Over the years several studies have tried to clarify the biological machinery involved in both innate and adaptive immune dysfunction in CKD patients in conservative and HD treatment.
The causes of this condition seem to be primarily related to: a) an abnormal accumulation of pro-inflammatory cytokines as a consequence of decreased renal elimination and/or increased generation of uremic toxins; b) elevated oxidative stress, c) volume overload and d) augmented rate of sepsis [10, 11].
Additionally, CKD-related immune dysfunction seems to be characterized by the coexistence of immune activation and immune suppression . In this setting, circulating uremic toxins and microinflammation play a critical role. However, the exact intricate mechanism associated to the immune alterations in CKD need to be more elucidated.
To better understand the biological bases underlying the immune alterations in patients with severe renal damages and to assess differences between CKD patients in pre-dialysis and those undergoing HD treatment, we used, for the first time, a transcriptomic strategy combined with classical bio-molecular approaches. This methodology allowed us to reduce biases due to the relatively small number of patients enrolled because of highly selective inclusion/exclusion study criteria (e.g., good demographic/clinical matching, exclusion of under-dialyzed patients) and to minimize confounding factors.
Furthermore, our study was performed using an oligonucleotide microarray technology able to evaluate simultaneously the expression of more than 15,000 genes. However, to take full advantage of the opportunities offered by this high throughput method, it is necessary to manage, integrate and interpret a huge amount of data correctly. Thus, we decided to use a pathway analysis to focus our research on candidate genes known to be associated with immune response in order to reduce the false positive rate and the puzzling factors not directly associated with the aims of our research.
Microarray analysis revealed that 213 immunological genes were able to discriminate CKD from HD patients. In particular, 71 genes resulted up-regulated and 142 down-regulated in HD compared to CKD. Although these differences were primarily related to the transcriptomic changes induced by the dialysis treatment, we cannot exclude that the different pharmacological treatments may have contributed to increase the immune-transcriptomic differences between CKD and HD patients. In fact, as extensively described [32–35], some medications (e.g., rHuEPO, VDR activators and IV iron), may influence both inflammatory and immune response. It is plausible that the deregulation of some immunological genes in our HD patients (for example the up-regulation of CD86 and the down-regulation of CD40/CD40LG) may have been, at least partially determined by the chronic use of rHuEPO and/or paracalcitol. However, at the state of art, there is a lack of pharmacogenomics study in this field. Further studies are warranted to address this important point.
Interestingly, the biological network generated by Ingenuity Pathway Analysis software using the top HD up-regulated genes, showed that pentraxin 3 (PTX3) and Interleukin 15 (IL-15) had an important functional role. PTX3 encodes for an acute phase protein produced by monocytes, macrophages and endothelial cells  and, as reported by several reports, its expression is high in plasma of patients with sepsis , acute myocardial infarction  and severe atherosclerosis .
Our results, confirming previous reports [40, 41], indicate that HD enhances PTX3 levels and point out that this inflammatory biomarker may be a valuable candidate to measure chronic microinflammatory state in our nephrology/dialyzed patients. In fact, in our subset of patients, C reactive protein (CRP) was not different between CKD and HD probably because less sensitive and specific biomarker for this patient population.
Then, we observed that IL-15 was highly expressed in our HD patients compared to CKD. This gene encodes for a 13 kDa cytokine produced by macrophages and other cell types in response to inflammatory and infective stimuli that shares many biological activities with IL-2 inducing the activation of JAK/STAT pathway and stimulating T and NK cells survival, proliferation and activation [42–45].
On the other hand, among the genes down-regulated in HD patients, we identified those encoding for the Human Leukocyte Antigen (HLA)-G. The HLA-G primary transcript encodes a transmembrane protein with a molecular weight of 39 kDa that may be classified as a nonclassical major histocompatibility complex class I molecule that differs from other HLA class I molecules with regard to its low polymorphism, restricted tissue distribution, slow turnover, immunosuppressive properties and limited peptide diversity [46–48].
Under physiological conditions, the production of HLA-G protein is restricted to trophoblast , thymic epithelial cells , first-trimester placental chorionic blood vessel endothelial cells , and IFN-γ-treated mononuclear phagocytes .
However, the up-regulation of this protein can be detected in several pathological conditions such as transplantation, tumors, viral infections and autoimmune diseases [53–57].
HLA-G gene expression is tightly regulated at both the transcriptional and post-transcriptional levels and through epigenetic mechanisms, which include DNA methylation and histone deacetylation . In addition, different researchers have shown that HLA-G mRNA expression does not systematically result in the presence of the protein [59, 60]. This implies a tight post-transcriptional regulation of HLA-G gene expression.
HLA-G possesses the capability to bind inhibitory receptors such as the Immunoglobulin-like transcript-2 and −4 (ILT2, ILT4) and the Killer Immunoglobulin-like Receptor (KIR)2DL4/CD158d with inhibitory effects [61, 62].
In particular, HLA-G may have a direct immune-inhibitory function through blocking effector cells and indirect immune-inhibitory activity by regulatory cell generation. Via the direct inhibitory functions, HLA-G is able to inhibit the cytolytic activity and proliferation of NK , the antigen-specific cytolytic functions of α/β and γ/δ T lymphocytes [63, 64], the alloproliferative response of T cells [65, 66], the proliferation of NK and T cells  and the DCs maturation . Therefore, the lower HLA-G expression in HD patients suggests that innate immune effector cells could be activated in this subset of patients confirming some reports showing that long-time dialysis enhances NK cytotoxic activity [68, 69].
Furthermore, since HD patients showed down-regulation of HLA-G, high level of IL-15, and inflammatory status as suggested by increased level of PTX3, we decided to evaluate protein expression of chemokine receptor CX3CR1.
CX3CR1 is the fractalkine (CX3CL1) receptor and is expressed in cytotoxic effector lymphocytes such as NK, cytotoxic T lymphocytes (CTL) and γ/δ T cells which possess high levels of intracellular perforin and granzyme B  suggesting that its activation could be involved in migration of the cells to sites of inflammation . Moreover, this receptor is up-regulated during chronic inflammation  and it is increased in monocytes, T lymphocytes and NK cells present in severe atherosclerosis and ruptured coronary plaques in patients with unstable angina pectoris .
As shown in Figure 4B, CX3CR1 levels were significantly higher in HD compared to CKD patients (p < 0.01).