3. Arginine-NO Metabolism during Sepsis and Inflammatory Conditions
Sepsis and inflammatory conditions are characterized by organ dysfunction [
124,
125], as a result of a misdistribution of blood flow and low peripheral vascular resistance especially at the microcirculatory level which in turn results from the inflammation and endotoxemia [
126,
127,
128]. Furthermore, sepsis is considered to be an arginine deficient state [
2,
3,
4,
129] with arginine becoming a semi-essential amino acid during stressed conditions such as sepsis [
2,
130] (see
Figure 2). Although more arginine is being released from protein breakdown [
131], it is also accompanied by an enhanced consumption of arginine, an impaired arginine
de novo synthesis and a decreased supply of arginine, as observed in septic patients compared with healthy adults and with non-septic intensive care unit patients, leading to an impaired availability [
1,
2,
131]. However, this does not necessarily mean that arginine deficiency occurs universally in all septic or inflammatory conditions. It is beyond the purpose of this review to determine whether arginine deficiency is present in all these conditions. Therefore, we focus on the available literature on arginine deficiency in human sepsis and animal models, using prolonged endotoxemia to mimic the arginine deficient state as observed in human septic patients. We previously observed decreased arginine concentrations in murine endotoxemia and in septic ICU patients [
3,
132]. During murine endotoxemia, arginine deficiency was present accompanied by a decreased NO production in the jejunal tissue of these animals. As a result, an impaired microcirculation in the jejunal villi was observed [
3]. In the septic ICU patients, the decreased arginine concentrations were present independent of the cause of sepsis [
132].
Several causes may be responsible for this impaired arginine production or the enhanced catabolism of arginine. The impaired arginine production can be the result of limited citrulline availability for arginine
de novo synthesis, which may result from decreased nutritional intake in the critically ill patient [
1,
133,
134,
135,
136], decreased uptake of protein by intestinal failure [
2] or an impaired glutamine-to-citrulline conversion despite adequate splanchnic glutamine uptake [
137]. Furthermore, renal failure can limit arginine
de novo production from citrulline [
39,
40,
41,
138].
Arginine utilization is enhanced by the catabolic activity of arginase [
2,
139,
140] and NOS2 or by an increased protein synthesis during inflammatory conditions [
71,
76,
131,
141,
142,
143,
144]. In the past, an enhanced NO synthesis during the initial phase of sepsis was suggested. This was considered as observed by enhanced plasma concentrations of nitrate/nitrite (NOx) [
145,
146]. Based on these increased plasma nitrite and nitrate levels in septic patients, and on the fact that cytokine-induced NOS2 expression releases more NO during experimental conditions compared to the other NOS enzymes, a key role for NOS2 in the hemodynamic changes of sepsis was expected [
147]. More recent studies indicate that the role of NOS2 expression rates and excessive NO production in causing hypotension is overrated [
148,
149], which will be discussed in more detail in
Section 3.4. To estimate the NO production in septic patients, measurement of NOx levels was frequently used. As recently observed, increased plasma levels of NOx were observed in septic ICU patients compared to control patients [
131,
146]. These concentrations were even significantly increased in septic patients with hemodynamic failure or fatal outcome [
146]. However, this enhanced nitrate/nitrite levels do not necessarily reflect an increased NO production during sepsis or endotoxemia, as several causes may result in these elevated plasma concentrations [
2,
150]. At first, a possible slower turnover of NOx in the plasma during sepsis may result in the increased concentrations of NOx, as observed in hypotensive septic patient [
150]. A second contributor to higher plasma NOx concentrations may be the impaired renal excretion of NOx, as a result of renal failure as present in sepsis [
151,
152]. Another important explanation may be the discrepancy in time-specific changes, per hour and day, in the NOS enzyme activity during the course of endotoxemia and sepsis [
153,
154], which may result in an increased plasma nitrate concentrations as result of the delay in conversion into nitrate and the renal excretion [
8]. Thus, the above-mentioned explanation has to be kept in mind when interpreting studies using NOx concentrations as marker for NO production.
Figure 2.
Arginine-citrulline-NO metabolism during inflammatory conditions. Abbreviations: NO, nitric oxide; NOS3, endothelial nitric oxide synthase; NOS2, inducible nitric oxide synthase; ASS, argininosuccinate synthase; ASL, argininosuccinate lyase; AS, argininosuccinate. Essential in developing multiple organ failure is the competition for arginine between the microcirculation and the inflammatory response. NOS3 is downregulated, while NOS2 is upregulated in response to pro-inflammatory cytokines. This will lead to more NOS2 induced NO production which is essential in the immune responses. Similar, arginase, mainly arginase I is upregulated by anti-inflammatory cytokines but is expressed in a later phase of infection and will therefore prevent NO overproduction by NOS2.
Figure 2.
Arginine-citrulline-NO metabolism during inflammatory conditions. Abbreviations: NO, nitric oxide; NOS3, endothelial nitric oxide synthase; NOS2, inducible nitric oxide synthase; ASS, argininosuccinate synthase; ASL, argininosuccinate lyase; AS, argininosuccinate. Essential in developing multiple organ failure is the competition for arginine between the microcirculation and the inflammatory response. NOS3 is downregulated, while NOS2 is upregulated in response to pro-inflammatory cytokines. This will lead to more NOS2 induced NO production which is essential in the immune responses. Similar, arginase, mainly arginase I is upregulated by anti-inflammatory cytokines but is expressed in a later phase of infection and will therefore prevent NO overproduction by NOS2.
Another method used to determine the NO production in sepsis is by using stable isotopes [
2,
131,
150]. In recent years a number of research groups used this method to measure the
in vivo whole-body NO production. Data from these studies indicate that the whole-body NO production was significantly reduced in sepsis compared to critically ill control patients and healthy subjects [
2,
131,
150]. However, the possible compartmentalization of arginine [
61], intracellular or within organs, may influence the NO production measurements with stable isotopes, as this is not used in the calculation of whole-body NO production [
155]. As observed, this may also contribute to differences measured in NO concentration with NOx or total body NO production [
2,
131,
150]. Furthermore, the location of the expressed NOS-isoforms within the organs may influence the measurement of the NO production. As described by Cauwels
et al. [
148], non-hematopoietic cells are essential for the systemic NO production during inflammation, such as the intestinal enterocytes, the Paneth cells or hepatocytes. Therefore, these different expression sites, which may be differently affected by inflammation and sepsis, can also lead to unparalleled concentrations of NO production. For example, nasal NO measurements is a good sensitive and specific biomarker for nosocomial infection acquisition in the upper airway and respiratory tract [
156,
157,
158]. In patients with maxillary sinusitis and sepsis, nasal NO production was significantly reduced in the sinus epithelium compared to control patients, suggested to be the result of reduced NOS2 mRNA expression [
157]. Also in patients with acute respiratory distress syndrome (ARDS) low NO levels were measured, which as suggested by the authors may be the result of the rapid reaction of NO with other molecular species, and therefore of less value to measure pulmonary inflammation [
159]. As for patients with pneumonia on mechanical ventilation, the NO levels were significantly increased, although basal concentrations were significantly lower compared to other studies [
151]. In line, in acute lung injury patients increased urine NO was measured, which was associated with a better outcome, which may reflect more perfused lung beds and associated decreased organ failure [
160].
Another suggestion is the time point at which the NO production is measured which may influence the differences in NO production and outcome of the abovementioned studies. This time-point is influenced by the expression of the enzymes, the possible plateau phase of NOS expression or the maximum NO production [
153,
161]. This plateau phase of NO production further depends on sufficient substrate availability, in other words arginine availability and its co-factors [
2,
3,
131]. As described in the clinical sepsis studies and experimental studies with endotoxemia, arginine deficiency is related to sepsis and also to a decreased NO production [
2,
3,
129,
131,
146]. All the abovementioned factors may lead to the discrepancy between the low NO production measured in tissue or plasma during sepsis and the increase in nitrite/nitrate plasma or urine concentrations as measured in experimental and human sepsis. Thus, measurements of total NO production during sepsis and endotoxemia may only be an estimation of the total production or presence of NO in the tissues during sepsis and endotoxemia.
On the other hand, the production of NO by NOS3, especially for the endothelial cell, is decreased during sepsis and prolonged endotoxemia [
3,
162]. Decreased arginine availability is suggested to play a role in this downregulation of NOS3 and thereby in the decreased bioavailability of NO. This reduced NO results in endothelial dysfunction [
163,
164].
The immune response also contributes to the arginine depletion. During infection and inflammation, macrophages become active in response to a range of stimuli including damaged cells, pathogens and inflammatory cytokines such as TNF-α [
165]. Non-activated macrophages exhibit minimal utilization of arginine and do not express NOS2 or arginase activity. Macrophages are classified based on their two main functions resulting in 2 phenotypes; the M1 macrophages which “fight and kill” and the M2 macrophages which “restore and heal tissue” [
166]. As part of the immune response, M1 and M2 macrophages stimulate T-lymphocytes to produce Th1 or Th2 cytokines to maintain the predominant type of immune phenotype [
166]. Upon activation by the immune response, M1-macrophages actively import extracellular arginine to synthesize NO by NOS2 [
50]. This enhanced NOS2 induced NO production in peripheral blood mononuclear cells is likely to be associated with an increased arginine transport across the cell membrane [
167]. Hypothetically, decreasing the arginine availability during sepsis may, therefore function as a protective mechanism to decrease excessive NO production by NOS2 and to regulate excessive adaptive (T-lymphocyte) immune responses, preventing possible excessive inflammation during sepsis. Arginase, expressed by M2-macrophages, which is part of the anti-inflammatory response, competes with NOS2 for arginine and therefore contributes to arginine deficiency for M1/Th1-type macrophages [
133,
168,
169] (see [
170] for an excellent review on the competing arginine pathways in macrophages). However, this not only results in less NO production by M1 macrophages, but also in an impaired T-cell function [
133,
169], which is not beneficial during sepsis. The capacity of intracellular arginine
de novo synthesis in M1 macrophages partly preserves the arginine availability for NO synthesis [
171], and as will be discussed in more detail below, also presences NOS2 derived NO synthesis, which is essential in the immune response as part of the host defense. As for the T cell function, arginase-induced arginine depletion results in suppression of T-cell activation, proliferation and differentiation
in vitro [
172,
173] and
in vivo [
174,
175,
176]. However, in human T-cells, this arginine depletion does not seem to impair the important aspects of T-cell function such as chemotaxis and cytotoxicity [
169]. Therefore, these findings, strengthen the believe that a decreased arginine availability may not be beneficial in sepsis, although a more balanced arginine availability may be essential, which preserves arginine for both pathways in the immune response, and so maintains the balance between NOS2 and arginase in inflammatory conditions.
As observed by our group, in tissue-specific arginase deficient mice, lacking arginase in hematopoietic cells, NOS2 produced significantly more NO during endotoxemia in the tissues and in macrophages compared to control [
76]. However, this was not accompanied with an improved microcirculation in these animals, which may suggest the importance of compartmentalization of arginine. Based on the different expression levels and tissues in which the expressed NOS isoforms exist, this may lead to tissue specific differences in arginine and NO production, and therefore a disbalance in the local (micro)circulation and immune response. Thus, we believe sepsis and endotoxemia are not characterized by too much NO, but by a maldistribution of the produced NO, leading to perfusion differences and eventually organ dysfunction as a result of the impaired microcirculation.
3.1. Alterations in the Arginine and Citrulline Transport Systems in Sepsis
During sepsis or endotoxemia a shift in the expression of CAT transporters is observed as the increased cytokine production and bacterial endotoxins result in a downregulation of CAT-1 transporters and upregulation of CAT-2, mainly CAT-2b [
56,
58,
167]. The upregulated CAT-2 is the main arginine transporter for activated macrophages [
50,
63,
65,
177], which import large amounts of extracellular arginine [
63,
65] for NOS2-derived NO synthesis [
50,
58,
63]. CAT-2 is co-induced with NOS2 in these macrophages, which results in shifting arginine towards NOS2 instead of NOS3 [
56], to maintain the required arginine availability for NOS2-derived NO production [
178]. This shift in arginine transport is suggested to be a part of the immune host response to produce increased amounts of NO, as a response to endotoxemia and the bacterial load, to control pathogens [
56,
167]. In addition, LPS-induced macrophages produce the cytokine TNF-α during infection or septicaemia, which is capable of activation of other macrophages to participate in the host defence system [
63,
165].
3.2. Decreased Citrulline Availability as Cause for Diminished Arginine de novo Synthesis in Sepsis/Infection
Besides the observed decreased arginine concentration in sepsis and endotoxemia [
2,
3,
4,
129,
132,
179,
180], endotoxemia and inflammatory conditions are characterized by a reduced citrulline production and bioavailability [
2,
131,
181,
182]. This reduced citrulline production and bioavailability may contribute to the decreased arginine
de novo synthesis during sepsis and endotoxemia [
2,
3,
131]. These low citrulline concentrations have been associated with higher mortality rates in this patient population [
35,
130,
135,
183]. Furthermore, lower citrulline concentrations are associated with decreased NO production [
2]. Indeed, supplementation of citrulline during endotoxemia led to an enhanced NO production in tissues of these animals [
3].
A number of mechanisms can be responsible for this reduced citrulline production. At first, a decreased nutritional intake may contribute to the observed low citrulline and arginine levels in the critically ill. Moreover, a reduced uptake of glutamine by the enterocyte as part of the intestinal failure [
2,
184] is suggested to contribute to the decreased citrulline synthesis [
137], although normal levels of glutamine and ornithine have also been reported in these patients [
136,
183]. Impaired absorption of citrulline, arginine and glutamine as a result from a decreased blood flow in the intestinal villi may be a factor in this reduced uptake by the enterocyte [
185]. Also an impaired glutamine-to-citrulline conversion plays a role in this citrulline deficiency [
137].
As a compensation, ornithine transcarbamylase (OTC) may help to restore the citrulline availability during conditions with low protein intake, such as sepsis and endotoxemia [
73]. OTC converts ornithine into citrulline, which can freely pass the liver to serve as a substrate for the arginine
de novo synthesis in the kidney [
186]. Also a reduction of protein synthesis and ureagenesis may aid in restoring the citrulline and arginine metabolism during these conditions [
187].
3.3. Decreased Arginine Availability by Enhanced Arginase Activity in Sepsis
During inflammatory conditions, both arginase-I activity and arginase-II activity are induced [
2] by specific cytokines [
188] in macrophages and neutrophils [
83,
179,
189] (see
Figure 2). The cytokines that induce arginase activity are T-helper II produced cytokines, such as interleukins 4, 10 and 13, which preferentially induce the expression of arginase-I [
77,
190,
191].
The expression of arginase occurs at different time points during the course of endotoxemia. As observed in an LPS-induced murine-endotoxemia model, arginase-I, already present in low levels before LPS induction, was upregulated in activated murine peritoneal macrophages at 12 h after the onset of endotoxemia. Arginase-I remained present during the total time course of the experiment with a peak expression at 36 h after the onset [
142]. On the contrary, arginase-II exhibited an increase in expression after 3 h of LPS stimulation with a peak expression at 6 h after induction and slowly decreased afterwards [
142].
As described above, arginase-I and NOS2 compete for the same substrate, which may suggest that the late onset of arginase-I expression is suggested to prevent the toxic effects of NOS2-induced overproduction of NO [
83]. Indeed, the different time points of peak expression of NOS2 and arginase in murine endotoxemia models, after, respectively, 20 min, 4–6 h and 12 h for NOS2 [
153] and 12–36 h for arginase may indicate this inhibitory effect [
142,
192,
193,
194]. Furthermore, the increased circulating neutrophils, with arginase containing granules [
83] which are capable of releasing arginase in the extracellular space in case of cell damage or phagocytosis, further contribute to the depletion of extracellular arginine availability for NO synthesis during endotoxemia [
133,
143,
179].
In endothelial cells both arginase-I and arginase-II are expressed during inflammatory conditions, which compete with NOS3 for arginine, resulting in an impaired NOS3-derived NO production and endothelial dysfunction [
78,
79,
80,
195,
196]. We recently demonstrated that tissue-specific absence of arginase-I, in endothelial cells and macrophages, resulted in increased arginine availability. However, due to the increased NOS2 induced NO production this did not result in beneficial effects upon the microcirculation during endotoxemia [
76]. In addition, an increased arginase-II activity in endothelial cells also resulted in enhanced arginine utilization. This led to limited substrate availability for NOS3; resulting in NOS3 uncoupling accompanied by a reduced NO production and augmented superoxide anions production [
78,
79,
80].
3.4. Regulation of NOS2 during Sepsis/Inflammation
Historically, cytokine-induced NOS2 expression and consequential enhanced NO production were suggested to be key factors in the development of sepsis-induced acute hemodynamic changes [
140,
197,
198,
199,
200] and end-organ damage [
146,
162,
199,
201]. The impact of NOS2 derived NO production was recently re-evaluated in an experimental LPS endotoxemia model indicating no association between NOS2 derived NO production and hypotension [
148].
LPS and T-helper I cytokines, such as interleukin-1, 6 and 8, TNF and interferon-γ, can induce NOS2 expression as part of the cellular immune response [
191,
202,
203,
204]. On cellular level, sepsis results in a cytokine-mediated induction of NOS2 in almost any cell type [
198], but especially in macrophages [
197]. Other cell types, including hepatocytes [
205], aortic smooth muscle cells [
51], vascular smooth muscle cells [
199,
205,
206], Paneth cells and enterocytes of the jejunum [
148] also participate. As mentioned above, the non-hematopoietic cells contribute to the systemic NO production, and not the hematopoietic cells such as macrophages or endothelial cells [
148,
149].
This enhanced NOS2 expression plays a major role in the host defense mechanisms against various intracellular pathogens [
197], and represents a major cytotoxic principle by accomplishing successful clearance and control of these intracellular pathogens [
97,
98]. Therefore, maintenance of NOS2 derived NO is essential during sepsis and endotoxemia as part of the host defense mechanism, as inhibition results in detrimental outcome, which will be discussed in
Section 4.1.
3.5. Regulation and Expression of Constitutive NOS during Infection/Sepsis
During inflammatory conditions and especially during sepsis [
207,
208], NOS1 exhibits an important immunoregulatory role, involving both pro-inflammatory and anti-inflammatory pathways [
209,
210,
211]. As part of the pro-inflammatory pathway, NOS1-induced NO production is suggested to act as a free radical and is prone to be converted into more reactive nitrogen species [
209,
212,
213,
214]. Endothelial cells express both NOS1 and NOS3, with NOS1 localized in the nucleus and NOS3 in the cytosol [
215]. Previous studies already indicated an important role for NOS1 in the maintenance of the microvascularisation [
102,
105,
106]. Furthermore, NOS1 localized in nucleus of endothelial cells, exhibits an anti-inflammatory role by preventing cytokine production after TNF stimulation in cultured endothelial cells [
106,
215,
216].
As for NOS3, inflammatory conditions and the decreased arginine availability result in NOS3 uncoupling [
48,
119,
217,
218], endothelial dysfunction [
219], with an increased superoxide production [
119,
220] and disturbed microcirculatory flow as a result [
221,
222,
223].
Another important contributor to NOS3 uncoupling is the induction of asymmetric dimethylarginine (ADMA). ADMA and
NG-methyl-
l-arginine (
l-NMMA) are the most powerful endogenous non-selective NOS inhibitors. ADMA competes with
l-arginine for the active site of NOS and for y
+-mediated uptake into cells (see [
224] for recent review). These methylarginines are eliminated from the body by enzymatic degradation by dimethylaminohydrolase (DDAH) and renal excretion. Along with arginine availability, endogenous inhibitors of NOS, including ADMA, may affect NO synthesis [
131]. During sepsis increased ADMA levels have been observed which inhibit NOS derived NO production resulting in an impaired microcirculation [
225]. Furthermore, increased mortality in critically ill patients has also been related to elevated ADMA levels [
226,
227]. The correlation between arginine and ADMA, defined as the
l-arginine/ADMA ratio, is decreased in adult sepsis patients [
228] indicating a role of ADMA in the modulation of NOS-regulated microcirculation.
3.6. ASS during Sepsis/Inflammation
Inflammation results in an upregulation of ASS expression, especially in macrophages [
229,
230] and vascular smooth muscle cells [
206,
231]. This may indicate stimulation of the intracellular arginine
de novo synthesis to maintain the NOS2-derived NO production [
47,
229]. During the early phase of inflammation and sepsis, with still sufficient extracellular arginine available, macrophages export approximately >98% of the intracellular produced citrulline into the plasma [
20,
50]. The intracellular regeneration of citrulline, therefore, only becomes important in case of arginine deficiency. During arginine deficiency, NO synthesis in macrophages depends on the import of citrulline to maintain the NOS2-induced NO production [
50,
232]. Since intracellular accumulation of citrulline is thought to facilitate excessive arginine
de novo synthesis, which results in abundant amounts of NO, exporting citrulline is suggested to regulate the NO production in macrophages [
47,
233,
234]. Another important role of ASS as part of the innate immune system’s defensive role, is the clearance and detoxification of LPS by binding to the active portion of LPS, lipid A, which result in inactivation of LPS [
235,
236]. In addition, ASS released from the liver into the systemic circulation neutralizes the extracellular LPS-induced cytotoxicity in response to inflammation [
230].
Until know, no studies on ASL function during endotoxemia or inflammatory conditions have been conducted.