Perspective Chapter: Utility of Injury Immunity Axis in Disease Phenotyping

Abstract

Organ injury is mediated by dysregulated inflammatory response of the host to invading organism or antigen. Dysregulated immune response can be more than or less than what is required to contain the organism or antigen. All disease states converge on inflammatory damage to tissue irrespective of what triggers the initial insult such as a transplanted organ, microbe, autoimmunity, and even a malignancy. Injury immunity axis can be used to phenotype a disease state to explaining its etiology, treatment options and possible disease trajectory. It will address the core issue of inflammation at cellular level guiding clinicians to tailor the treatment on case to case basis. This chapter brings immunity to center-stage in diagnosis and management of diseases due to various causes. This can be accomplished by phenotyping diseases across injury immunity axis to ascertain the status of immune system forefront. It is indeed a novel concept by which we look at different manifestations of a disease through a unique perspective. It is also an attempt to acknowledge the fact that immune system work-up and immune biomarkers need better representation in the list of investigations. The importance of immunological basis of diseases needs significant amount of research and robust data to translate this knowledge into the standard of care.

1. Introduction

During the COVID-19 pandemic, we observed that it’s hosts immune response to the invading SARS CoV2 that determines the clinical presentation and consequent outcomes. If immune response is appropriate, patients remain asymptomatic. If immune response is not appropriate, a cytokine storm becomes evident. And there are cases between these two extremes. It appears that COVID-19 is a disease of immune system.

This cannot be true for COVID-19 only. If we have a closer look, most if not all disease states are disorders of self-defense or immunity. Syndromes like sepsis, acute kidney injury can be explained by different immune responses by different hosts or by the same host at different time lines in the disease trajectory. These myriads of immune responses invariably present with various clinical scenarios. In fine, a single disease entity can present with different phenotypes depending on which type of immune response it has evoked in the host.

This manuscript is an attempt at having a look at immune system as a pivotal system in tissue injury. All disease states converge on inflammation and immune damages to tissue irrespective of what triggers the initial insult such as a transplanted organ, microbe, autoimmunity and even a malignancy. This manuscript is aimed at addressing the basic culprit and finding opportunities to address it for better treatment outcomes.

As of now we treat these immune system abnormalities without robust immune function tests. Tests like white cell count, differential count, procalcitonin, ferritin, hs-CRP, cytokine and chemokine assays are utilized as immune function tests. We need to understand disease states as abnormalities of immune system and disease phenotyping as per on one or more immune function test. Till we find better indices which may function as immune function tests, let’s rely on whatever we have got and keep looking for better indicators of immune activation and suppression.

2. Basic concept

Organ injury is mediated by dysregulated immune/inflammatory response of the host to invading organism or antigen. Dysregulated immune response can be more than or less than what is required to contain the organism/antigen. Either way, dysregulated immune response causes tissue injury. But, the type of immune dysregulation will dictate the phenotype of injury. Medical or surgical management of tissue injury depends on the injury phenotype. In other words, we may need to typify the kind of immune dysregulation a patient has before initiating treatment.

Tissue injury can be prevented or resisted with optimal immune response. To complicate things further, immune response of the host during recovery determines if tissue recovers with regeneration or sclerosis (irreversible organ dysfunction). Hence, immune response of host to invading pathogen determines disease trajectory, treatment chosen and outcomes. With this background, we can have four different phenotype of tissue injury after an insult with invading organism/antigen. These four quadrants tell us if tissue injury is due to immune hyper-activation, immune deficiency or otherwise. Once we understand status of immune system, it’s easier to manipulate the immune responses best suited for the host. This 2/2 model of injury immunity interplay can be exploited in a number of disease states where host has to deal with a foreign antigen. The foreign antigen may be a bacterium, virus, transplanted organ, malignant cell or even a self-antigen to which tolerance is lost (Figure 1).

Figure 1.

Injury immunity axis is plotted as a 2/2 model. Right upper quadrant has hyperactive immune response causing tissue injury. Right lower quadrant shows optimal immune response with no tissue injury, hence host stays protected. Left upper quadrant shows deficient immune response that leads to tissue damage as direct cytotoxicity of invading organism/antigens. Left lower quadrant belongs to an unexposed host or an exposed host deficient in receptors for the invading organism to adhere and internalize.

2.1 COVID-19

In response to SARS-CoV-2 infection, both innate and adaptive immune systems are involved. SARS-Co-V applies several mechanisms to overcome the immune response. First, it inhibits the rapid expression of interferon type 1 (IFN-1). Moreover, SARS-CoV-2 interferes with IFN-1 signaling through inhibition of STAT-1 phosphorylation. The second defensive mechanism of SARS-CoV-2 is immune exhaustion through exaggerated and prolonged IFN-1 production by plasmacytoid dendritic cells (pDCs). This process leads to the influx of activated neutrophils and inflammatory monocytes/macrophages, that in turn, results in lung immunopathology (e.g. acute respiratory distress syndrome). Finally, it results so-called “cytokine storm” further weakens the immune system through IFN-1 mediated T cell apoptosis [1, 2].

Immune classification of patients with SARS-CoV-2 was performed by using the tools suggested for bacterial sepsis, i.e. ferritin more than 4420 ng/ml for MAS, and HLA-DR molecules on CD14 monocytes lower than 5,000 in the absence of elevated ferritin, for the immune dysregulation phenotype. It was found that contrary to the patients with bacterial community acquired pneumonitis and severe respiratory failure (SRF), all patients with SRF and SARS-CoV-2 had either immune dysregulation or macrophage activation syndrome (MAS) [3, 4].

It looks like COVID-19 is a disorder of immune system and host immune dysregulation determines disease phenotype from asymptomatic carrier state to florid pneumonia. Different disease phenotypes are not only important for diagnosis but gives us a chance to modify treatment on case to case basis. It is more than obvious that one disease phenotype may not get appropriate benefits from treatment aimed at other phenotypes (Figure 2).

Figure 2.

This diagram shows clinical spectrum of COVID 19 presentations integrated on injury immunity axis. Right upper quadrant indicates a patient with cytokine storm with hyperactive immune system causing tissue injury which is managed with immune suppression and extracorporeal cytokine/chemokine removal. Right lower quadrant shows optimal host immune response which is protective with appropriate antibody production and preventing tissue injury. Left upper quadrant belongs to a host unable to mount immune response (hypoactive immune response), hence shows extensive viral replication, direct cytotoxicity requiring immune augmentation, antiviral and anti-bacterial. Left lower quadrant belongs to a person not exposed to SARSCoV2 or who is theoretically deprived of receptor for viral attachment and internalization (ACE II). In either way, such a person remains protected with no tissue injury.

2.2 Sepsis

Immune responses of critically ill patients with sepsis is classified into three patterns: macrophage-activation syndrome (MAS), sepsis-induced immune-paralysis characterized by low expression of the human leukocyte antigen D (HLA-DR) on CD14 monocytes and an intermediate functional state of the immune system lacking obvious dysregulation [3].

In contrast with sepsis-1, in sepsis-3, the “systemic inflammatory response” is replaced with “dysregulated host response”, and SIRS was changed to SOFA. The dysregulation of host responses is a complicated process and includes inflammation, the neuroendocrine response, coagulation, and metabolic responses. In fact, the neuroendocrine response and coagulation are closely linked to inflammation [5].

Hyper-inflammation-induced organ failure is thought to be the most common cause of death during the first days of sepsis. In the chronic phase of sepsis, persistent inflammation, immunosuppression, and catabolic syndrome (PICS) becomes the main cause of secondary ICU-acquired infections and long-term mortality. However, it is difficult to distinguish hyper-inflammation and immunosuppression in patients with sepsis. Therefore, the timing and course of anti-inflammatory treatments also require discussion. Assessing the inflammatory and immune status of sepsis calls for a precise stage-dependent therapy [6].

Current sepsis observations suggest that multiple organ failure occurs even in the context of preserved cell morphology. In addition, organ dysfunction is often reversible, even in organs that regenerate poorly (heart, lung, central nervous system, kidneys). Therefore, it is apparent that sepsis-induced organ dysfunction occurs primarily though cellular and molecular dysregulation, as opposed to gross tissue damage. By this principle, immune dysfunction in sepsis is also associated with molecular alterations that alter cellular phenotype and function.

Moreover, not only acquired but innate immune system is activated in sepsis and contributes to tissue damage. There is well established evidence that activation of the complement system is often linked to activation of both the clotting and the fibrinolytic systems. Development of neutralizing C5a antibodies in murine models dramatically attenuated the intensity of sepsis, including greatly improved 7^th day survival, reduced levels of plasma cytokines, and decreased multiple organ failure. The vast majority of patients who die from sepsis have ongoing infections, suggesting that defects in innate immunity in general and neutrophil-mediated bacterial clearance in particular, could serve as potential therapeutic targets to regulate neutrophil apoptosis, production, maturation and function [7]. Given the profound immunosuppression induced by depletion of immune cells that occurs during sepsis, the ability to sequentially follow the uncontrolled lymphocyte apoptosis as a means to evaluate the efficacy of immune adjuvant therapies provides promising novel therapeutic opportunities. Furthermore, an increasing number of immune-adjuvant therapies to prevent sepsis-induced immune paralysis have been identified as apoptosis dependent. IL-7 and anti-PD-L1 have been found to have potent effects to prevent lymphocyte apoptosis [8].

In fine, unless we know the phenotype of sepsis manifestation, choosing appropriate therapy for the immune system dysfunction is difficult to practice (Figure 3).

Figure 3.

It shows sepsis phenotypes integrated on the injury immunity axis. Right upper quadrant shows hyperactive immune response seen in SIRS, hypercytokinemia and macrophage activation which is dealt with immunosuppression and/or extracorporeal cytokines removal. Right lower quadrant belongs to appropriate immune activation hence no tissue injury. Left upper quadrant is sepsis induce immune deficiency (so called immune paresis/paralysis). Tissue damage in patients belonging to this quadrant is due to direct cytotoxicity, disseminated infection and needs immune augmentation. Left lower quadrant is a curious case of someone tolerant to invading pathogen/antigen owing to lack of exposure to antigen or theoretically absent pathogen specific receptors.

2.3 Organ transplantation

Organ transplantation is threatened by the possibility of graft loss due to rejection. Rejection is the immune response by the recipient immune system that injures graft parenchyma. Based on the chronology, severity and insidiousness, rejections are popularly known as hyperacute, acute or chronic. But, end result of all rejections is graft loss unless treated with appropriate immunosuppression. Appropriateness of immunosuppression is gauged by graft organ function and not by immune function tests. We know that graft function decline is a late feature compared to immune activation and invasion of graft tissue. But, management of allograft dysfunction is not yet based on immune function tests (immunometer).

The survival of ABO incompatible-transplanted (ABOi) organs in coexistence with anti-allograft antibodies and complement which originally results in graft rejection was described as accommodation. Is this successful engraftment of ABOi allografts (accommodation) a certain level or type of allograft tolerance, or does it just reflect some other biological condition of allografts? Thus, the mechanism investigation of accommodation and tolerance could be significant for conquering humoral barriers to transplantation and promoting long-term survival of allografts. Tolerance is a state of the immune system unresponsiveness to substances or tissue that are capable of eliciting an immune response in a given organism, in contrast with traditional immune-mediated elimination of foreign antigens. Accommodation is a unique immunologic condition that is different from immune tolerance. It is defined operationally as a state in which the transplanted organ works normally under the existence of antibodies in the recipient specifically targeting the allograft [9]. As of now we do not have any specific tests that might tell us occurrence and sustenance of either accommodation or tolerance.

Another issue that crops up in graft dysfunction is infections, drug toxicities and ischemia. In the absence of immune function tests (read immunometer), differentiating drug toxicities, viral infections (BKVN, CMV) from rejection becomes difficult. Needless to mention that these entities represent excessive immunosuppression on most of the occasions.

As organ transplantations and its sequelae are determined by immune response of recipient, we need to base treatments on immune function tests rather than wait till graft dysfunction becomes evident and at times insurmountable. Management of allograft recipient can be tailor-made if the patient is allotted to any of the injury immunity axis quadrants (diagram 4). This may give us an opportunity to alter immunosuppression before the graft shows decline in function or recipient shows adverse effects of immunosuppression (Figure 4).

Figure 4.

It shows sequelae to organ transplantation with 2/2 injury immunity axis model. Right upper quadrant shows graft injury which is immune mediated (rejection) which needs immunosuppression as therapy. Right lower quadrant highlights graft injury due to causes other than rejection (drugs, infections, ischaemia) where immune suppression may or may not be excessive. Left upper quadrant show no injury to graft despite host mounting immune response. This would arise as the graft is able to withstand the immune activation (accommodation). Left lower quadrant shows inactive host immunity (spontaneous or induced) and consequent no injury to grafted tissue (tolerance). But it should be apparent that these are not water tight compartments and patient can move from one to another quadrant. Clinicians may aim to reach left upper quadrant phenotype for their patients with all possible therapies.

2.4 Acute kidney injury

Acute kidney injury is a clinical syndrome resulting from multiple etiologies. Presently, KDIGO classification for defining AKI is based on functional parameters of rising serum creatinine and drop in urine output. With evolution in understanding of AKI pathogenesis, it is well understood that damage to kidney precedes decline in function. Hence, there are efforts ongoing on how to integrate functional markers and damage markers in defining AKI. As it holds true for other organ systems, kidney damage is mediated and sustained by inappropriate inflammation and immune activation. This immune mediated damage is equally sustained by cortex and medullary compartments. Hence, if we intend to reverse/modify or transform the immune activation, treatment should be based on immune mediated damage phenotype. In other words, immune dysfunction preceded damage to kidney and consequent AKI.

Let’s try to come up with a model where immune biomarkers (cause), damage biomarkers (effect) and functional biomarkers (presentation) throw light on AKI phenotype which will guide management of this syndrome easier.

Inflammation is a complex biologic response that is essential for eliminating microbial pathogens and repairing tissue after injury. AKI associates with intrarenal and systemic inflammation; thus, improved understanding of the cellular and molecular mechanisms underlying the inflammatory response has high potential for identifying effective therapies to prevent or ameliorate AKI. Coupled to this is the emerging concept that mechanisms of intrarenal inflammation during AKI also exert potentially harmful effects on distant organs and tissues through release of soluble mediators or re-entry of activated leukocytes into the bloodstream [10].

Research focused on identifying and measuring inflammatory phases in the setting of human AKI is required to target inflammation modifying therapies and identify optimal times to start and stop such therapies. Additionally, great emphasis should be given to comparative analysis regarding the nature and kinetics of inflammatory response in AKI (Figure 5) [11].

Figure 5.

AKI phenotyping is described using three sets of biomarkers 1. functional 2. damage 3. immune biomarkers. This gives us eight different AKI phenotypes and their possible management options. 2/2 model of functional and damage biomarkers is superimposed on injury immunity axis to further understand the chain of events in AKI syndrome. Functional biomarker positivity indicates CKD or advanced AKI. Damage biomarker positivity indicates early AKI which may be immune mediated (IB positive) or non-immune mediated (IB negative). Theoretically, let’s presume IB appears earlier than DB and disappears late in the disease trajectory of AKI. FB: functional biomarker; DB: damage biomarker; IB: immune biomarker.

2.5 Autoimmune disorders

Tolerance is the failure of the immune system to respond to an epitope in an aggressive way. Most self-tolerance results from the deliberate inactivation or destruction of lymphocytes bearing BCRs or TCRs (B cell and T cell receptors) that recognize and bind self-epitopes. Inactivation or destruction may occur during early development (central tolerance) or may be imposed on lymphocytes in the periphery (peripheral tolerance).

Under normal circumstances, autoreactive cells in the body are not activated by contact with self-molecules. Unless they are interacting with APCs, they are not also receiving cytokine signals necessary for activation. However, in inflammatory sites, local cytokine levels may be sufficient to activate auto reactive T cells when they are binding to self-epitopes on non-APCs [12].

Thus, in a patient with autoimmune disorder, two mutually independent immune systems are at work. Autoimmune system is aberrant, unchecked and harmful to the host, hence needs to be suppressed. Alloimmunity is trigger operated, responds to feedbacks and protective to host. But, when immunosuppression is started for a person with autoimmune disorder, none of the immune function tests are used to guide standard of care for various reasons such as no confirmed benefits, lack of cost effectiveness and inability to transform into clinical outcomes. In these situations, immune system aberrations are dealt, not according to immune status of host (which may be described by an immunometer) but according to target organ function. This leads to excessive or inadequate immunosuppression. In other words, it’s preferable to suppress autoimmunity selectively and preserve alloimmunity with the help of best possible immune function tests.

Interleukin-6 (IL 6) is elevated in the sera of SLE patients and is considered a sensitive marker of disease activity and nephritic flares. It is a potent stimulator of the differentiation and activation of lymphoid and myeloid cells and the production of acute phase proteins within the liver [13, 14]. But as we know, none of the management guidelines on autoimmune disorders recommend immune function tests as a part of standard of care (Figure 6).

Figure 6.

It shows injury immunity axis for autoimmune disorders. There are two different immune systems at work 1. Alloimmune and 2. Autoimmune. Right upper quadrant shows unchecked hyperactive autoimmunity causing tissue damage. Post immunosuppressive therapy patient may migrate to right lower quadrant with reversal of tissue injury with preserved alloimmune response. If immune suppression is continued or escalated, patient reaches left lower quadrant with suppressed alloimmune response making host susceptible for infections, drug toxicities and consequent tissue injury (left upper quadrant). Clinicians may prefer to hold patients in right lower quadrant as long as possible (state of disease remission).

3. Quest for immunometer

Immunometer, as we may call it, is a number that tells us status of immune system (innate immunity, acquired immunity and autoimmunity). It is essentially at a conceptual level and may be a vital component of immune system if proves validated. Multiple molecules are candidate for immunometer but none stands proven as of now. It could be because of lack of robust data on tissue injury phenotyping or traditional reliance on target organ function parameters to access immune system status and obvious complexity of immune system itself. We observed that in above mentioned 2/2 models for different disease states injury immunity axis gave us desired phenotypes. Injury immunity axis can be exploited to find different disease phenotypes explaining etiology, treatment options and possible disease trajectory. It will address the core issue of inflammation at cellular level guiding clinicians to tailor treatment case to case basis and avoid unnecessary interventions. The immunometer can consist of three types of biomarkers describing current status of immune system. First type of biomarkers can describe dampened immune response (immune paralysis) and the prototype biomarker can be IL-10. Second type can be a set of biomarkers exhibiting excessive immune response (immune hyperactivation) and the prototype biomarker can be interleukin 6/12, TNF alpha. The third type can describe immune protection (appropriate immune response) to the host and the prototype biomarker can be IgG against the particular antigen or transforming growth factor beta. Although produced by a wide variety of cell types, macrophages and T lymphocytes (T cells) are the primary producers of cytokines, which may have predominantly pro-inflammatory (inflammation-promoting; IL1α, IL1β, IL2, IL6, IL8, IL12, TNFα, IFNγ) or anti-inflammatory (inflammation-suppressive; IL4, IL5, IL10, TGFβ) abilities [15, 16]. The relative contribution of various biomarkers can be explained with the help of immunometer. This is a novel concept and needs further research. The immunometer concept will stimulate further research and pave way for the discovery of novel biomarkers.

To explain the concept, let us see an example of how important immune function testing can be. 53-years-old male patient presented with complaints of fever, dysuria and malaise for 1 week. On evaluation, he was diagnosed as bilateral pyelonephritis with right sided emphysematous pyelonephritis. As a result, patient developed urosepsis, acute kidney injury (AKI) on chronic kidney disease (CKD). Due to severe azotaemia and acidosis, patient required dialytic support (SLED). Though there was partial improvement initially, he again had reappearance of high-grade intermittent fever. This warranted for a right sided PCNL and drainage of perinephric collection. To look for other causes of non-responding sepsis, serum ferritin and interleukin 6 (IL-6) were tested. Both of these biomarkers were significantly elevated. (Ferritin 4532 ng/ml and IL-6 531 pg/ml on 1.8.19). Bone marrow aspiration and biopsy was done which revealed acquired hemophagocytosis with no evidence of myelodysplasia, granulomas or infiltration. Patient was started with hydrocortisone 200 mg/day. Gradually fever subsided and hemodynamic improved. As patient showed adequate respiratory attempts and acceptable oxygenation, he was extubated [17].

Seemingly, treating immunological disorders and sepsis is seen antagonistic but might be mutually complementary as is seen in this particular case with acquired HLH (hemophagocytic lymphohistiocytosis) where patient responded to immune suppression with corticosteroid. Hence, biomarkers like ferritin, IL 6 can constitute the immunometer and can help diagnose underlying immune dysfunction.

Moreover, antibiotic resistance and scarcity of new antibiotics are two more reasons to search for immune dysregulation in sepsis. As many of these cases could be immune dysregulation and not infection per se (Figure 7).

Figure 7.

It shows a model of immunometer. It is akin to a galvanometer. On left hand side of center, it shows extent of immune suppression (dampened immune response) and on right hand side, it shows immune hyperactivation (excessive immune response or a cytokine storm). The central part is an appropriate immune response of the host which is either protective or reparative. The knowledge of the type of immune response a particular patient exhibits on the immunometer scale can be helpful in diagnosis of disease phenotype, choosing treatment modality and prognostication as well. Immunometer concept can be applied to multiple biomarkers at a time that may change the outlook of multiple clinical presentations of one particular disease.

4. Candidate molecules/cells for constructing an immunometer

Multiple of molecules and cells contribute to immune functions. Many of these are specific for a particular arm of immune system. We tried to compile most of these molecules/cells for finding out appropriate candidate/s for describing immune system as immunometer (Table 1).

Innate immune system		Acquired immune system
Cells	Substances	Cells	Substances
aAPC, aDC, Macrophages (M1, M2),	CH 50, C3, C4, Perforins, Granzymes, soluble fas ligands	CD14, Monocyte HLA DR, Eosinophis, actiavated T cELLS, Treg, Breg, TH17, CD 4+, cd 8+, neutrophil to lymphocyes ratio (NLR), platelet to lymphocyte ratio (PLR)	IL 1, IL6, IL 10, sTNF receptor, TNF, TGF-b, chemokines, resolvin, protectin D1, Heat shock proteins
Autoimmunine system
Post organ transplantation (alloantigen)		Native tissue antigen (autoantigen)
Cells	Substances	Cells	Substances
Immunophenotyping Organ biopsy,	Donor specific antibodies (HLA/NON-HLA), urine perforins, CD antigens (14, 3, 4)	TH1/TH2 ratio, CD 4+/25+ cells, neutrophil to lymphocyes ratio (NLR), platelet to lymphocyte ratio (PLR)	ANA, ANCA, DS-DNA, anti GBM antibody,

Table 1.

Candidate molecules/cells for constructing an immunometer.

The Table shows three types of immune systems at work. The innate, acquired and auto immune systems are closely related and affect each other in many ways. To understand their respective contribution to a particular disease state, we need distinct biomarkers. These biomarkers will constitute the hypothetical immunometer helping clinicians decide on targeted treatment to modify the particular immune dysregulation. A lot of work and data is needed before we bring the immunometer hypothesis to reality.

5. Conclusions

This article is an attempt to understand multiple pathways for a particular disease state to manifest in different phenotypes. Each particular phenotype having a distinct immune function abnormality requiring a precise targeted treatment. Unless we understand the immune interplay among different phenotypes of a single disease, it may not be possible to address the underlying immune function abnormality for achieving better outcomes.