Inflammation usually serves a protective role in minimizing injury or infection. Pathogen-Associated Molecular Patterns (PAMPs) from microbial structures or endogenous Damage-Associated Molecular Patterns (DAMPs) released in tissue damage initiate the inflammatory response through interaction with Pattern Recognition Receptors (PRRs)[1] on immune cell surfaces to activate inflammatory pathways. Both innate and adaptive immune cells are orchestrated to initiate, regulate, and resolve inflammatory responses via secreting and responding to a profile of inflammatory signaling molecules, i.e. cytokines, to activate and recruit more immune cells to the site of inflammation[2]. Cytokines bind to cytokine receptors on immune cells in an autocrine or paracrine manner to further manipulate the inflammatory response. Pro-inflammatory cytokines induce further inflammatory responses, while anti-inflammatory cytokines act as regulatory signals to restore immune homeostasis.

Activation of immune cells induces a shift from oxidative phosphorylation towards aerobic glycolysis with an increase in lactate production[3], like the Warburg effect observed in tumor cells[3], [4]. The local increases in lactic acid and protons result in an acidic microenvironment[5]. Acidotic extracellular pH levels, which can range from pH 5.5-7.0, are commonly found in inflammatory conditions such as tumors[6], autoimmune diseases[7], and sites of infection[8]. It has been well documented that acidosis results in complex changes of immune cell activity[9]. In neutrophils, extracellular acidosis has been shown to increase cell activation, trans-differentiation, endocytosis/phagocytosis, and antigen presenting capacity, while decreasing neutrophil extracellular trap (NET) formation, apoptosis, reactive oxygen species (ROS) production, and cell migration[10], [11], [12], [13], [14], [15]. In the case of monocytes and macrophages, acidosis has a complex effect on cellular activity. On one hand, decreased extracellular pH increases inflammasome activation and IL-1β production[16], [17], however, it also reduces monocyte recruitment and production of inflammatory mediators such as TNFα and IL-6[18] and reduces responsiveness of macrophages to LPS stimulation[19], [20]. Similarly, acidosis has both pro-[21], [22] and anti-inflammatory effects[23], [24] on dendritic cells. In the case of natural killer cells, low pH acts in an anti-inflammatory manner through inhibition of critical functions, including release of perforin and granzyme granules, secretion of pro-inflammatory cytokines, and cytotoxic response including anti-tumor immunity[25], [26]. In summary, inflammatory acidosis and lactate have pleiotropic effects on immune cells in inflammation.

Cytokines are classified as interleukins, colony stimulating factors, interferons, tumor necrosis factors, tumor growth factors, and chemokines[27]. Cytokines are extracellular signaling proteins, which can be affected by environmental changes such as alterations in pH. Specifically, pH changes modify the charge distribution of a protein, and thus may alter protein geometry and interfere with the electrostatic interactions in protein binding[28], [29]. The slight decrease in pH from 6.7 to 6.0 significantly reduces the solubility of negatively charged protein, e.g. insulin[30]. In particular, imidazole in histidine residues has a pKa around 6.0, which can be very sensitive to inflammatory acidosis in local tissue (pH 5.5-7.0) and even systemic pH changes in sepsis (pH 7.0-7.2)[31]. The surface charges of cytokines, indicated by the isoelectric point (pI), stabilize proteins in aqueous solution and can contribute to the receptor binding. Thus, inflammatory acidosis has the potential to modify cytokines in terms of their stability, biodistribution, and affinity for receptor binding. Surprisingly, this phenomenon has not been discussed in the literature. In addition, abundant serum proteins and plasma membranes are mostly negatively charged, and the extracellular matrix and cell surface are intrinsically decorated with negatively charged polysaccharides, e.g., heparin and hyaluronic acids. This negatively charged physiological matrix constantly influences the trafficking and biodistribution of cytokines as they make their way to their targets. Hence, cytokines may acquire distinct surface charges as part of their evolutionary adaptation, allowing them to efficiently carry out and regulate their functions through interacting with extracellular matrices (ECMs) and responding to immunological and pathological acidosis.

Interestingly, we have observed a significant charge disparity between major pro-inflammatory and anti-inflammatory cytokines, which provides us an opportunity for precise immune modulation in sepsis treatment[32]. Major pro-inflammatory cytokines, e.g., TNF-α, IL-1β and IL-6, have negative charges. This may be important for pro-inflammatory cytokines to avoid nonspecific interactions with extracellular molecules/matrix allowing for the effective initiation of an immune response against infection and limit tissue damage. In contrast, anti-inflammatory cytokines, e.g., IL-10, IL-4, and TGF-β, are predominantly positively charged resulting in longer residence via charge interactions with negatively charged ECMs for prolonged and effective anti-inflammatory effects. Overwhelming inflammation induced by sepsis or other inflammatory diseases can have detrimental effects such as multiple organ failure. Thus, multiple mechanisms for effective immune regulation are essential to control the side effects of inflammation. Additionally, inflammatory acidosis may serve as an intrinsic regulatory mechanism to prevent overwhelming inflammation through suppressing immune cell activity and decreasing the activity of a broad spectrum of the negatively charged proinflammatory cytokines. However, prolonged acidosis may increase anti-inflammatory cytokine activity and residency to result in immune suppression in the later stage of sepsis and contribute to the immune suppressive microenvironment in solid tumors.

It is difficult to separate the activity of cytokines from the cell responses that occur from contact with environments undergoing immunological and pathological pH changes. In addition, many cytokines share overlapping and redundant signals during inflammation[33]. Bioinformatic analysis of intracellular proteomes revealed an interesting correlation between protein PI and subcellular localization, which is defined by the local pH and membrane charge[34]. Thus, we would like to investigate whether cytokines with overlapping and similar functions also share a similar surface charge, which allows for a universal pathway for immune regulation in response to acidosis. Although there are no specific studies exploring the potential significance of cytokine charge disparities, it is conceivable that if crucial pro-inflammatory cytokines displayed highly positive charges instead of the observed negative charges, our immune system could be compromised due to the entrapment of these cytokines within ECM resulting in ineffective initiation of immune responses. Thus, we propose a novel hypothesis that cytokine charge plays a critical role in regulating biodistribution and activity, contributing to immune regulation in response to the physiological and immunopathological microenvironment. To test this hypothesis, we review whether there is indeed a trend of charge disparity across different types of cytokines, which may provide a novel insight in immune regulation and lay out the foundation for the further investigation. In this review, we took a comprehensive survey of cytokine charge across different species (human, mouse, and rat) based on theoretical predication and measured isoelectric points (pIs). We further discussed the link between cytokine charge disparity and in vivo biodistribution, circulating time, and spatiotemporal activities in regulating the immune status in different diseases with acidosis.