Annex 1 to The Cell’s Autonomous Response Window for Advanced Healing

The long held notion that inflammation is a process that autonomously “initiates” or “clears”, is debatable according to recent knowledge. The worn-out assertion of inflammation being the first line of defense, by a myriad of authors, consequently is incorrect. The statement simply ignores innate immunity with all its humoral, cellular and barrier defenses that come first and prior to resorting to inflammation; governed by innate immunity in the first place. Titles of articles like “Inflammation–Nature’s Way to Efficiently Respond to All Types of Challenges: Implications for Understanding and Managing “the Epidemic” of Chronic Diseases”¹, are simply to be taken as a misnomer. When early defenses are not sufficient to conquer a threat, then signaling at perivascular microenvironments, present in the outermost abluminal layer of blood vessels, will favor formation of a fibrotic and stiff extracellular matrix (ECM) (ECM = absolutely required for normal cellular maintenance)* typically associated with abnormal endothelial adhesion, leaky microvascular capillaries driven by higher regulation of inflammatory/remodeling genes, regulated via NOTCH3, a known mediator of endothelial-perivascular cell communication². This will favor engaging into rolling of immune cells across the capillary wall, through CD44 a hyaluronan HA receptor found on most leukocytes, to start inflammation and perhaps also adaptive immunity, depending on the magnitude of the tissue niche disruption that is being confronted. As described in the manuscript, immunity is a continuum where opposing extremes mutually possess characteristics of the other and functioning by means of simply an early and late response, that mutually balance for maintenance of a health environment.

Perivascular niches are specialized microenvironments where stromal and immune cells interact with vasculature to monitor tissue status. Common niche regulatory programs modulating immune functions exist for several tissues and species and confirm how tissue topology directs gene or protein expression in various cell types, even of different lineages, in a coordinated fashion. These site-specific fingerprints might represent a conserved mechanism to efficiently orchestrate responses to inflammatory stimuli at the tissue level. Mesenchymal cells are located within or in proximity to the blood vessels wall, which include pericytes, adventitial fibroblasts and mesenchymal stromal cells. Perivascular stromal cells play a role in tissue homeostasis, immunity and inflammatory pathologies by multiple mechanisms, including vascular modulation, leucocyte migration, activation or survival in the perivascular space and differentiation into specialized ‘effector’ mesenchymal cells subsets essential for tissue repair and immunity, such as myofibroblasts and lymphoid stromal cells. Among these, mesenchymal stem cells (MSCs) play a critical role in response to stress such as infection. They initiate the removal of cell debris, exert major immunoregulatory activities, control pathogens, and lead to a “remodeling/scarring = regeneration/fibrosis” phase. Thus, host-derived ‘danger’ factors released from damaged/infected cells (called alarmins, e.g., HMGB1, ATP, DNA) as well as pathogen-associated molecular patterns (LPS, single strand RNA) can activate MSCs located in the parenchyma and around vessels to upregulate the expression of growth factors and chemoattractant molecules that influence immune cell recruitment and stem cell mobilization. MSCs, in an ultimate contribution to tissue repair, may also directly trans- or de-differentiate into specific cellular phenotypes such as osteoblasts, chondrocytes, lipofibroblasts, myofibroblasts, Schwann cells, and they may somehow recapitulate their neural crest embryonic origin. On the dark side, many viruses and particularly those associated with chronic infection and inflammation may hijack and polarize MSC’s immune regulatory activities. Eventually if the response shortfalls, dysregulated circumstances ensue and it is when the responses may contribute to inflammatory and fibrotic diseases³,⁴,⁵,⁶. Restitution of homeostasis using the same structures, cells and functions used by innate immunity to elicit inflammation while sparing tissue damage or fibrosis, doesn’t mean that inflammation contributes to maintenance of homeostasis; it means that the perivascular microenvironments have sensed the threat and responded in a physiological fashion and reinstated homeostasis, as stated in the manuscript.

Since the manuscript unveils how healing does occur within a constitutive and evolutionary conserved process only available during a short period that follows wounding and which renders superior healing remained unknown for millenia and dormant for 125 years, it is sensible, in view of this newly acquired knowledge and the scientific facts of sequela arising from established practice and not profiting from the innate immunity constitutive window benefits, to downgrade healing with fibrosis since comparatively, it yields tissue of inferior quality and of diminished function. Not only, when inflammation is invoked, a trade-off is confronted by the organism, between fighting the threat effectively and managing available energy, to ensure survival. This trade-off is especially apparent during stress, inflammation and infections, where the immune system requires augmented energy⁷. As a result, the organism reallocates resources by temporarily suppressing other energy-intensive physiological processes, such as growth, reproduction, and basal metabolic functions, giving priority to the immune system and to maintenance of overall physiological homeostasis⁸. Two main strategies may be identified, which organisms adopt to survive; either to resist or tolerate. Resistance is an active, energy-consuming process that protects the organism against external threats, such as pathogens when early defenses have been overturned. Tolerance is an energy-conserving state that enhances an organism’s tolerance to environmental stressors, particularly during periods of nutrient deprivation and extreme conditions and at the same time, pursuing to diminish tissue damage. Balancing energy between dormancy and defense has remained an ongoing evolutionary challenge. As a result, the organism as described above, reallocates resources in favor of immune system prioritization. Mice infected with Escherichia coli provides direct evidence. This research showed that upon infection, mice with high hepatic expression of B-cell lymphoma 6 (BCL6) exhibited a significant survival advantage, but concurrently developed metabolic abnormalities, such as fatty liver and glucose intolerance⁹. BCL6, a critical transcriptional repressor, plays a key role in balancing immune responses and metabolic processes. Its elevated expression evidently enhances the immune defense during infection. However, this prioritization of immune function comes at a significant metabolic cost, including visceral adiposity and impaired glucose metabolism. Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ) is another critical regulator of the immune-metabolic trade-off. PPAR-γ in macrophages is indispensable for alternative activation (AA), functioning as a metabolic checkpoint that suppresses inflammation and supports AA through the facilitation of glutamine metabolism¹⁰. In macrophage-specific PPAR-γ knockout (Mac-PPAR-γ KO) mice, the absence of PPAR-γ impairs AA, resulting in a pro-inflammatory macrophage phenotype. The altered immune response of Mac-PPAR-γ KO mice makes them less susceptible to infection by Leishmania major. However, this immune advantage comes at a high metabolic cost. Mac-PPAR-γ KO mice exhibit pronounced alterations in adipose tissue mass and function, as well as impaired glucose homeostasis, particularly under a high-fat diet¹¹.

Sex-specific differences in the immune- metabolic trade-off represent evolutionary adaptations shaped by distinct reproductive strategies and survival priorities. Nikkanen et al. (2022) demonstrated that male mice achieve higher survival rates during acute infections through BCL6- mediated suppression of fatty acid oxidation and reprogramming of triglyceride metabolism. Prioritizing immune activation to combat pathogens, comes at a metabolic cost, eventually manifesting as fatty liver and impaired glucose metabolism. In contrast, females appear to adopt a more metabolically conservative approach. Supported by estrogen’s dual role in enhancing immune responses while simultaneously mitigating metabolic risks. As researched by Caputa et al. (2022)¹², estrogen regulates lipid metabolism, reduces chronic inflammation, and improves insulin sensitivity, thereby conferring metabolic protection during the reproductive years. This protective balance, though, is disrupted after menopause, when declining estrogen levels result in heightened inflammation and metabolic dysregulation.

As the body ages, the cumulative effect of the trade-off becomes more pronounced, accelerating the development of metabolic diseases and contributing to the overall decline in metabolic flexibility. Exploring IgG accumulation and the reactivation of endogenous retroviruses, have shed light on the mechanisms by which the trade-off exacerbates tissue damage and metabolic dysfunction in aging. Qiang and his team (Yu et al., 2024)¹³ demonstrate that the accumulation of immunoglobulin IgG in adipose tissue with age, promotes tissue fibrosis and contributes to metabolic decline. This increase disrupts the body’s metabolic balance, through a direct mechanism by which immune components exacerbate age-associated metabolic challenges. In parallel, the reactivation of ancient viral elements, such as endogenous retroviruses, enhance immune responses in aging organisms¹⁴. These dormant viruses can activate immune pathways, such as the cGAS signaling cascade, sustaining inflammation even after viral threats have subsided. This demands continuous metabolic resources, thereby causing further tissue damage and accelerating the aging process. The interplay underscores the importance of maintaining a balance between immune function and metabolic health to mitigate the effects of accelerated aging and chronic metabolic diseases¹⁵.

Up to this point in the answer, resistance is depicted as it has classically and generally been envisioned. But if a role for innate immunity is admitted in understanding immunological defense, a different and contrasting scenario emerges. That innate immunity can balance a defense response metabolically, has not been quantitatively determined. However, important qualitative information has accumulated increasingly, pinpointing a metabolic balancing role that can be identified in innate immunity. The manuscript deals in detail with mechanisms that are involved in autophagy, here complementary nutrient and metabolical roles will be presented. It is understood from the manuscript that autophagy is a cardinal feature regarding such roles. Autophagy can target and degrade different types of nutrient stores and produce a variety of metabolites and fuels, including amino acids, nucleotides, lipids and carbohydrates. Autophagy balances cellular nutrient and energy demand and supply — specifically, how energy deprivation switches on autophagic catabolism, how autophagy halts anabolism by degrading the protein synthesis machinery, and how bulk and selective autophagy-derived metabolites recycle and feed into a variety of bioenergetic and anabolic pathways during stress conditions. These catabolic pathways crosstalk with one another to provide building blocks for biosynthetic activities and feed into glycolysis and mitochondria for energy production. powering three outcomes for autophagy-derived metabolites, namely: the provision of building blocks for biosynthetic pathways; ATP production; and thermogenesis A contrasting foresightful picture emerges in contrast to the thermodynamic dissipative scenario that the right hand side of the continuum has to offer. This is particularly striking regarding glycophagy. Autophagy plays an important role in glucose metabolism via autophagic degradation of glycogen (glycophagy)96,97. Glycogen is a polysaccharide of glucose and a main source of intracellular glucose storage in the liver and muscles. In the cytosol, glycogen is broken down via glycogenolysis, which results in the sequential release of glucose-1-phosphate by the cytosolic enzyme glycogen phosphorylase98. Cytosolic glucose-1-phosphate is then isomerized by phosphoglucomutases into glucose 6-phosphate, which can be used intracellularly, or further transported into the ER via the glucose 6-phosphate transporter (G6PT). Inside the ER, glucose 6-phosphate is dephosphorylated by glucose 6-phosphatase to produce free glucose that can be released extracellularly via glucose transporters as fuel for other organs. In glycophagy, glycogen is delivered to lysosomes by autophagosomes, and then hydrolyzed into single α-glucose molecules by lysosomal acid α-glucosidase (GAA). In glycophagy, glycogen is delivered to lysosomes by autophagosomes, and then hydrolyzed into single α-glucose molecules by lysosomal acid α-glucosidase (GAA). In comparison, lysosomal glycogen degradation generates non-phosphorylated free glucose that can be readily released. Under normal conditions lysosomes are responsible for degrading only 1–3% of cellular glycogen, and the bulk of glycogen may be degraded by cytosolic glycogenolysis. Under stress conditions, when the glucose demand increases, glycophagy may play a more important role in glycogen degradation¹⁶. After 24 hours of starvation, for example, glycophagy may almost completely compensate for glycogenolysis in Drosophila larval muscle¹⁷. Contrariwise to glucose release from glycogenolysis, which requires coordinated activities of many enzymes and transporters for (de)phosphorylation and trafficking, glycophagy directly produces free glucose and may therefore be a more efficient mechanism to supply glucose to the circulation under stress conditions, since it is achieved in homeothermy, while under inflammatory settings, tolerance to limit tissue damage is only possible to achieve by lowering body temperature¹⁸.

One of the most fundamental challenges faced by the immune system is the efficient recognition and clearance of the body’s own cells, which because of senescence or injury enter programmed cell death pathways. While cells dying of apoptotic death pathways do not pose an immediate risk to the host, if these cell corpses are not efficiently removed there is the risk of progression to secondary necrosis. This can lead to the loss of integrity of cell membranes with release of cytoplasmic and nuclear components that can serve as ligands for proinflammatory cellular receptors, and the triggering of autoimmune responses. Hence, there is an absolute need for the clearance of the immense number of cell corpses generated each day throughout the lifespan of multicellular organisms, even in health. The immune system has developed a redundant layering of superimposed mechanisms as a direct consequence. It is quite meaningful that the control of apoptotic clearance is intertwined with the regulation and resolution of inflammatory responses.

The composition of the early immune repertoire is biased with prominent expression of spontaneously arising B cell clones that produce IgM with recurrent and often autoreactive binding specificities. Amongst these naturally arising antibodies (NAbs) are IgM antibodies that specifically recognized damaged and senescent cells, often via oxidation-associated neo-determinants. These NAbs are present from birth and can be further boosted by apoptotic cell challenge. Natural antibodies (nAbs) are most commonly defined as immunoglobulins present in the absence of pathological conditions or deliberate immunizations. Occurrence of nAbs in germ- and antigen-free mice suggest that their production is driven, at least in part, by self-antigens can belong to the IgM, IgG, or IgAsubclasses. Recent studies have shown that IgM NAb to apoptotic cells can enhance phagocytic clearance, as well as suppress proinflammatory responses induced via Toll-like receptors, and block pathogenic IgG-immune complex (IC)-mediated inflammatory responses. Specific antibody effector functions appear to be involved, as these anti-inflammatory properties are dependent on IgM-mediated recruitment of the early recognition factors of complement. Clinical surveys have suggested that anti-apoptotic cell (AC) IgM NAbs may modulate disease activity in some patients with autoimmune disease. In mechanistic studies, anti-AC NAbs were shown to act in dendritic cells by inhibition of the mitogen-activated protein kinase (MAPK) pathway, a primary signal transduction pathway that controls inflammatory responses. This immunomodulatory pathway has an absolute requirement for the induction of MAPK phosphatase-1. This disregarded component of immunity can be further reviewed by consulting the following works²¹,²²,²³.

So, to ears that have not internalized what lies on the left hand side of the continuum, which simply is contrasting superior healing that can be elicited constantly and safely, “inflammation = pathology” sounds absolute. However regarding denial of the participation of innate immunity in healing, despite a plethora of mechanisms available to reinstate homeostasis and that accomplishes marvelous healing, sooner or later, the opposing stance will be undermined by their very supporters gradually sensing farfetched the avouchement that inflammation mediates quality defense and repair; veritably a non-other than poor quality result at an unduly metabolic and functional costly price, especially onerous when healing in organs is involved. Not only, it is accepted by many now¹⁹,²⁰, that there is not a disease where inflammation is not involved and once the process has advanced towards the right hand side of the continuum, it becomes a forceful drive towards disease (the manuscript deals with hardships of resolving inflammation once it is unleashed), strengthening support for “inflammation = pathology”. The manuscript addresses this topic and also reference¹ deals in depth with the subject. Moreover, the fact that natural antibodies (nAbs) beyond providing immediate protection from infection, have been shown to play various functional roles in the immune system, which include clearance of apoptotic debris, suppression of autoimmune and inflammatory responses, regulation of B cell responses, selection of the B cell repertoires, and regulation of B cell development, is quite compelling in understanding the implications of inflammation. These functions are given by the nAbs’ reactivity, which is broad, cross-reactive, and demonstrated to identify evolutionarily fixed epitopes shared between foreign and self-antigens. Furthermore, nAbs possess characteristics which are unique and that also contribute to their functional roles which set them apart from antigen-specific antibodies. Of the latter and in further support for their beneficial role in the protection against infections and in the maintenance of immune homeostasis, is the fact that preparation of polyclonal immunoglobulins, an intravenous immunoglobulin (IVIG) rich in nAbs, is best effectively used in the replacement therapy of primary and secondary immunodeficiencies and in the immunotherapy of a large number of autoimmune and inflammatory diseases²⁴. This evidence, identifies them as rheostats for restriction of inflammation, described in the manuscript as an early response of the immunity continuum. 

References of reports on experimental evidence demonstrating that superior healing takes place in absence of inflammation²⁵ and that scarring severity is commensurate to the intensity of inflammation²⁶, are quoted as further proof of “inflammation = pathology”.

Finally, constant clinical observations that healing with tissue/microenvironment control does take place regularly without infection, vanishes fears of increased risk of defense to infection despite absence of a pro-inflammatory microenvironment. Namely, sacral decubitus ulcers (high risk area) have been kept free of infection for extended time without using antibiotics plus all other types of wounds treated in humans remain consistently free of infection. Depending on the gravity of tissue damage, this newly discovered healing does evolve either entirely without, or with markedly modulated inflammation. In essence, it is the spawning of a different class of experience, which, perhaps will not impact on mere awareness about it, but it will profoundly do so, when felt in its own flesh.

This novelty is a promising practical and affordable opportunity for easing the tragedy of antimicrobial resistance mortality that for the year 2050 will rank among the prospected prominent determinants of death , and should merit mindful consideration.

References

1. Bennett Jeanette M, Reeves Glenn, Billman George E. Inflammation–Nature’s Way to Efficiently Respond to All Types of Challenges: Implications for Understanding and Managing “the Epidemic” of Chronic Diseases. Frontiers in Medicine 2018; 5.

2. Franca Cristiane M, Verde Maria Elisa Lima, Silva-Sousa Alice Correa. Perivascular cells function as key mediators of mechanical and structural changes in vascular capillaries. Science Advances 2025; 11(2):eadp3789.

3, Wang Yanhui, Zhang Xiaoyun, Li Xin. The vascular microenvironment and its stem cells regulate vascular homeostasis. Frontiers in Cell and Developmental Biology 2025; Volume 13 – 2025

4. Lebeau Grégorie, Ah-Pine Franck, Daniel Matthieu. Perivascular Mesenchymal Stem/Stromal Cells, an Immune Privileged Niche for Viruses? International Journal of Molecular Sciences 2022; 23(8038).

5. Benabid Adam, Peduto Lucie. Mesenchymal perivascular cells in immunity and disease. Current Opinion in Immunology 2020; 64:50–55.

6. Pascual-Reguant Anna, Kroh Sandy, Hauser Anja E. Tissue niches and immunopathology through the lens of spatial tissue profiling techniques. European Journal of Immunology 2024; 54(2):2350484.

7. Wang Andrew, Luan Harding H, Medzhitov Ruslan. An evolutionary perspective on immunometabolism. Science 2019; 363(6423):eaar3932.

8. Liu Jingjing, Zhou Gaosheng, Wang Xiaoting. Metabolic reprogramming consequences of sepsis: Adaptations and contradictions. Cellular and Molecular Life Sciences 2022; 79(8):1–15

9, Nikkanen Joni, Leong Yew Ann, Krause William C. An evolutionary trade-off between host immunity and metabolism drives fatty liver in male mice. Science 2022; 378(6617):290–295

10. Nelson Victoria L, Nguyen Hoang CB, Garcìa-Cañaveras. PPARγ is a nexus controlling alternative activation of macrophages via glutamine metabolism. Genes & Development 2018; 32(15-16):1035–1044

11. Odegaard, J., Ricardo-Gonzalez, R., Goforth, M. et al. Macrophage-specific PPARγ controls alternative activation and improves insulin resistance. Nature 447, 1116–1120 (2007)

12. Caputa George, Matsushita Mai, Sanin David E. Intracellular infection and immune system cues rewire adipocytes to acquire immune function. Cell Metabolism 2022; 34(8):747–760.

13. Yu Lexiang, Wan Qianfen, Liu Qiongming. IgG is an aging factor that drives adipose tissue fibrosis and metabolic decline. Cell Metabolism 2024; 36(4):793–50

14. Liu Xiaoqian, Liu Zunpeng, Wu Zeming. Resurrection of endogenous retroviruses during aging reinforces senescence. Cell 2024; 36(4):793–507.

15. Aging Biomarker Consortium., Bao, H., Cao, J. et al. Biomarkers of aging. Sci. China Life Sci. 66, 893–1066 (2023). https://doi.org/10.1007/s11427-023-2305-0

16. He Congcong. Balancing nutrient and energy demand and supply via autophagy. Current Biology 2022; 32(12):R684–R696

17. Role of Autophagy in Glycogen Breakdown and Its Relevance to Chloroquine Myopathy Zirin J, Nieuwenhuis J, Perrimon N (2013) Role of Autophagy in Glycogen Breakdown and Its Relevance to Chloroquine Myopathy. PLOS Biology 11(11): e1001708. https://doi.org/10.1371/journal.pbio.1001708

18. Ganeshan Kirthana, Nikkanen Joni, Man Kevin. Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance. Cell 2019; 177(2):399–413 E12.

19. Chavda Vivek P, Feehan Jack, Apostolopoulos Vasso. Inflammation: The cause of all diseases. Cells 2024; 13(1906)

20. Mendes Alexandrina Ferreira, Cruz Maria Teresa, Gualillo Oreste. Editorial: The physiology of inflammation—the final common pathway to disease. Frontiers in physiology 2018; 9

21. Holodick Nichol E, Rodríguez-Zhurbenko Nely, Hernández Ana María. Defining Natural Antibodies. Frontiers in immunology 2017; Volume 8 – 2017.

22.Reyneveld IJsbrand G, Savelkoul Huub FJ, Parmentier Henk K. Current Understanding of Natural Antibodies and Exploring the Possibilities of Modulation Using Veterinary Models. A Review. Frontiers in immunology 2020; Volume 11 – 2020.

23. Vas Jaya, Grönwall Caroline, Silverman Gregg. Fundamental roles of the innate-like repertoire of natural antibodies in immune homeostasis. Frontiers in immunology 2013; Volume 4 – 2013

24. Maddur, M.S., Lacroix-Desmazes, S., Dimitrov, J.D. et al. Natural Antibodies: from First-Line Defense Against Pathogens to Perpetual Immune Homeostasis. Clinic Rev Allerg Immunol 58, 213–228 (2020). https://doi.org/10.1007/s12016-019-08746-925. Martin Paul, D’Souza Deana, Martin Julie. Wound Healing in the PU.1 Null Mouse—Tissue Repair Is Not Dependent on Inflammatory Cells. Current Biology 2003; 13(13).

26. Wilgus Traci A. Inflammation as an orchestrator of cutaneous scar formation: A review of the literature. Plastic and Aesthetic Research 2020; 7.