We investigated a classical conditioning paradigm in human macrophages using streptomycin as CS and LPS as UCS. Conditioning was not shown. Rather, the groups that showed an IL-6 response at evocation were those that had been exposed to LPS in the learning phase, irrespective of presence of the CS in either the learning or the evocation phase. The reason for this is not clear, but the effect could possibly be due to sustained activation. Markedly lower responses to UCS were observed after repeated exposures to LPS (and streptomycin) in a subsample of cultures. This indicates that habituation has occurred, and agrees well with previous studies in human macrophage cultures , experimental animals  and humans [22, 23]. Because habituation enables the organism to save energy by down-scaling the response to known stimuli, or to protect the organism from e.g. inflammatory damage, this mechanism could be advantageous also in immune cells.
One of several limitations of this pilot experiment is the scant knowledge of what might constitute a suitable stimulus repertoire to test simple learning in immune cells. While LPS is a canonical activating stimulus for macrophages, it is less obvious what sort of substances, if any, could constitute conditioned stimuli without interfering with the biology of the unconditioned response. We argued that streptomycin fulfills fundamental criteria for a CS, as it is non-cytotoxic and not known to induce specific functional responses in macrophages, while being able to bind to ACh receptors on the cell surface and thus being detected by the cell . Binding of streptomycin to ACh receptors has been shown in the context of neuromuscular junctions, where streptomycin has inhibitory activity [18, 19]. Streptomycin is not considered to invoke specific responses in macrophages, and is frequently used in culture of macrophages to prevent bacterial infection.
Whether conditioning may be achieved in macrophages or other immune cells using a different stimulus set, or different CS/UCS duration and intensity, remain open questions. Another area of obscurity concerns the appropriate number of pairings in the acquisition phase to obtain a conditioned response. Along with a strong tradition of effective aversive taste conditioning  and in vivo immune conditioning , and in line with some prior work on unicellular organisms , we used only one pairing between the CS and the UCS. Another limitation is that habituation was tested in a group of cells that were simultaneously exposed to streptomycin. However, our results do not indicate that streptomycin in itself affected IL-6 secretion, nor that it interacted synergistically or antagonistically with the IL-6 response to LPS after one pairing.
When classical conditioning of the immune system is investigated in mammals, it is presumed that a conditioned stimulus such as a taste is sensed by the CNS via neural afferents, while the neural or immunological changes induced by the US is sensed via neural or humoral afferent pathways . In simple models such as the present using single cells, albeit co-cultured with peers, corresponding sensory signaling tasks need to be carried out within the unicellular system. The surprising flexibility observed in simple systems through e.g. epigenetic mechanisms, and evidence for conditioning in simple organisms  or even in photoreceptors , suggests that highly competent cells in the immune system could potentially show associative learning capacities even in the absence of a nervous system. Hypothetically, if mechanisms of simple learning exist in non-neural cells, it is possible that a hierarchically organized regulation of behavioral responses to actual or anticipated stimuli is performed by the nervous system in conjunction with local circuits that also include cells of the immune system. Thus, the abilities attributed to the immune system to serve sensory functions [27, 28] may include aspects of plasticity, so that facilitation and inhibition of signaling from the micro (e.g. detection of danger associated molecular patterns) to the macro level occurs in a way analogous with how neural responses are modulated by e.g. serotonin release in micro circuits after learning in behavioral models . The implications of understanding learning at the cellular, immune or epigenetic level may therefore have bearings on how we understand learning on a more general level, as the brain response to molecular signals detected in the periphery, and therefore also on regulation of human behavior.
Future studies might explore the possibility to investigate simple learning in immune cells ex vivo by varying e.g. the conditioned stimulus, the number of pairings and by testing other leukocytes, e.g. NK cells which display immunological memory  and which can be cultured without proliferation. If associative learning capacities, as suggested from recent findings in cell biology and gene regulation, could be demonstrated in leukocytes in vitro, the old analogy of the immune system as a mobile brain  would appear even more warranted.