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LL-37

LL-37, which is also known as cathelicidin. Chemically, it is a peptide, i.e., a short sequence of amino acids (AA), which is made up of only 37 amino acids.  Its particular sequence of AA (also called the primary structure) is illustrated in Figure 1.  As you can see in Figure 1, its primary structure has two leucine residues and “L” is the alphabetical representation of leucine, therefore it is named as LL-37 (Yang et al., 2020).  LL-37 has anti-microbial properties against a broad spectrum of pathogenic microbes, like, bacteria (both, gram positive and negative), viruses and fungi (Turner et al., 1998).  It is naturally synthesized in the body, upon the microbial invasion (Chen et al., 2018).  It links the two major domains of the immune system, i.e., innate and adaptive. It does so by employing the cells of immune system at the site of infection, and thereby stimulating only the specific receptors on these cells.  As it regulates the expression of both distinct types of inflammatory cytokines, i.e., pro- and anti-, normal expression and activity of LL-37 is not only essential but critical to normal physiology of the body.

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Figure 1 The Primary S tructure of the LL-37; in which the residual amino acids are represented by the letters

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Figure 2  The alpha helix of the LL-37 (Burton and Steel, 2009)

Talking about the secondary structure of LL-37, although it is highly dependent on the external environment conditions, primarily pH.  When it forms an alpha helical structure, it gains a net charge of +6, thus a cation.  The secondary structure of LL-37 is diagrammatically illustrated in Figure 2.  For the sake of its molecular analysis, it can be viewed a continuous structure having three distinct parts. As the structure of alpha helix is not centred on a signal axis, therefore it would be viewed as two distinct helices instead of one, ranging from 2 to 30 peptides. The second alpha helix is responsible for the antimicrobial activity. And, the third part is the tail, that is not well structured, ranging from 31 to 37 peptides (Burton and Steel, 2009).

Being an antimicrobial peptide (AMP)/ host defense peptide, LL-37 is part of a body’s first line of defense.  Apart from this, it has regulatory roles in several biological activities; like, the neutralization of lipopolysaccharide (LPS) thereby the lipopolysaccharide (LPS)-induced apoptosis in the endothelial cells is inhibited, and the regulation of inflammatory responses.

In a research study based on the sepsis model, especially cecal ligation and puncture (CLP) model of murine, LL-37 is found to cause improvements not only in the pathology but also in the survival rate of mice having sepsis.  The latter is graphically presented in Figure 2.  Moreover, LL-37 also lowers the level of bacterial population in the blood and peritoneal fluid of the murine model. Thus, LL-37 can be thought as a novel therapeutic approach to the treatment of sepsis.

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Figure 3 The Survival Rate of sepsis murine model when administrated with LL-37.

This happens as LL-37 suppress the pyroptosis (a pathway that results in the cell death) in macrophages of the sepsis murine model, which can be induced via lipopolysaccharide (LPS) from gram negative bacteria or ATP from the dead/ dying cells.  LPS acts on CD14/TLR4, while ATP acts on P2X7, to initiate the pyroptosis.  For both the initiating factors, the caspase-1 is activated by the activity of inflammasome, leading to the release of Interleukin-1 beta (IL-1β) and ultimately to the cell death via pyroptosis.  LL-37 suppress the pyroptosis by blocking the interaction of CD14/TLR4 and P2X7 with LPS and ATP, respectively.  Also, all the steps, following these interactions are automatically stopped via LL-37 administration.  This whole mechanism of suppressing the pyroptosis at the molecular scale, is diagrammatically presented in Figure 3.  However, the exact mechanism by which Caspase-1 activation and pyroptosis are suppressed is yet to be discovered. 

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Figure 4 LL-37 suppress the pyroptosis in the macrophages by blocking the action of both of the initiating factor, i.e., lipopolysaccharide (LPS) and ATP. The alpha helix (secondary structure) of LL-37 peptide is shown in green colour for this figure

As it can be seen in Figure 3, the activation of Caspase-1 is directly linked to the initiation of pyroptosis.  The comparison of these two closely related steps in terms of percentage, is graphically presented in Figure 4.  From Figure 4, there exists a direct relationship between the activation of Caspase-1 and the initiation of pyroptosis, which can be clearly seen. 

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Figure 5 The activation of Caspase-1 (%) and the initiation of pyroptosis (%) in the sepsis murine model. Both have been decreased by the administration of LL-37

Moreover, LL-37 induces NETosis in the neutrophils, which is essentially the release of Neutrophil Extracellular Traps (NETs).  NETosis is normally induced by different types of microbes, like, bacteria, fungal hyphae, protozoa, in the neutrophils.  During NETosis, irrespective of the inducing factor, first of all, some structural changes happen in the nucleus, like, chromatin condensation, followed by the swelling and fragmentation of the nuclear membrane. Second, the fragmentation of nuclear membrane enables the nuclear chromatin to get linked to the proteins in the cytoplasm of the neutrophils.  Thereby, NETs come out of the neutrophils as extracellular structures, and they trap and kill the microbes by the activity of NET-associated Components, which are essentially the chromatin DNA associated with the protein (granular and the cytoplasmic ones).  The inducing factors along with the major landmarks of NETosis are diagrammatically depicted in Figure 5.  This mechanism is indeed responsible for the lowered level of bacterial population in the blood and peritoneal fluid of the sepsis model of murine. Besides this phenomenon, the higher level of ectosomes release containing the lactoferrin, Myeloperoxidase (MPO) and cathelicidin-related antimicrobial peptide (CRAMP).  Interestingly, these molecules have the antimicrobial activity.  By releasing them, the level of bacterial population is dramatically reduced; this is especially true when the dose/ concentration of LL-37 is kept higher.  This is evident from the Figure 6.  The reduction of the bacterial load was the primary reason behind the improved survival rates in the sepsis murine model, when treated with LL-37. 

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Figure 6 The induction of NETosis, via either microbes or LL-37, followed by the release of Neutrophil Extracellular Traps (NETs) and thereby trapping and killing of different types of microbes. The Neutrophil Extracellular Traps (NETs) are the structures extended out of a neutrophil and highlighted within a red box.

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Figure 7 The reduced level of bacterial population is represented by the Relative bacteria viability (%). The level is dramatically reduced, especially with higher dose/ concentration of LL-37

The overview of the positive roles of LL-37 in the sepsis murine model, which have been previously discussed in detail, are summarized and briefly presented in Figure 7.  In short, on one side LL-37 protects the body cells from cell death, while on other sides kills the harmful microbes (Nagaoka et al., 2020).

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Figure 8 A brief overview of the positive therapeutic roles of LL-37 in the sepsis murine model.  These roles collectively are responsible for the improvement in the sepsis symptoms in the murine model