Their Last Will and Testament: Dying Immune Cells Protect the Urinary System with Extracellular DNA Traps

Abstract

Like most epithelial organs, bladder and kidney can be directly accessed by bacteria evolved for invasion. Epithelia and immune cells attempt to stymie this infection with biophysical and chemical mechanisms. Goldspink and colleagues connected the Na⁺ gradient in the kidney medulla with an immune defense mounted by dead cells, namely, the explosive death of neutrophils and macrophages resulting in Extracellular DNA Traps. The pathway from Na⁺ concentration to immune death is depicted.

Ascending invasion of the kidney by bacteria is met with biophysical, chemical and even defense by suicide. Among these, a physiologic process is repurposed for immune defense¹. The transport of Na⁺ from the urinary lumen into the interstitial space not only generates osmotic gradients critical for water balance, but in addition provides Na⁺ dependent signals sensed by collecting duct epithelia. The Na⁺ gradient signals via BRX-p38 MAPK and upregulates NFAT5, which in turn drives CX3CL1 and CCL2 expression and the recruitment of CD14⁺ mononuclear cells to the interstitium^2,3. The pathway is essential because washout of the medullary gradient by diabetes insipidus, by vasopressin antagonists, or by prolonged diuretics suppresses CX3CL1 and CCL2 expression and promotes urosepsis in mice and humans⁴.

The medullary Na⁺ gradient not only regulates cytokine gene expression but also immune cell activity. Na⁺ priming includes enhanced chemoattraction of medullary macrophages and CD14⁺ mononuclear cells towards CX3CL1 and CCL2, and enhanced bacterial phagocytosis. Parallel responses to the Na⁺ gradient occur in T cells. Increased Na⁺ enhanced the TH17 phenotype and the production of cytokines IL-2, TNF-α, IL-9, GM-CSF, TBX21, and CCR6⁵. In sum, medullary Na⁺ gradients regulate not only water balance but also the immune response to ascending uropathogenic infection.

Goldspink and colleagues have identified the next steps in Na⁺ mediated immune defenses. They discovered an easily overlooked principle of immunity—that the ‘explosive’ death of immune cells promotes immune clearance by generating stringy Extracellular DNA Traps (ET’s⁶). It is the Na⁺ concentration in the kidney medulla that primes monocytes and PMNs to produce ETs.

The authors substantiated this sequence by targeting each step of the pathway. They varied the Na⁺ concentrations in vitro, and they manipulated the Na⁺ gradient in vivo using loop diuretics. They compared Na⁺ to other osmoles such as urea and they combined Na⁺ with different anions to rule out their effect. Na⁺ provided a dose dependent effect on myeloid cell death and ET formation; reduced Na⁺ or medullary washout suppressed ET and enhanced pyelonephritis. Na⁺ responses depended on Ca²⁺ influx but not on NCX or the Na/K ATPase.

The authors then identified Peptidylarginine deiminase 4 (PAD4) as the proximal regulator of the ETs in conjunction with Na⁺ hyperosmolarity. PAD4 is expressed in granulocytes, predominantly neutrophils, and is capable of converting arginine residues into citrulline. PAD4 is critical in generating neutrophil extracellular traps (NETs) by citrullinating histones, reducing their charged based interactions with DNA, causing chromatin decondensation. As a result, citrullinization promotes the expulsion of chromosomal DNA, carrying cytotoxic and antimicrobial molecules from the cell into the extracellular space. The NETS also trap serum complement.

Numerous stimuli including bacteria, yeast and fungi, and LPS activate PAD4. For example, Escherichia coli (the bacteria species responsible for the majority of UTI) has been shown to activate PAD4 induced NETs⁷. PAD4 subsequently coordinates complex cellular remodeling, resulting in NETosis. The steps include the disassembly of the actin cytoskeleton, shedding of plasma membrane vesicles, remodeling of the cytoskeleton, then endoplasmic reticulum vesiculation, chromatin decondensation, nuclear rounding, and nuclear envelope rupture, and finally the release of DNA into the extracellular space as a result of plasma membrane rupture. PAD4 orchestrates NETosis when it locates in the cell nucleus, particularly regulating chromatin decondensation, nuclear envelope rupture and the extracellular release of DNA, as shown by genetic permutations of PAD4⁸.

Goldspink and colleagues have shown that resident innate immune cells sacrifice themselves to create ET’s in response to pyelonephritis, both in human and mouse kidneys infected with UPEC. Indeed, tissue resident macrophages can produce ETs (known as METs) dependent on PAD4, containing unique MET contents (matrix metalloproteinase 12 in human alveolar macrophages, myeloperoxidase in human glomerular macrophages and histone H4 in human monocyte derived macrophages and peripheral monocytes) not necessarily found in NETs.

The authors describe enhanced bactericidal activity in assays containing autologous serum in the presence of Na⁺ - induced ET, indicating a role for opsonization. Likewise, Palmer et al 2012⁹, demonstrated opsinized Staphlyococcus aureus in the presence of NETs and reciprocally, opsonization localized immune activation to the site of infection by amplifying the production of NETS. It is consistent that LPS both amplifies Na⁺ induced ET and increased bactericidal activity.

Goldspink and colleagues have demonstrated a rapid innate immune response designed for the entrapment of bacteria, and the recruitment of additional innate and adaptive immune cells. Hence, ETs are thought to have bacteriostatic rather bactericidal effect, which results from circulating complement subsequently binding entrapped bacteria. Indeed, bacteria may express strategies to evade each step of ETosis, confirming their importance in immune defense. Bacteria express DNA binding proteins, extracellular adherence proteins, nucleases and toxins (preventing the release of NETS) in order to escape the ETs and thus prevent effective bacterial clearance which would have otherwise limited bacteremia and mortality.

Many questions remain (Figure 1):

Figure 1:

Components of the ET pathway highlighted by Goldspink et al. Qn1–6 refers to areas for future analysis. Adapted from Kidney International, Vol /edition number, Goldspink et al, Renal medullary NaCl concentrations induce neutrophil and monocyte extracellular DNA traps that defend against pyelonephritis in vivo, In Press Journal Pre-Proof, Copyright (2023), with permission from Elsevier.

1. The dynamics of the infiltrating immune cells: resident vs recruited?

Tissue resident macrophages are highly heterogeneous. They consist of multiple sub-populations, required for tissue homeostasis and immune responses. It is of great interest to determine if there are sub-populations of macrophages in the kidney primed for the expulsion of METs upon encounter with pathogens. To identify these cells we suggest specific lineage tracing studies (including using Csf1R-MerCreMer to label embryonic derived resident macrophages)¹⁰, as well as applying markers of circulating cells such as anti-CD45 and monocyte Ms4a3-Cre. Tamoxifen induced Cre’s allow one to express fluorescent tags at different stages of lineage development to identify yolk sac, then fetal liver, and then bone marrow derived macrophages. Fluorescent tags and adoptive transfer experiments in vivo can identify the lifespan of different cell populations. Perhaps the brief life span of granulocytes/monocytes identified by Goldspink is a key mechanism controlling the breadth and extent of inflammation preventing excessive tissue damage.

2. How do immune cells survive in the kidney medulla?

The short half-life of immune cells in hyperosmotic states raises the question as to how resident macrophages survive in the medulla of the kidney. It may be that they are transcriptionally reprogrammed to express different Na⁺ or Ca²⁺ channels. Perhaps, as in the collecting ducts (see above), NFAT5 is a key regulator of macrophages located in hyperosmotic conditions. Or perhaps once in the kidney, these cells are protected by initially localizing in less salty niches. Alternatively, no such mitigation applies and resident macrophages must be continuously replenished from bone marrow derived monocytes.

3. What is the exact relationship between Apoptosis and NET or METosis?

Goldspink et al document that high salt concentrations induce immune cell apoptosis as well as ETosis. Do these outcomes co-occur, or is there a decisive fork in the road in the selection of type of cell death? The requirement for PAD4 is indicated by the authors, but the use of either PAD4 or Caspase3 null mice or CRISPR/Cas9 modified cell lines would be helpful in determining the relationship between Caspase3 and PAD4 activation.

4. How does high Na⁺ activate these two cell death regulators?

Attempting to answer this question has been challenging because cellular assays have mixed outcomes. High salt concentrations has been shown to interrupt the direct antimicrobial activity of human neutrophils such as phagocytosis, however, opposing findings have demonstrated that high salt concentration can modulate the production of NETs in ocular disease and the release of interleukin 8¹¹. The depicted assays provide simplified models of apoptosis and ET formation which can help to untangle the Na⁺ pathway.

5. Do other PAD family members assist in N/METosis?

Additional PADs have been implicated in macrophage cell development, including PAD2. As the authors discuss, PAD2 promotes monocyte, macrophage and possibly neutrophil ET formation. Hence while many types of microbes are known to stimulate PAD4 dependent NET formation, it remains possible that additional PADs are stimulated by subclasses of microbes or in different neutrophil and macrophage subpopulations.

6. Refining the Na⁺ gradient in the kidney.

Examination of ET’s in the setting of different diuretics has important medical implications because of their widespread use and differential effects on the medullary Na⁺ gradient.

In summary, the defense of the urinary system depends on many modalities. The urinary tract deploys biophysical forces such as peristalsis, urine flow, bizarre decoy molecules such as uromodulin, iron-siderophore chelators (Siderocalin-NGAL), and chemical attack by RNases, Defensins, Cathelecidins and urinary H⁺. When these mechanisms fail, infected urothelia are sacrificed. Goldspink shows that not only do infected bladder urothelia commit suicide, but that the urinary track is also protected by the altruistic sacrifice of immune cells.