Typically two quantitative proteomic platforms are used for profiling plasma: gel based 2-dimensional electrophoresis usually with difference gel electrophoresis (2DE DIGE) 20–22
and mass spectrometry based quantitation using isobaric Tags for Absolute and Relative Quantitation (iTRAQ) 23, 24
. In our previous investigations of plasma samples we used 2DE DIGE platform which resulted in identification of previously not reported differential expression of proteins such as afamin and gelsolin 18, 25, 26
. Differential expression of these proteins were further confirmed in similar analysis performed with SIV infected rhesus macaques 9, 27
. While select proteins in the current study (gelsolin, ceruloplasmin, complement C3, vitronectin, for example) were previously documented being differentially expressed in human samples 18, 25, 26
, the evaluation of these proteins in a broad range of interdisciplinary studies under distinct pathobiologic settings provides further validation for their importance in HIV-1 disease. Here we have expanded such studies by using the iTRAQ platform to quantitatively analyze differentially expressed plasma proteins during the course of SIV infection, with successful validation of selected statistically significant proteins.
Biomarkers for monitoring the progression of immunodeficiency virus infection are still limited to measurements of viral loads and T cell subtypes 4, 28
. There are a handful of other cellular markers that correlate with systemic viral infection however they do not have adequate sensitivity and specificity to be used in clinical practice. Progression of disease over many years is a very complex process and includes distinct host responses occurring during the acute and chronic phases. Acute response to viral infection occurs within days to a few weeks after viral exposure and is characterized by rapid elevations in viral load and substantive decreases in the numbers and function of CD4+ T lymphocytes. Transition from acute to chronic phase is a complex process and one of reasons is that during acute infection, members of the complement cascade remain at the same level or decreased as prior to infection. Acute infection leads to humoral and cellular immune responses specific to the infecting viral strain that typically suppresses viral replication and partially restores CD4+ T cells. The resulting chronic viral disease stage becomes symptomatic for the host after several years when sufficient numbers of T cells are destroyed. SIV infection of rhesus macaques reflects this process and infected animals after initial acute phase enter chronic infection and eventually progress to terminal stage of AIDS 5–7
. In this study we used this non-human primate model to investigate changes in plasma proteomes at three time points: before, during acute and chronic phases of infection to get an insight of organism’s response to viral infection in a very well controlled experiment.
Acute phase proteins (APPs) belong to a biochemically and functionally diverse group of proteins that are typically produced in liver as a response to inflammation. The observed changes in APPs likely represent host response to initial SIV infection. We observed that several positive acute phase proteins, including, serine proteinase inhibitors (SERPINS), complement, plasminogen and ceruloplasmin, as well as negative acute phase proteins such as transthyretin, retinol binding protein and histidine rich glycoprotein were affected by SIV infection.
SERPINS are a group of proteins that inhibit proteases and play key roles in controlling inflammation. Here we identify 2 SERPINS, A1 and A3 that are differentially regulated through the course of SIV infection. During the course of acute and chronic infection, SERPINA1 increased 81% and 232%, respectively, when compared to baseline levels. SERPINA1 inhibits the formation of gp120, as well as it inhibits HIV protease thus preventing HIV p55(gal) processing to p24, which are critical processes for HIV-1 morphogenesis 29, 30
. Recently, it was demonstrated that a C-terminal portion of SERPINA1 inhibits HIV-1 infection by as much as 99%, while full length SERPINA1 only inhibited HIV-1 infection up to 70% 31, 32
. These results provide supporting evidence that the host is unsuccessfully attempting to compensate for increased viral replication through many mechanisms and an increase in SERPINA1 might be one of them.
Leucine rich alpha 2-glycoprotein (LRG) expression has been shown to increase in various diseases 33, 34
, however it’s physiological function has yet to be defined. Shirai et al propose LRG as a potential biomarker in certain inflammatory conditions as their data suggests that LRG is an up-regulated APP in response to stimulation of hepatocytes by proinflammatory cytokines 35
. While we observed no significant change of LRG during acute infection, we observed a 103% increase during sustained chronic infection. Another study using iTRAQ platform also identified LRG as a novel inflammatory serum biomarker for autoimmune diseases 36
. Our results, along with other published studies, provide evidence that while LRG may not be a biomarker for a specific disease, but a more universal marker for inflammation.
Histidine rich glycoprotein (HRG) is a negative APP 37–39
and has been documented to decrease in the acute states of AIDS 40
. Our observation of a 45% decrease of HRG during acute infection, while rebounding to baseline levels in chronic infection supports these findings. HRG also interacts with several components of the complement system and assist in maintaining normal immune function, however, it’s full affect on the complement cascade is unclear 41
We posit a relationship between plasminogen activity and vitronectin in affecting HIV-1 infection. The up-regulation of vitronectin during the chronic phase of SIV infection may indicate an effect on cell-associated viral transfers based on interactions with integrins and in cell adhesion as it has been shown that attachment of virus to its host cells is a critical early event for infection 42
. It was previously shown that urokinase-type plasminogen activator (uPA) inhibits HIV-1 replication in macrophages and that this inhibition is directly linked to vitronectin-mediated cell adhesion 43
. uPA activates plasminogen into the active serine protease plasmin 44
and both uPA and it’s receptor are expressed in immunocytes 45
and affect inflammatory processes 46
. That plasminogen levels decreased without change in vitronectin during the acute phase of infection with both being upregulated during the chronic phase of infection, further supports the observation that plasminogen activator is dependent on vitronectin activity 43
Other differentially expressed proteins identified in this study is comprised of several members of apolipoprotein family, hemopexin, vitamin D binding protein (VDBP), fibronectin, and vitamin K dependent Protein S, all which are also primarily made in the liver in response to inflammation. However, we observed changes in different directions e.g., no change from baseline to acute phase and increase as infection progressed to chronic phase or decrease during acute phase and restoration to pre-infection levels later in infection. These changes indicate that various components of organism’s response to viral infection are differentially regulated and defining a pattern will help us better understand the course of virus survival from immunesurveillance.
Interestingly most proteins of the complement cascade are down-regulated or not changed during acute phase however they are significantly up-regulated in chronic phase, which suggests that during initial infection, the complement cascade is suppressed by the virus and re-bounds as infection chronically progresses. The complement inflammatory cascade is part of the innate immune response bridging with acquired immunity by enhancing antibody responses and immunologic memory, lysing foreign cells and clearing immune complexes and apoptotic cells. Activation of the complement cascade is initiated through three pathways: classical, alternative and lectin. All pathways meet at the “hub” point, which is C3 and it, is cleaved to C3a and C3b triggering downstream reactions to produce anaphylatoxins C5b and C5a. The latter subsequently triggers formation of Membrane Attack Complex (MAC). In addition, C3 shows multiple pro-inflammatory functions involving histamine release from mast cells, smooth muscle contraction, increased vascular permeability 47
, chemotaxis of dendritic cells and mast cells 48
Complement C3 also modulates adaptive immunity at various levels, including cytokine release, T-cell proliferation, regulatory T cell development and B cell activation/differentiation 49–51
. It has been reported that C3b and C3c can also control antigen proteolysis, which could be related to C3-mediated enhancement of antigen presentation, thus, aiding in the establishment of a specific adaptive immune response 52
Complement components C5b, C6, C7, C8 and C9 create the MAC. Out of these five proteins we observed up-regulation of C6, C8 and C9, as well as C5, which is directly upstream of this complex. The host immune system may up-regulate C5 convertase to initiate formations of more MACs in response to viral infection, however we did not observe increased levels of C5b fragment which initiate formation of MAC. Because MAC is formed in a 1:1:1:1:1 ratio, up-regulation of C6 and C8 might be a confounding effect and seems unlikely that over-expression of these two elements will increase MAC formation. On the other hand the net level of any component circulating in blood might be affected by the fact that other cells outside of liver also produce some complement proteins.
Function and role of complement C4a has been mostly studies in the context of lupus erythromatosus and deficiency in either C4a or C4b is associated with autoimmunity. Recent study showed that patients with AIDS have persistent elevated levels of C4a which does not change with ART treatment while C4b levels remain unchanged 53
. In our current approach using a monkey model, we report that levels of C4a increase 91% in chronic infection, which correlates with plasma samples from humans 18, 25
, the biological consequence of this observation and previous reports that C4a, is strong inhibitor of blood monocytes chemotaxis at concentrations as low as 10(−16) mol/L 54
Ceruloplasmin is a 132 kDa protein mostly synthesized in the liver, but also in the brain 55
. It is a copper containing protein with ferroxidase activity oxidizing Fe+2
to less toxic Fe+3
, which contributes to homeostasis. Defense against oxidant stress by scavenging superoxide radicals and oxidation of low density lipoproteins and its compensatory role to oxidative stress 55
has been postulated as beneficial in several pathological conditions including HIV infection 56
. Unchanged (statistical analysis) or lower (Western blot analysis) levels of ceruloplasmin during acute phase of SIV infection may help the virus to escape from immune surveillance. Increased levels of ceruloplasmin (confirmed by statistical and Western blot analyses), which could be linked to increasing inflammation during late stages of disease, were also observed in sera of patients with HIV-associated dementia (HAD).
Gelsolin and its secretory form plasma gelsolin (pGELS) are distributed throughout body with body fluids. pGELS is not synthesized in liver and very little is known about regulation of its expression. Clinical studies have shown that pGELS is linked to a number of pathological conditions such as inflammation, cancer and amyloidosis 57
. Increasing experimental evidence indicates that pGELS plays an important role in acute as well as chronic inflammatory responses although both mechanisms are different. For example in chronic rheumatoid arthritis there is crosstalk between cytoskeleton and immune system mediated by cytokines and transcriptional factors 58
involving pGELS. Spinardi and Witke concluded that modulation of pGELS expression might have beneficial effects such as inhibition of pGELS during acute inflammation and boosting pGELS in chronic inflammatory response 57
. According to this interpretation, our observed down-regulation in gelsolin levels during acute phase may reflect host’s effort to combat viral infection.