**3. The determinants of immunoparalysis**

Only recently it became clear that the CARS does not represent only a physiologic counterbalance to the inflammatory response to PAMP and DAMP but that it can determine a critical condition in and by itself [13, 15].

Actually, different experimental and clinical studies indicate that the advanced stage of sepsis and SS is characterized by a reduction of both the innate and adaptive immune responses (**Table 4**). Extensive evidence supports this model, even if large inter-patient differences exist. First, monocytes present a reduced expression of membrane HLA-DR in association to either a decreased secretion of inflammatory mediators when stimulated or a diminished antigen presentation. Second, different membrane-bound receptors able to potentiate the immune response, including IL-2α, IL-7R α, CD86, etc., are reduced. Third, the production of immunosuppressant substances, such and programmed death 1 (PD1) and its ligand (PD-L1), is increased in antigenpresenting cells, thus inhibiting the activation of T lymphocytes. Fourth, there is an increased appearance of immunosuppressive T-cell subpopulations, such as myeloid-derived suppressor cell and CD4<sup>+</sup> and CD25<sup>+</sup> T-regulatory cells (Treg), which suppress adaptive immunity. These appear to be particularly relevant, as Treg (a) actively produce anti-inflammatory cytokines including TGF-β and IL-10, (b) downregulate the secretion of pro-inflammatory mediators, (c)

neutralize cytotoxic T cells, and (d) deactivate the monocytes. Fourth, immune cells present an increased apoptosis, and their loss is not replaced enough by the production of new ones. Finally, the phagocytosis of apoptotic cells by fixed and circulating macrophages leads to a switch of the latter to the M2 phenotype, whose feature is an increased production of the antiinflammatory substances IL-10 and IL-1ra. Put briefly, all these mechanisms exert their action via relatively few common pathways, which include the increased apoptosis determining the reduction of immune cells, the loss of antigen presentation, the blunted response to PAMP, and the reduction of energy production caused by the impairment of the glucose metabolism (**Table 5**) [16, 17]. All these reactions are driven by epigenetic changes causing in different time frames the activation or deactivation of genes involved in the immune response, and the resulting phenotype is an intense inflammatory response or, conversely, an immunoparalysis.

Endotoxin tolerance ↑ Anti-inflammatory mediators, ↓ pro-inflammatory mediators

Apoptosis

↓ Antigen presentation

Immune cell number anergy

Immunoparalysis in Septic Shock Patients http://dx.doi.org/10.5772/intechopen.88866 13

The recognition of sepsis-induced immunoparalysis is not straightforward because the clinical manifestations associated with the switch from the hyperinflammatory state to CARS and the full-blown depression of the immune capabilities are not so protean as the symptoms of SS [18]. Moreover, the SSC guidelines focus almost exclusively on the former and pay much less attention, if any, to the latter. From a practical and clinical point view, some issues appear

The transition from the hyperinflammatory phase to immunoparalysis can be challenging to identify and to monitor at the bedside and represents a kind of no man's land in the clinical

The onset is highly variable. Actually, although the secretion of immunomodulatory substances can occur relatively early, their clinical consequences present wide variations. Some authors [19] observed a substantial difference of mHLA-DR starting from 3 to 7 days in a small group of surgical septic patients, and other authors demonstrated that significant decrease of

**4. The diagnosis of immunoparalysis**

**Mechanisms Effect**

**Table 5.** Mechanisms of immunoparalysis.

Apoptosis ↓ Immune cell number

Energy failure Immune cell anergy

Epigenetic regulation ↓ Pro-inflammatory mediators

course of patients which survived from the initial phase of SS.

particularly relevant.

**4.1. Timing of onset**


mHLA-DR, human leukocyte antigen on the monocyte surface; PD-(L1), programmed death ligand; CTLA-4, cytotoxic lymphocyte antigen 4; BTLA, B and T lymphocyte attenuator; LAG-3, lymphocyte activation gene 3; TIM-3, T lymphocyte immunoglobulin protein 3; sFAS, soluble FAS ligand; TGF-β, transforming growth factor-β.

**Table 4.** Factors of immunosuppression.


**Table 5.** Mechanisms of immunoparalysis.

toward the baseline immune function; and in the third one, the CARS prevails and causes the

Only recently it became clear that the CARS does not represent only a physiologic counterbalance to the inflammatory response to PAMP and DAMP but that it can determine a critical

Actually, different experimental and clinical studies indicate that the advanced stage of sepsis and SS is characterized by a reduction of both the innate and adaptive immune responses (**Table 4**). Extensive evidence supports this model, even if large inter-patient differences exist. First, monocytes present a reduced expression of membrane HLA-DR in association to either a decreased secretion of inflammatory mediators when stimulated or a diminished antigen presentation. Second, different membrane-bound receptors able to potentiate the immune response, including IL-2α, IL-7R α, CD86, etc., are reduced. Third, the production of immunosuppressant substances, such and programmed death 1 (PD1) and its ligand (PD-L1), is increased in antigenpresenting cells, thus inhibiting the activation of T lymphocytes. Fourth, there is an increased appearance of immunosuppressive T-cell subpopulations, such as myeloid-derived suppressor

appear to be particularly relevant, as Treg (a) actively produce anti-inflammatory cytokines including TGF-β and IL-10, (b) downregulate the secretion of pro-inflammatory mediators, (c)

T-regulatory cells (Treg), which suppress adaptive immunity. These

↓ TNF-α production

↓ lymphocytes

↑ IL-10/TNF-α

Anti-inflammatory cytokines ↑ IL-10, IL-13, IL-4, IL1 receptor antagonists, TGF-β

immunoglobulin protein 3; sFAS, soluble FAS ligand; TGF-β, transforming growth factor-β.

mHLA-DR, human leukocyte antigen on the monocyte surface; PD-(L1), programmed death ligand; CTLA-4, cytotoxic lymphocyte antigen 4; BTLA, B and T lymphocyte attenuator; LAG-3, lymphocyte activation gene 3; TIM-3, T lymphocyte

↑ CTL-4, BTLA expression ↑ LAG-3 and TIM-3 expression

↑ myeloid-derived suppressor cells

loss of the immune capabilities.

12 Infectious Process and Sepsis

condition in and by itself [13, 15].

and CD25<sup>+</sup>

**Factors involved Marker**

Tissue macrophage dysfunction Presently none Negative regulatory mediators ↑ PD-(L)1 expression

Receptors downregulation ↓ IL-7 receptor

Suppression of immune cells ↑ CD-4, CD-25

Apoptosis ↑ FAS

**Table 4.** Factors of immunosuppression.

Monocyte deactivation ↓ mHLA-DR expression

cell and CD4<sup>+</sup>

**3. The determinants of immunoparalysis**

neutralize cytotoxic T cells, and (d) deactivate the monocytes. Fourth, immune cells present an increased apoptosis, and their loss is not replaced enough by the production of new ones. Finally, the phagocytosis of apoptotic cells by fixed and circulating macrophages leads to a switch of the latter to the M2 phenotype, whose feature is an increased production of the antiinflammatory substances IL-10 and IL-1ra. Put briefly, all these mechanisms exert their action via relatively few common pathways, which include the increased apoptosis determining the reduction of immune cells, the loss of antigen presentation, the blunted response to PAMP, and the reduction of energy production caused by the impairment of the glucose metabolism (**Table 5**) [16, 17]. All these reactions are driven by epigenetic changes causing in different time frames the activation or deactivation of genes involved in the immune response, and the resulting phenotype is an intense inflammatory response or, conversely, an immunoparalysis.

### **4. The diagnosis of immunoparalysis**

The recognition of sepsis-induced immunoparalysis is not straightforward because the clinical manifestations associated with the switch from the hyperinflammatory state to CARS and the full-blown depression of the immune capabilities are not so protean as the symptoms of SS [18]. Moreover, the SSC guidelines focus almost exclusively on the former and pay much less attention, if any, to the latter. From a practical and clinical point view, some issues appear particularly relevant.

### **4.1. Timing of onset**

The transition from the hyperinflammatory phase to immunoparalysis can be challenging to identify and to monitor at the bedside and represents a kind of no man's land in the clinical course of patients which survived from the initial phase of SS.

The onset is highly variable. Actually, although the secretion of immunomodulatory substances can occur relatively early, their clinical consequences present wide variations. Some authors [19] observed a substantial difference of mHLA-DR starting from 3 to 7 days in a small group of surgical septic patients, and other authors demonstrated that significant decrease of the CD14/HLA-DR and of heat-shock proteins (HSP) 70 and 90 was present already within 24 hours from the onset of sepsis [5]; in both studies, these alterations were more marked in patients who developed SS later on. More recently, Morris et al. [20] in association with raised percentage of regulatory T cells (Treg) were predictive for infections occurring between 3 and 9 days after ICU admission, and a similar timing has been demonstrated also in another study in which the mortally rate of secondary infection was ~14% [17]. On the basis of these findings, it is reasonable to hypothesize that (a) a combination of cellular and soluble factors able to blunt the immune response is present since the very initial phase of sepsis; (b) their effects on the clinical course, namely, the appearance of secondary infections and/or viral reactivation, can occur within the initial 10 days from the admission; and (c) these are associated with a substantial mortality of patients surviving the initial insult.

challenges, and the measurement of the blood concentrations of soluble mediators involved in the different clinical frames [14, 15, 21, 22]. It could be useful to describe separately those currently available and those which will be used likely in the next future. Most of the former (**Table 6**) can be obtained cheaply and on a daily basis; among all, the neutrophil-to-lymphocyte ratio has been indicated as the less costly and more rapidly available monitoring tool [23, 24]. Other advanced, expensive, and not yet widely available monitoring tools take advantage of more sophisticated lab techniques (**Table 7**) requiring lab expertise and financial resources putting them at risk of not being used outside the research center. Another dynamic approach, which shares the very same limitations of the previously described advanced techniques, consists in challenging the immune cells with substances able to trigger their activation, including LPS, other PAMP, and phytohemoagglutinin; actually, a number of investigators demonstrated that a blunted response to the stimulation is associated with an increased rate of severe infec-

Independently from the systems used, it should be clear that the monitoring of the immune response in septic as well in other clinical conditions (a) is based on the time variations of a panel of indicators and not on a single one and (b) due to their direct and indirect costs,

**Function Cell Marker Outcome Lab technique Runaround (h)**

↑ PD-L1 Secondary

All lymphocytes ↑ CTLA 4, BTLA Not clear FC, IHC 1.5

Treg ↑ Treg Death FC 1.5

FC, flow cytometry; IHC, immunohistochemistry; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent

CD 127 Death,

Both Transcriptomic CD 74, CX3CR1 Not clear PCR, microarray 72

secondary infections

infections

secondary infections

secondary infections

secondary infections MODS

IL10/TNF ratio Death ELISA 5

↑ PD Death FC, IHC 1.5

Not clear Cell culture,

FC, PCR, IHC, ELISA

ELISA, FC, IHC

FC, IHC 1.5

FC 1.5

FC, IHC 1.5

Cell culture + FC 72

5

Immunoparalysis in Septic Shock Patients http://dx.doi.org/10.5772/intechopen.88866 15

72

tious complications in different patient populations [25–27].

Monocytes ↓ sCD127 Death,

Dendritic cells ↓ Count Death,

T cells Proliferation Death,

**Table 7.** Some promising, yet not currently available, markers of immunoparalysis.

Endotoxin tolerance

Innate immunity

Adaptive immunity

assay.

### **4.2. Monitoring of the immune function**

In ICU patients, every organ system is monitored to allow a change in the treatment tailored on the variation observed. An ideal monitoring system should be accurate, cheap, and not labor-intensive, and the information gathered should be readily if not continuously available. Since it has become clear that the immune system in sepsis undergoes modifications not reflected by the commonly measured biological variables such as the arterial pressure, the heart rate, the urinary output, etc., different investigations aimed to identify one or more markers of changes of its functions whose follow-up could be valuable to modify the therapy according to its changes: as an example, the occurrence of immunoparalysis contraindicates the administration of steroids whose use is recommended by the SSC guidelines.

Several monitoring systems exploring both legs of the immune response have been developed so far, based on the repeated assessments of the cells involved, their response to different


**Table 6.** Some currently available indicators of immune function.

challenges, and the measurement of the blood concentrations of soluble mediators involved in the different clinical frames [14, 15, 21, 22]. It could be useful to describe separately those currently available and those which will be used likely in the next future. Most of the former (**Table 6**) can be obtained cheaply and on a daily basis; among all, the neutrophil-to-lymphocyte ratio has been indicated as the less costly and more rapidly available monitoring tool [23, 24]. Other advanced, expensive, and not yet widely available monitoring tools take advantage of more sophisticated lab techniques (**Table 7**) requiring lab expertise and financial resources putting them at risk of not being used outside the research center. Another dynamic approach, which shares the very same limitations of the previously described advanced techniques, consists in challenging the immune cells with substances able to trigger their activation, including LPS, other PAMP, and phytohemoagglutinin; actually, a number of investigators demonstrated that a blunted response to the stimulation is associated with an increased rate of severe infectious complications in different patient populations [25–27].

the CD14/HLA-DR and of heat-shock proteins (HSP) 70 and 90 was present already within 24 hours from the onset of sepsis [5]; in both studies, these alterations were more marked in patients who developed SS later on. More recently, Morris et al. [20] in association with raised percentage of regulatory T cells (Treg) were predictive for infections occurring between 3 and 9 days after ICU admission, and a similar timing has been demonstrated also in another study in which the mortally rate of secondary infection was ~14% [17]. On the basis of these findings, it is reasonable to hypothesize that (a) a combination of cellular and soluble factors able to blunt the immune response is present since the very initial phase of sepsis; (b) their effects on the clinical course, namely, the appearance of secondary infections and/or viral reactivation, can occur within the initial 10 days from the admission; and (c) these are associated with

In ICU patients, every organ system is monitored to allow a change in the treatment tailored on the variation observed. An ideal monitoring system should be accurate, cheap, and not labor-intensive, and the information gathered should be readily if not continuously available. Since it has become clear that the immune system in sepsis undergoes modifications not reflected by the commonly measured biological variables such as the arterial pressure, the heart rate, the urinary output, etc., different investigations aimed to identify one or more markers of changes of its functions whose follow-up could be valuable to modify the therapy according to its changes: as an example, the occurrence of immunoparalysis contraindicates the administration of steroids whose use is recommended by the SSC guidelines. Several monitoring systems exploring both legs of the immune response have been developed so far, based on the repeated assessments of the cells involved, their response to different

**Function Cell Marker Outcome Lab technique Runaround (h)**

Secondary infections

Secondary infections

Secondary infections

Secondary infections

FC. Hematology analyzer

FC. Hematology analyzer

FC. Hematology analyzer

FC, IHC, PCR 1.5

1.5

0.5

0.5

Neutrophils ↑ Immature forms Death

Monocytes ↓ HLA-DR Death

All lymphocytes Lymphopenia Death

White blood cells NTL Death

**Table 6.** Some currently available indicators of immune function.

Both Lymphocytes Viral reactivation Death PCR 12

FC, flow cytometry; IHC, immunohistochemistry; PCR, polymerase chain reaction; NTL, neutrophil/lymphocyte ratio.

a substantial mortality of patients surviving the initial insult.

**4.2. Monitoring of the immune function**

14 Infectious Process and Sepsis

Innate immunity

Adaptive immunity Independently from the systems used, it should be clear that the monitoring of the immune response in septic as well in other clinical conditions (a) is based on the time variations of a panel of indicators and not on a single one and (b) due to their direct and indirect costs,


FC, flow cytometry; IHC, immunohistochemistry; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay.

**Table 7.** Some promising, yet not currently available, markers of immunoparalysis.

demonstrating the tumor cells are able to suppress in many different ways the host's immune response against themselves. Independently from the substance use and the molecular target, these innovative treatments have been demonstrated to be effective but somehow difficult to handle, as they are associated with a number of side effects ranging from mild to life-threatening [31]. As several similarities exist between tumor- and sepsis-induced blunting of the immune response [32], it is likely that in the next future the immune-boosting treatments will be developed to treat the latter, aiming to develop a precision medicine also in ICU patients

Presently, according to the SSC guidelines [2], the immune-targeted approaches are limited to the administration of steroids in not fluid and catecholamine-responding SS, whereas the use of intravenous immunoglobulins (IvIg) is discouraged. Actually, this latter position is questionable as a number of trials performed in several thousands of patients demonstrated that (a) the administration of IvIg is associated with the reduction of mortality in different subsets of SS patients; (b) among the different preparations available, the only ones containing supranormal concentrations of IgM and IgA appears more effective, and (c) the improvement of survival is time-dependent, as a ~6% increase of mortality has been observed for every day of

Besides steroids and IvIg, other treatments aimed to modulate the immune response include

GM-CSF

FTL3L TNF

Anti-PD1 ab Anti-PDL 1 ab

GM-CSF TLR agonists FT3L TNF

Toll-like receptor antagonists

Immunoparalysis in Septic Shock Patients http://dx.doi.org/10.5772/intechopen.88866 17

Anti CTLA4, TIM3, LAG3 ab

blood purification (BPT) techniques and a number of substances able to boost it.

**Cells/factors involved Alterations Possible therapies**

↑ Tolerant dendritic cells

Altered metabolism ↓ Proliferation

↑ PGE 2 ↑ TGFβ

**Table 8.** Immunosuppressive pathways shared by cancer and sepsis.

↑ Myeloid-derived suppressor cells ↓ Monocyte HLA-DR expression

↑ Immune checkpoint inhibitors Malfunction of NKT cells ↑ Treg and Breg cells ↑ CD 155 expression

GMC-SF, granulocyte-macrophage colony-stimulating factor; FTL3L, FMS-related tyrosine kinase 3 ligand; PD, programmed death; PDL1, programmed cell death ligand 1; CTL4, cytotoxic T-cell protein 4; TIM3, T-cell immunoglobulin mucin receptor 3; Treg Breg, regulatory T and B cells; TGFβ, transforming growth factor-β; PGE, prostaglandin E2.

[33] (**Table 8**).

delay in the administration [34].

Myeloid cells ↑ Immature neutrophils

Lymphocytes ↓ Cytokine production

↑ Systemic cytokine release ↑ IL-10

**Figure 2.** The multiple hits phenomenon ultimately leading to the exhaustion of the immune response.

it should be limited to the subjects at risk; as an example, it is worthwhile to monitor the immune function in patients undergoing multiple abdominal surgical procedures for suture dehiscence but not in another one safely recovering after peritonitis.

### **4.3. The identification of patients at risk of immunoparalysis**

Even with the exclusion of clinical conditions and/or treatments known to cause an immunoparalysis (i.e., solid and hematologic cancers, autoimmune disorders), etc., this circumstance can occur in virtually all ICU patients; however, different studies identified some predisposing factors that should be considered particularly relevant, including septic shock, advanced age, health care-associated infections, elevated Charlson's score indicating a substantial underlying fragility, comorbidities, prolonged hospital and ICU length of stay, and multiple surgical procedures [17, 28, 29]. The latter, which are associated with the repeated activation of the inflammatory and anti-inflammatory responses, according to the multiple hits model, ultimately lead to the exhaustion of the immune response [30] (**Figure 2**).
