventions.
Our final paper concerns HIV. Despite the narrative originating by Fauci and endlessly pushed by the MSM, you can be non-vaccinally immune to being infected by HIV. Actually it’s the base case with newborns from mothers with HIV, and is a great example of the benefits of subinfectious exposure and priming.
From: “Human Immunodeficiency Virus (HIV)–Specific Cellular Immune Responses in Newborns Exposed to HIV In Utero”11 (2002) by Kuhn et al.:
Abstract
Significant immunological changes are associated with intrauterine human immunodeficiency virus (HIV) encounter among uninfected infants of HIV-infected mothers. Peripheral blood cells of more than one-third of these exposed-uninfected infants proliferate and produce IL-2 after stimulation with HIV, and HIV-specific CD4+ T helper cell responses can be quantified in nearly all when sensitive intracellular cytokine assays are used. HIV-specific CD8+ cytotoxic T lymphocyte responses can be elicited in some, although less frequently. It is difficult to demonstrate that these responses are components of protective immunity and not simply epiphenomena of exposure. However, HIV-specific responses are associated with lack of infection, even with prolonged reexposure through breast-feeding. Elevations in nonspecific markers of immune activation provide further corroboration, as do similar findings in adults, consistent across all known routes of HIV transmission. Many questions remain, but much can be learned from this special population that may be informative for development of effective immunity in response to HIV vaccines.
No need for big pharma. Your immune system may be primed without vaccines, and mothers don’t even need to be on antiretroviral drugs to protect the child:
It has intrigued the scientific community that kinds of apparently HIV-specific anamnestic cellular immune responses can be detected in individuals exposed to HIV but who remain uninfected [1, 2]. Uninfected infants of HIV-infected mothers are a special case of such an exposed-uninfected population and offer a unique opportunity to investigate the significance of these observations. Even in the absence of antiretroviral drugs, most infants of HIV-infected mothers (65%–85%) do not acquire HIV infection. However, a proportion of uninfected infants may have been sensitized or primed to HIV in utero.
From a South African study:
Just under 10% of infants with envelope-specific responses had HIV RNA detected in venous blood collected on the day of birth, which implies intrauterine transmission. Those not already infected were followed prospectively to quantify the subsequent risk of transmission through intrapartum routes and breast-feeding (two-thirds were breast-fed). None of the infants responsive to HIV envelope peptides in cord blood was found to be HIV-infected on subsequent tests, whereas 17% of infants unresponsive to envelope peptides acquired HIV infection intrapartum or postpartum (P = .02) (figure 1). Responses to non-HIV antigens were not associated with transmission [11].
Figure 1. Evidence of lower risk of intrapartum and breast-feeding HIV transmission associated with HIV envelope–stimulated IL-2 production in cord blood leukocytes. Shown are the Kaplan-Meier probability curves of detecting HIV RNA by the age (up to 1 year) among 59 breast-fed infants born to HIV-infected mothers stratified by their HIV-stimulated response in cord blood. No mothers were treated with antiretroviral drugs. IL-2 production in cord blood leukocytes was measured after stimulation with HIV envelope peptides by bioassay. Those with stimulation indices >3 were considered responsive to envelope, and those with stimulation indices <3 were considered unresponsive [11].
SIV is a virus that Macaques may be primed for. Macrophages present foreign antigens via MHCI and II (major histocompatibility complexes) to T cell receptors:
Macaques inoculated with doses of simian immunodeficiency virus (SIV) below the threshold required for seroconversion exhibited T cell proliferation to SIV peptides and, when subsequently challenged with higher SIV doses, did not become infected. Animals never previously exposed to SIV all became infected [85].
You don’t hear this widely discussed, but antiretroviral drugs paradoxically led to higher viral loads in both parent and child and therefore worse outcomes:
In 1 study of HIV-infected pregnant women treated with short-course combinations of zidovudine-lamivudine in Soweto, South Africa, HIV envelope peptide–stimulated IL-2 production in cord blood leukocytes was reduced >10-fold compared with that in cord blood from HIV-infected untreated women [92]. These findings are puzzling, because although antiretroviral drugs could theoretically have reduced HIV from maternal blood and other fluids, effectively reducing antigenic exposure, all women on treatment had levels of HIV RNA in plasma at delivery well above the threshold of detection. The effects of the drugs were dramatic, even when they were started only shortly before delivery (figure 2). In at least 1 other exposed-uninfected population (health care workers with occupational exposures to HIV), reductions in HIV-specific responses were observed with postexposure prophylaxis with zidovudine [93].
Figure 2. Reduced HIV-stimulated IL-2 production in cord blood leukocytes from infants of HIV-infected mothers treated with antiretroviral drugs. Shown are the stimulation indices quantifying the amount of IL-2 produced after stimulation with HIV envelope peptides among group 1, HIV-infected mothers not treated with any antiretroviral drugs during pregnancy or peripartum; group 2, HIV-infected mothers treated with zidovudine-lamivudine started only at the onset of labor; group 3, HIV-infected mothers treated with zidovudine-lamivudine at ∼36 weeks gestation; and group 4, HIV-uninfected control mothers [92].
In IgG4-RD, infiltrating M2 macrophages are thought to play an important role in the generation of the characteristic pathophysiology such as Type 2 helper T lymphocytes (Th2) immune responses and fibrosis through the production of pro-fibrotic cytokines (IL-10, IL-33) and chemokines (The cc-chemokine ligand 18,CCL18).
From: “Immunological mechanism of IgG4-related disease” (2020)
I’ve written before about the galectin-3 fold on the NTD region of S1 Spike. One of the pathways to IgG4 is hypothesised to be:
S1 Gal-3 > Polarised Th2 responses12 > Th2 interleukins (e.g. IL-4, IL-5, and IL-13) > IgG1 to IgG413
Interleukins 4, 5, and 13 help to induce typical allergy-associated IgE and eosinophilic responses, as well as IgG4-RD pathologies.
In our paper “Review: N1-methyl-pseudouridine (m1Ψ): Friend or foe of cancer?”14(2024) we discussed another. This was how 100% m1Ψ-modified mRNA suppressed interferon alpha signalling, and how this was associated with macrophage polarization to the IgG4 and tumour-promoting M2 phenotype:
Interestingly, IFN alpha (IFN-α) (a Type-I IFN) reprograms M2 macrophages to the M1 phenotype, inhibiting tumor growth and metastasis [78]. Conversely, M2 macrophages facilitate invasion, proliferation, and migration as well as apoptosis inhibition of glioma cells (the commonest and deadly tumor of the central nervous system), thus promoting immune escape [79]. Regarding COVID-19 vaccines, even though the BNT162b2 vaccination effectively produced cellular and humoral immunity against SARS-CoV-2, it waned at six months and also reduced IFN-α and IFN-γ levels [49]. We propose that the reduction of IFN-α and IFN-γ levels after BNT162b2 vaccination could favor a shift from M1 to the M2 phenotype, thus promoting cancer growth and metastasis.
Impaired toll-like receptor (TLR) signalling was another factor associated with a less favourable cancer prognosis due to reduced inflammation by M1 macrophages.
Of relevance to this Substack, in 2017 Furukawa et al. published the study “Interleukin-33 produced by M2 macrophages and other immune cells contributes to Th2 immune reaction of IgG4-related disease”.15
Note the positive feedback here, due to the Th2 cytokines as discussed earlier.
Therefore, not only is synthetic Spike associated with IgG4 class switching but engineered mRNA-induced polarisation to M2 macrophages and T helper cell type 2 (Th2) immune responses. This is the kind of immune priming you would wish to avoid.
It would be instructive to have both post-vax histology and cytokine reports from our case studies:
Interleukin-33 (IL-33) is a recently described cytokine that is secreted by damaged epithelial cells, macrophages, and dendritic cells, and potently activates helper T type 2 (Th2) immune responses, which have been suggested to play a major role in IgG4 production of IgG4-RD.
Here, we assessed the expression of IL-33 and related molecules in the salivary glands (SGs) of patients with IgG4-RD versus that in patients with Sjögren’s syndrome (SS) and controls.
Expression of IL-33 and its receptor (ST2) was strongly detected around ectopic germinal centers (GCs) in the SGs from patients with IgG4-RD, whereas IL-33 was expressed only in epithelial cells in patients with SS and controls.
Moreover, IL-33 and CD68+/CD163+ macrophages were mainly distributed around ectopic GCs in patients with IgG4-RD. Double immunofluorescence staining showed that IL-33 expression co-localized with CD68+/CD163+ macrophages.
Finally, mRNA expression levels of IL-33 showed a positive correlation to those of Th2 cytokines (IL-4 and IL-13) in patients with IgG4-RD.
Our data suggest that IL-33 produced by M2 macrophages might contribute to the pathogenesis of IgG4-RD via aberrant activation of Th2 immune responses.
From: “Figure 1: The expression of IL-33 and related molecules was higher in patients with IgG4-related disease (IgG4-RD). (A) mRNA expression levels of IL-33 and related molecules was examined in salivary glands (SGs) from controls (n = 10), and patients with Sjögren’s syndrome (SS) (n = 10) and IgG4-RD (n = 7). Molecules were quantitatively estimated as described in the Methods section, and significant differences between groups were determined by the Mann–Whitney U test (*p < 0.05, **p < 0.01). (B) Correlation between mRNA expression levels of IL-33 and related molecules in SGs from controls, as well as patients with SS and IgG4-RD. Statistical significance of differences between groups was determined by Spearman’s rank correlation (p < 0.05). (C) Distribution of IL-33 and related molecules in SGs from representative controls and patients with SS and IgG4-RD. Counterstaining was performed with Mayer’s hematoxylin (blue). IL-33 positive ductal epithelial cells are indicated by block arrows, while IL-33 positive cells are indicated by yellow arrows. Scale bars, 100 μm.” Source: https://www.nature.com/articles/srep42413
Immunosuppression caused by macrophage polarisation, PD-1 and IgG4 class switching is one type of vaccine-associated pathology. At the other end of the spectrum, we have positive feedback loops of macrophage activation leading to severe hyperinflammation, and this may be deadly.
Fajiloun et al. discussed the phenomena in a recently published paper from July ‘24.
Key takes from “Unveiling the Role of SARS-CoV-2 or mRNA Vaccine Spike Protein in Macrophage Activation Syndrome (MAS)“16
There is a caution from the journal:
“Note! Please note that this article is currently in the "Article in Press" stage and is not the final "Version of record". While it has been accepted, copy-edited, and formatted, however, it is still undergoing proofreading and corrections by the authors. Therefore, the text may still change before the final publication. Although "Articles in Press" may not have all bibliographic details available, the DOI and the year of online publication can still be used to cite them. The article title, DOI, publication year, and author(s) should all be included in the citation format. Once the final "Version of record" becomes available the "Article in Press" will be replaced by that.”
Hemophagocytic lymphohistiocytosis:“A rare disorder in which histiocytes and lymphocytes (types of white blood cells) build up in organs including the skin, spleen, and liver, and destroy other blood cells.”17
Macrophage Activation Syndrome (MAS), a severe and life-threatening disorder, is a subtype of secondary hemophagocytic lymphohistiocytosis [1,2]. MAS is characterized by an excessive and uncontrolled immune response involving the activation and proliferation of macrophages and T lymphocytes, leading to a hyperinflammatory state marked by elevated levels of proinflammatory cytokines known as a “cytokine storm,” linked to hemophagocytosis [3].
Pre-2021 incidence?
MAS is an old syndrome with an incidence estimated at 0.16-0.4/100,000 inhabitants, a prevalence of 1-24/million inhabitants, and the median age at diagnosis varying from 27 to 36 years [6].
Classically activated macrophages are induced by interferon-γ (IFNγ), which is generated during adaptive immune responses by CD4+ or CD8+ T cells, innate immune responses by natural killer (NK) cells, and by tumor necrosis factor (TNF), which is produced by antigen-presenting cells [7].
Hence, MAS can occur spontaneously (primary MAS) or as a secondary complication of microbial infections, rheumatism, tumors, or medications (secondary MAS), with rheumatism (Systemic Juvenile Idiopathic Arthritis (SJIA)) being the most frequent cause of MAS [3,8,9].
Explain it like I’m 5: Disease or vax-induced inflammation can cause your immune system to overreact, leading to overproduction of antivirals, which further amplifies your immune responses, leading to MAS. This may also cause your white cells to start attacking your red cells.
In the case of arthritis, elevated levels of cytokines, such as IL1, IL-6, IL-18, and TNFα are produced. On the other hand, in cases of infections (bacterial, parasitic, viral, or fungal), pathogenassociated molecular patterns are sensed by toll-like receptors (TLRs), also leading to the production of inflammatory cytokines. This increased production of proinflammatory mediators (IL-2, IL-6, IL-8, IFNγ, TNFα, GM-CSF, and M-CSF) in both scenarios can amplify TLR signaling.
Moreover, infections also stimulate the activation and proliferation of CD8+ , CD4+ T cells, and NK cells, resulting in IFNγ secretion. Elevated levels of IL-18 further boost IFNγ production from activated lymphocytes. The resulting IFNγ binds to the IFNγ receptor (IFNGR) and subsequently induces phosphorylation of STAT1 by JAK1 and JAK2 in the cytoplasm.
The STAT1 dimer binds to the γ-interferon activation site (GAS) and enhances the transcription of interferonstimulated genes, such as interferon regulatory factor 1 (IRF1), thereby causing MAS. The activation of STAT1 by IFNγ can also induce micropinocytosis, leading to engulfment and degradation of erythrocytes (Fig. 1) [10-12].
As with IgG4, misdiagnosis is common:
The symptoms of MAS are often similar and confused with several active autoimmune diseases or sepsis, leading to delayed diagnosis, worsening outcomes, and increased mortality and morbidity [6,8]. This confusion is exacerbated by the absence of specific diagnostic tests or established criteria for distinguishing MAS from SJIA [10].
Part of the reason for misdiagnosis is heterogeneous conditions. It’s a long list!
Manifestations of this illness typically include persistent high-grade fever, skin rash and hemorrhagic manifestations (such as petechiae, ecchymosis, purpura, bruising, intravascular coagulation, gastrointestinal and mucosal bleeding), hepatosplenomegaly, lymphadenopathy, abnormal liver function, lung failure (including pleural effusion, respiratory failure, pneumonia, interstitial infiltrates, pleurisy, pulmonary hemorrhage, acute respiratory distress syndrome), icterus, renal failure, proteinuria, hematuria, neurological impairment (manifesting as irritability, disorientation, lethargy, headache, seizures, coma, mood swings, confusion) and cardiac failure (pericardial involvement, arrhythmia, heart failure, cardiomegaly) [6,8,13,14].
Laboratory findings of MAS typically show elevated ferritin and aspartate aminotransferase levels, decreased platelet and white blood cell counts (lymphopenia), hypofibrinogenemia, neutropenia, anemia, and low NK cell counts. Additionally, in many cases, checking for bone marrow hemophagocytosis is necessary [8,13,15].
Mechanisms include disruption of the renin–angiotensin system (RAS), leading to elevated Angiotensin II (Ang II), ROS production, DNA damage, and stimulation of the NF-κB pathway.
Note: Engineered spike also does this by binding to ACE2. Even without MAS you suffer accelerated ageing through ROS, DNA and endothelial damage.
This is taken from “SARS-COV-2 spike protein promotes RPE cell senescence via the ROS/P53/P21 pathway”18(2023) by Zhang et al.
“ARPE-19 is a spontaneously arising retinal pigment epithelia (RPE) cell line derived from the normal eyes of a 19-year-old male who died from head trauma in a motor vehicle accident. This cell line is a suitable transfection host.”19
They found that ARPE-19 cells express ACE2, and transfected Spike induced their senescence, and NAC can massively reduce ROS generation. I still have some standing by.
ROS levels increase very quickly in response to Spike, leading to retinopathy and age-related macular degeneration (AMD).
DCFH-DA is a fluorescent dye which they used as a marker for ROS generation.
SA-β-Gal is a marker for senescence. D and E demonstrate the effects of NAC:
From: “Figure 4. Removing ROS by NAC down-regulates S-protein-induced senescence of ARPE-19 cells. A Flow cytometry assay measuring DCFH-DA-degenerated fluorescence level in ARPE-19 cells that were treated with S-protein for 24 h (upper panel) or transfected with plasmid p3xFlag-S (lower panel). B and C Quantitation of DCFH-DA fluorescence in A. The data represent mean ± SD (n = 3), ***p < 0.001. D SA-β-Gal stain of ARPE cells that were treated with S-protein or S-protein + NAC for 48 h. E Quantitation of the percentage of SA-β-Gal positive cells in D. The data represent mean ± SD (n = 3), ***p < 0.001. F SA-β-Gal stain of ARPE-19 cells that were transfected with p3xflag-S followed by NAC treatment for 48 h. G, Quantitation of percentage of SA-β-Gal positive cells in F. The data represent means ± SD (n = 3), ***p < 0.001. H, immunoblot the expression of P53, P21 and GAPDH in ARPE-19 cells that were treated with media containing PBS (lane 1), S-protein (lane 2) and S-protein + NAC (lane 3)”. Source: https://link.springer.com/article/10.1007/s10522-023-10019-0
Returning to the MAS paper, Ang II also stimulates the disintegrin and metallopeptidase ADAM17 via the p38/MAPK pathway, which is involved in generating soluble forms of proinflammatory TNF-α. This then further stimulates NF-κB in a positive feedback loop leading to cytokine storm, lymphopenia of CD4+ and CD8+T cells and, in the case of viral infection, less antiviral NK and interferon production.
The increased viral load, proinflammatory cytokines and hyperactivated macrophages and neutrophils cause further tissue damage. This includes endothelial dysfunction, ARDS, and coagulopathy in severe cases, with the risk of multi-organ failure and death.
From: “Fig. (1). Pathophysiological pathways contributing to Macrophage Activation Syndrome (MAS). MAS can develop due to various causes, including rheumatic diseases, drugs, tumors, antibodies, immune complexes, and infections. These triggers activate macrophages by stimulating cytokine production. Regarding SARS-CoV-2 infection, it can induce MAS through multiple mechanisms, including the activation of TLR, NOX, and ADAM-17 pathways. MAS can lead to several complications, such as endothelial dysfunction, coagulopathy, respiratory illnesses, and potentially contribute to death.” Source: https://www.eurekaselect.com/article/141832
In conclusion:
MAS is a very severe condition that can be fatal. Following infection with SARS-CoV-2 or vaccination, particularly with mRNA encoding the viral spike protein, many cases of MAS have been reported in published studies. We reiterate our suggestion regarding the importance of the impairment of the RAS in contributing to numerous non-genetic human diseases, including immunological syndromes [35].
Treatment overlaps with those administered for IgG4-RD:
Management MAS typically involves high-dose corticosteroids, which can occasionally lead to fatal outcomes.
Alternative treatments such as cyclosporin A (CyA), TNFα inhibitors, and IL-1 blockers like anakinra have shown varying degrees of success, with CyA being notably effective in severe or corticosteroid-resistant cases.
Early in 2020, there was evidence that those on ACE inhibitors for hypertension had better COVID outcomes20. There appear to be grounds for this, but it was controversial at the time:
Additionally, drugs that inhibit the RAS (such as ACE inhibitors, renin inhibitors, and AT1R antagonists) and blockers of the TLR4 receptor show promise in managing MAS [15,35].
Given the severity of MAS and its potentially life-threatening complications, further studies are warranted on this syndrome, particularly regarding its diagnosis and management, especially in the context of SARSCoV-2 and/or an anti-COVID-19 vaccine-induced pathogenesis.
Rather like an over-fed macrophage, I feel it prudent to finish here as I have already surpassed my self-imposed review length limit. Given their array of receptors, you could spend your whole career just studying macrophages.
Part III will continue a deep dive into IgG4-RD mechanisms, including how IgG4 Abs redirect macrophages to the immunosuppressive subtype M2b, and also inhibits anti-tumour T cells.
I will also be removing the paywall on the Whernside walk Substack on September 11th, having given paid subscribers advanced access for a month.
DoorlessCarp’s Scientific Literature Reviews is a reader-supported publication. To receive new posts and support my work, consider becoming a free or paid subscriber.
DoorlessCarp’s Scientific Literature Reviews is a reader-supported publication. To receive new posts and support my work, consider becoming a free or paid subscriber.
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Divangahi M, Aaby P, Khader SA, et al. Trained immunity, tolerance, priming and differentiation: distinct immunological processes.Nat Immunol. 2021;22(1):2-6. doi:10.1038/s41590-020-00845-6
Stone MB, Shelby SA, Núñez MF, Wisser K, Veatch SL. Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes. Dustin ML, ed. eLife. 2017;6:e19891. doi:10.7554/eLife.19891
Mócsai A, Ruland J, Tybulewicz VLJ. The SYK tyrosine kinase: a crucial player in diverse biological functions.Nat Rev Immunol. 2010;10(6):387-402. doi:10.1038/nri2765
Karnasuta C, Akapirat S, Madnote S, et al. Comparison of Antibody Responses Induced by RV144, VAX003, and VAX004 Vaccination Regimens.AIDS Res Hum Retroviruses. 2017;33(5):410-423. doi:10.1089/aid.2016.0204
De Angelis ML, Francescangeli F, Rossi R, Giuliani A, De Maria R, Zeuner A. Repeated Exposure to Subinfectious Doses of SARS-CoV-2 May Promote T Cell Immunity and Protection against Severe COVID-19.Viruses. 2021;13(6):961. doi:10.3390/v13060961
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Saegusa J, Hsu DK, Chen HY, et al. Galectin-3 is critical for the development of the allergic inflammatory response in a mouse model of atopic dermatitis.Am J Pathol. 2009;174(3):922-931. doi:10.2353/ajpath.2009.080500
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Rubio-Casillas A, Cowley D, Raszek M, Uversky VN, Redwan EM. Review: N1-methyl-pseudouridine (m1Ψ): Friend or foe of cancer?Int J Biol Macromol. Published online April 5, 2024:131427. doi:10.1016/j.ijbiomac.2024.131427
Furukawa S, Moriyama M, Miyake K, et al. Interleukin-33 produced by M2 macrophages and other immune cells contributes to Th2 immune reaction of IgG4-related disease.Sci Rep. 2017;7(1):42413. doi:10.1038/srep42413
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I appreciate your comment in the first paragraph of your conclusion.
We have an immunologist (see <hervk102.substack.com>) who has spent her career studying one particular flavour of macrophage.
There may be some integration of your science with her findings which would explain some of the immunological observations.
I always think first of an energetic mechanism in your earlier examples- a nibbling lion may just represent a cell with insufficient energy to ingest the entire particle.
Thank you for your expertise and explanations- I particularly like the 5-year old model!
![Evidence of lower risk of intrapartum and breast-feeding HIV transmission associated with HIV envelope–stimulated IL-2 production in cord blood leukocytes. Shown are the Kaplan-Meier probability curves of detecting HIV RNA by the age (up to 1 year) among 59 breast-fed infants born to HIV-infected mothers stratified by their HIV-stimulated response in cord blood. No mothers were treated with antiretroviral drugs. IL-2 production in cord blood leukocytes was measured after stimulation with HIV envelope peptides by bioassay. Those with stimulation indices >3 were considered responsive to envelope, and those with stimulation indices <3 were considered unresponsive [11].](https://substackcdn.com/image/fetch/$s_!kNk_!,f_auto,q_auto:good,fl_progressive:steep/https%3A%2F%2Fsubstack-post-media.s3.amazonaws.com%2Fpublic%2Fimages%2Ffef5dbc1-f94d-4bac-9ff6-d26c6d08c456_397x320.gif "Evidence of lower risk of intrapartum and breast-feeding HIV transmission associated with HIV envelope–stimulated IL-2 production in cord blood leukocytes. Shown are the Kaplan-Meier probability curves of detecting HIV RNA by the age (up to 1 year) among 59 breast-fed infants born to HIV-infected mothers stratified by their HIV-stimulated response in cord blood. No mothers were treated with antiretroviral drugs. IL-2 production in cord blood leukocytes was measured after stimulation with HIV envelope peptides by bioassay. Those with stimulation indices >3 were considered responsive to envelope, and those with stimulation indices <3 were considered unresponsive [11].")
![Reduced HIV-stimulated IL-2 production in cord blood leukocytes from infants of HIV-infected mothers treated with antiretroviral drugs. Shown are the stimulation indices quantifying the amount of IL-2 produced after stimulation with HIV envelope peptides among group 1, HIV-infected mothers not treated with any antiretroviral drugs during pregnancy or peripartum; group 2, HIV-infected mothers treated with zidovudine-lamivudine started only at the onset of labor; group 3, HIV-infected mothers treated with zidovudine-lamivudine at ∼36 weeks gestation; and group 4, HIV-uninfected control mothers [92].](https://substackcdn.com/image/fetch/$s_!jqis!,f_auto,q_auto:good,fl_progressive:steep/https%3A%2F%2Fsubstack-post-media.s3.amazonaws.com%2Fpublic%2Fimages%2Faf58101b-14a9-4460-a2ff-bd4aae59162f_395x312.gif "Reduced HIV-stimulated IL-2 production in cord blood leukocytes from infants of HIV-infected mothers treated with antiretroviral drugs. Shown are the stimulation indices quantifying the amount of IL-2 produced after stimulation with HIV envelope peptides among group 1, HIV-infected mothers not treated with any antiretroviral drugs during pregnancy or peripartum; group 2, HIV-infected mothers treated with zidovudine-lamivudine started only at the onset of labor; group 3, HIV-infected mothers treated with zidovudine-lamivudine at ∼36 weeks gestation; and group 4, HIV-uninfected control mothers [92].")
Cyprinus Carpio - a magnum opus, as you keep taking it to another level.
I appreciate your comment in the first paragraph of your conclusion.
We have an immunologist (see <hervk102.substack.com>) who has spent her career studying one particular flavour of macrophage.
There may be some integration of your science with her findings which would explain some of the immunological observations.
I always think first of an energetic mechanism in your earlier examples- a nibbling lion may just represent a cell with insufficient energy to ingest the entire particle.
Thank you for your expertise and explanations- I particularly like the 5-year old model!