Paulo,

in our hands using Listeira monocytogenes to infect macrophages, this is a normal behaviour of macrophages when they become overloaded with bacteria, no matter whether they are gram-positive or gram-negative, intracellular or extracellular, pathogenic or not. The problem is always "numbers". When you do controlled infections for your regular experiments, depending on the infection rate of your pathogen, you will always use a ratio. In the case of Listeria and macrophages, 5:1 bacteria: macrophages or 10:1 bacteria: macrophages. If you use higher doses, such as 25:1 or higher 30: 1 or 50:1, then you will have the effect that you describe: loss of adherence. This effect is simple, it has to do with the number of receptors used for the bacteria and of course the toxic effects of the different bacterial products, that may use several receptors on the surface of macrophages. At the end, this will lead to macrophage pyroptosis, that is, a especial way of macrophages apoptosis, that involves decrease of integrins expression in the surface of the macrophages, maximal increase of the ROS mechanisms and induction of acute-phase cytokines, such as IL-1, TNF-a and IL-6. These also leads to the effects observed.

So, contaminations are usually high number of bacteria on top of your macrophages monolayer that it will behave as the infections with high ratios, such as 25:1 and higher.

Hope this can answer your question at least a little!!!

Dear Paulo,

Good question indeed. In our lab, in vivo interactions of tissue macrophages with invading Salmonella, reveal communication from bacteria to macrophages via outer membrane vesicles, which have been suggested to sort of hijack macrophage defence in favour of bacterial multiplication. This has been discussed in a numbers research articles given at my researchGate page.

Paulo,

It will depend more upon the bacterial species involved than anything else. Some species actively 'kill' phagocytes as part of their disease mechanisms-Yersinia is an example of this which induces cell death vain its Type III secretion system. Others, including Salmonella, Mycobacteria, Listeria and Brucella have evolved varying mechanisms to survive in cells-Salmonella for example will inhibit arrest the cell cycle to maintain intracellular persistence. Salmonella-infected macrophages ex vivo can still adhere though-a trick I used about 15 years ago.

If the contamination may also, as you say, lead to activation via whole raft of TLR and other PRR. SImply the activation, which is likely to be pro-inflammatory will start to drive the cell cycle towards necrosis, apoptosis to pyrroptosis and in time lead to loss of adherence. Best tip is to avoid contamination-it happens to us all though.

Dear Paulo,

The first thing that i may suggest you is that what level contamination it was? because if it is greater than MOI of 5-10, then usually some of the macrophages can die and release DAMPs (Damage associated molecular patterns) in the medium which can affect macrophage adherence properties. So, you cant compare such level contamination with the MOI u may use for your infection studies.

Paulo, I guess a lot of the response depends on the intracellular "fine tuning" of the response after bacterial attachment/ingestion. In general (again, slightly depending on the species as well), macrophages mature to a certain extend and would in vivo start migrating away from the place of infection and enter the regional draining lymphnodes. This would involve a different set of adhesion molecules being expressed. However, depending the the speed of bacterial growth, and whether the bugs have the ability to secret proteins either into the phagolysosome or into the cytosol (using specific secretion systems). the macrophages may just undergo apoptosis (and therefore start floating). This is for example triggered by autocrine TNF loops, where the initial pro-inflammatory (positive) response can turn nasty if stimuli persists too long. This can be the result, for example, of NOD receptor activation, in addition to TLR activation (mainly surface). So, it really very much depends on amount of bugs, strain/species of bugs, source of macrophages (for example, peritoneal and alveolar macrophages behave very different to bacterial stimuli compared to monocyte-derived macrophages). Hope this helps?

Paulo,

in our hands using Listeira monocytogenes to infect macrophages, this is a normal behaviour of macrophages when they become overloaded with bacteria, no matter whether they are gram-positive or gram-negative, intracellular or extracellular, pathogenic or not. The problem is always "numbers". When you do controlled infections for your regular experiments, depending on the infection rate of your pathogen, you will always use a ratio. In the case of Listeria and macrophages, 5:1 bacteria: macrophages or 10:1 bacteria: macrophages. If you use higher doses, such as 25:1 or higher 30: 1 or 50:1, then you will have the effect that you describe: loss of adherence. This effect is simple, it has to do with the number of receptors used for the bacteria and of course the toxic effects of the different bacterial products, that may use several receptors on the surface of macrophages. At the end, this will lead to macrophage pyroptosis, that is, a especial way of macrophages apoptosis, that involves decrease of integrins expression in the surface of the macrophages, maximal increase of the ROS mechanisms and induction of acute-phase cytokines, such as IL-1, TNF-a and IL-6. These also leads to the effects observed.

So, contaminations are usually high number of bacteria on top of your macrophages monolayer that it will behave as the infections with high ratios, such as 25:1 and higher.

Hope this can answer your question at least a little!!!

Paulo

Several mechanisms can occuring among of them the kind of contamination what is the bacteria? It can killing macrophages or inducing cell maturation downregulating adhesion molecules

Dear Paulo

Some of the previous responses are Ok although I am surprised that the mannose receptor, glycan receptors, as well as th combinations of Toll receptors plus complement receptors were not documented. I simple assements pf phagocytosis using flow cytometry, the amount of ingested bacteria is far less tan the one which is phagocyted and practically the maount of bacteria surround the cells makes it imposible to adhere at any surface. One can decrease bacteria binding using 2 mM EDTA plus gelatin, whash the cells two-three times at 90 x g and then centrifuge a cell pellet layered above a 1- 2 ml of serum, human or bovine not de complemented, and the cells that are then in the infranantant are able to adhere normally to tissue culture flasks. Thus, one may envision that oclusión of adhesión receptors and glycan receptors are resposible for this effect.

regards,

Paulo, as Dirk Werling and others mentioned a lot of events play a role. In my experience, whether bacteria or particle - the number do play a role, however, engulfing may be the same mechanism, but processing involves several factors. In case of bacteria the endotoxins play a major role but keep in mind the macs response - the oxygen burst and NOS2 production in the macs, may cause increase in ROS/RNS. Some bacteria are known to tolerate may be by using the aerobic milieu to grow inside the macrophages. As the microbes propagate they tend to dissolve the cell wall to let themselves out and are candidates for engulfing back by new macs, thus, the process of multiplication progresses (so called - incubation period!). To be brief, during these processes, nutrients for the bacterial growth are derived from the intracellular milieu of the mac. Expressions of chemokine and chemotactic receptors – CCR2, FPRs and MCP-1 are repressed. An endo lysosomal aspartic proteinase, CatE present in macs are inhibited by the bacterial endotoxins or it may get oxidized, as a result, surface expression of adhesion receptors CD 29 (integrin beta 1) and CD18 (integrin beta 2) receptors loose its moiety. You can test this by dose dependent response. Once you know the titer, isolate the bacterial proteins and screen for the specific protein that is responsible for inhibiting CatE and see if there is a dependency associated with adherence. GoodLuck!

Note adherence to macrophage is antigen-epitope specific (classical immunology) and hence contamination with different species makes it lose its epitope specificity.

Good morning Paulo,

So this is a very interesting question, and does relate in a way to work we conduct in the lab. So for us its the question is how does phagocytic behaviour affect the overall output of cell motility (migration)? This relates to your question as both cell behaviours stem from the cytoskeleton and, as you know, migration is an output of dynamic adhesion, correct spreading and polarisation all leading to correct membrane edge dynamics.

I would say, most certainly, that the comments from above would apply as the simple mechanics of receptor-ligand binding should not be ignored. Plus it would be a simple thing to over-look when dealing with such a question of mechanical cell behaviour and cellular function. But with that in mind I have taken a slightly out of the box thought by incorporating the basic units to the cell such as membrane (PiPs), pH (Ca2+, Na+...), cytoskeleton dynamics, and vesicular trafficking.

So if we ignore the toxicity in this assay as a factor that affects cell adhesion, mentioned briefly in the earlier part of your question as cells adhere happily in both low and high concentrations of bacteria, we now only consider the mechanics. The mechanics can be broken down into phagocytic cup formation, polarisation of the cytoskeleton, phagosome formation, phagosome trafficking, late phago-lysosome homeostasis, microtubule dynamics and actin dynamics, and ion flux.

When you overwhelm the cells with too much bacteria I can imagine that the overall turnover of membrane would be fast at first but then will slow down...like a traffic jam effect due to the fact that a single cell would have a maximum limit to how much membrane it uses. This has also been a very large question in the field, how much membrane does a cell have? Answers have varied simply due to the changes in experimental tech available over time.

I can also imagine that continual ion flux across membranous compartments will eventual slow down due to an overall net decrease in available ATPs and GTPs, thus affecting pH. pH shift would then in turn affect both vesicular trafficking and cytoskeletal dynamics as both systems require continual turnover of the core constituents that make up each system. Lastly, pH and active vesicular trafficking, which act along stabilised microtubules (supported the actin cytoskeleton) are all factors that affect integrin stabilisation, detachment, and trafficking. So if you already have a cell that is totally overwhelmed with multiple membranous phagosomes, trying to lyse its bacterial content, whilst still trying to go about its basal functions...I can imagine that cell adhesion would be affected. I do believe that trafficking was brought up in an earlier post, but trafficking strongly relies upon a stable cytoskeleton.

Anyway, I shall leave it at that. We have managed to answer some of these questions in the lab but not the whole story. The whole story does require quite elaborate experimental set-ups that to be honest cannot be supported with the current tech available to us. But I hope this helps you, as an outside perspective to your question. Sorry about the essay!

All the best and good luck,

Chris

Well, besides all the factual explanation, many scientific reasoning, and the time invested, one simple difference in bacterial inoculation in culture versus bacterial contamination may be due to the fact that the defined strain of bacteria when introduced in culture gets well mixed in the medium and there is a homogeneous dispersion of the strain where cells usually outnumber. However, in the contamination, it usually initiates in the surface with the surface effect, competing for the nutrient in the medium, it usually takes longer for the cell to recognize its initial establishment of contamination. As it senses, lamelliopodia stretches to engulf the contaminant particle, especially in alveolar macs, I’ve observed, in live cell scanning, the stretching of lamellipodia, once it engulfs the particle (those that sink versus floaters), it attaches itself to adjacent cells. The cell behavior is interesting to observe but during the process n number of molecular interventions can occur, I believe every 1/100th of a second is important in the rapid signaling process. All said, cellular activation once initiated, the cells from the resting stage gets activated as they are the sentinels of the immune system.

Real quickly, and just a guess - but it could be that the contaminating bacteria have sugar moieties on their surface binding to associated receptors on the macrophages. This might preclude adherence.

One more thought. Take some of the non adherent macrophages and run them through a Gram stain. If bacteria are present you will see them on the surface of the macrophages.

Hi Richard, have bacteria shown this strategy before? Its quite interesting to think that some bacteria may act as a physical barrier on the extra-cellular cell surface. And also how would sugar moieties affect surface binding? All very interesting!

Best,

Chris

I appreciate responses given so far. I did like to make this submission. Various pathogens have different virulence factors, which are released against macrophage invasion, though it gives an SOS signal for macrophage activation and secretion of inflammatory cytokines. However, some predisposing host factors can influence non-adhesion of macrophages when in contact with an immunogen such as age and genetic factors.