The total numbers of monocytes did not differ between groups and neither did the fraction of CD14+ CD16+ positive cells in the monocyte subset differ between groups (Table 2). No difference between TLR2 or TLR4 expression on monocytes or on the CD14+ CD16+ monocyte subset was found between groups.
The production of IL-10 to PHA was markedly attenuated in cases in univariate and age-adjusted analysis (P < 0.0001/P < 0.0001, Table 3). The production of IL-10 to endotoxin was also decreased in the case group (P = 0.0192/P = 0.0439). The production of IL-2 to PHA was also markedly lower in the case group (P = 0.0019/P < 0.0001). Adjusting for the leukocyte subsets best correlating with the respective cytokines did not change conclusions.
To test if the balance between proinflammatory and anti-inflammatory potential differed between patients with impaired CD4 cell recovery and controls, we compared the relationship between IL-10 production with PHA stimulation, primarily from lymphocytes, and TNF-α production to endotoxin, primarily from monocytes (Fig. 2). In regression analysis, we found that there was an interaction between group and TNF-α production (P = 0.013). Thus, the slope of the IL-10/TNF-α association differs between groups. Similar findings applied when TNF-α to endotoxin was substituted with TNF-α to PHA or plasma TNF-α.
For all cytokines, the production from unstimulated whole blood was undetectable for several patients and cytokine production did not differ between the case and control groups (data not shown).
For the cytokines, TNF-α, IL-1β, IL-6, IL-8, and monocyte chemoattractant protein-1, the plasma levels were compared between groups by T tests and by analysis of covariance adjusted for age (Table 3). There were no differences between groups. IL-10 and IL-1β were only detectable in a fraction of samples (47% and 81%, respectively) and, hence, these parameters were dichotomized and compared between groups by logistic regression. IL-10 detection was higher among cases in unadjusted but not in the adjusted analysis (unadjusted: P < 0.046, odds ratio for detectable IL-10, case vs. control group: 3.4; CI: 1.0 to 11; adjusted for age: P = 0.06, odds ratio: 3.6, CI: 0.96 to 13). Plasma TNF-α levels or the interaction between group and plasma TNF-α did not affect plasma IL-10 levels in logistic regression analysis (P = 0.96). IL-1β did not differ between groups (unadjusted: P = 0.11, odds ratio for detectable IL-1β, case vs. control group: 5.9, CI: 0.68 to 51; adjusted: P = 0.10, odds ratio: 6.8, CI: 0.72 to 65).
An impaired recovery of CD4 T cells was associated with increased immune activation and reduced numbers of regulatory T cells.36 Furthermore, CD4 and CD8 T-cell activation levels (HLA-DR+/CD38+) before cART initiation were negatively correlated with subsequent CD4 cell recovery.37 We found higher levels of HLA-DR on CD4 and CD8 T cells and as expected CD28 expression correlated negatively with HLA-DR expression. Surprisingly, the CD38 expression on CD4+ or CD8+ cells did not differ between groups. Moreover, the total number of CD8 cells was lower among cases. However, taken together, our results show that peripheral CD4 and CD8 T cells from the individuals in the case group share a more activated phenotype. This finding is corroborated by the reduced production of IL-10 among cases. IL-10 is an important anti-inflammatory cytokine that may down regulate immune activation during HIV infection. Thus, we reported that mortality was reduced among treatment-naive HIV-infected individuals carrying a genetic polymorphism associated with high production of IL-10, that is, the G-allele in position -1082 in the promoter to the IL-10 gene.38
In apparent contradiction, cases tended to have higher plasma levels of IL-10 than normal responders. This is, however, in accordance with higher plasma IL-10 levels and a positive correlation to viral load among HIV-infected individuals.38,39 However, as an even stronger correlation between soluble TNF receptor II and HIV RNA was observed, we proposed that plasma IL-10 levels are only increased by HIV because of ongoing immune activation with high levels of circulating TNF-α and subsequent up regulation of IL-10.38
Attention has recently been drawn to the profound depletion of CD4 T cells in GALT described in SIV-infected rhesus macaques40 and human HIV-infected patients.41,42 The GALT depletion has been linked to endotoxin translocation, and it was proposed that endotoxin translocation could be a mediator of immune activation during HIV infection.43 In a recent study, IL-10 in the gut was reported to be crucial to avoid IFN-γ driven inflammation due to endotoxin translocation. Without IL-10, the homeostasis of the gut mucosa was disturbed.44 Interestingly, endotoxin tended to be increased in patients with impaired CD4 recovery.45 Hence, diminished IL-10 production could be associated with endotoxin translocation.
In addition to lower IL-10 production in cases, we found an interesting difference in the relationship between IL-10 production to PHA and TNF-α production to endotoxin. Whereas the full responding patients displayed a positive correlation between TNF-α and IL-10 potential, this was not found among cases. Taken together, our findings suggest that lack of IL-10 enhances immune activation and thereby may impair CD4 recovery.
As for IL-10, the IL-2 production from PHA-stimulated whole blood was decreased among cases. IL-2 is an important growth factor for T cells,46 and HIV-specific CD4 cells producing IL-2 are decreased in HIV progressors but increase with treatment.47 We previously reported that impaired production of cytokines, among them IL-2 and IL-10, was an independent predictor of death among HIV-infected individuals.26 Moreover, studies indicate that IL-2 adjuvance could improve immunological recovery during cART.48 Hence, low production of IL-2 among cases may contribute to the impaired CD4 recovery. Alternatively, it reflects that cases have higher numbers of terminally differentiated CD4+ TEM but fewer TCM and naive cells.
In this study, cases were older than patients with a normal CD4 recovery. Age has previously been associated with an impaired immunological recovery after initiation of cART.49 This is a limitation of the study, however, all group comparisons were supplemented with age adjusted analyses. In these analyses, only CD38 expression on naive CD4 T cells correlated (positively) with age (data not shown). The cases in this study had undetectable HIV RNA and survived for more than 3 years despite a low CD4 cell count. Thus, it should be noted that cases in this study represent a selected subgroup of patients with low CD4 counts. It should also be noted that the CD4 cell count of case patients had not reached a plateau phase; conversely, they did experience immune reconstitution although at a slow rate. According to the case definition, the CD4 cell count after 3 years of full viral suppression was below 200 cells per microliter, and the slightly higher mean CD4 count was due to a subsequent increase from identification until recruitment at routine visits.
Hepatitis B and C infections combined tended to be over-represented among cases. Among a subgroup of cases coinfections may thus be part of the explanation for the exaggerated immune activation. Thus, analyses were performed with adjustments for hepatitis B and C infection (data not shown); this did not change conclusions. It should also be noted that we did not check if earlier treatment with zidovudine was overrepresented among cases.
According to earlier studies and our findings, old age, low nadir CD4 cell counts, high levels of proviral DNA, high levels of immune activation, and possibly coinfections are associated with the risk of an impaired immunological recovery.50 Even though we do not know the cause of these associations, clinicians may still use these as risk factors to identify patients who require closer monitoring. Our findings of lower production of IL-2 and IL-10 point to the existence of an imbalance in the cytokine environment. IL-2 and also IL-7 have been proposed as adjuvances to support the immune reconstitution during cART, but their place in the routine treatment of patients with impaired CD4 cell recovery has not been determined.48,50,51
We did not adjust for multiple comparisons in this study. However, as all findings pointed in the same direction, that is, cases displayed increased immune activation, we considered adjustment for multiple comparisons not to be appropriate.
Patients with an impaired recovery of CD4 cells shared a dysregulated phenotype including low levels of CD28 expression on CD4 and CD8 T cells, increased expression of the activation marker HLA-DR on T cells, and decreased production of the anti-inflammatory IL-10 and the T-cell growth factor IL-2. Persistent immune activation with an unopposed proinflammatory immune response despite the low rate of viral replication may be the reason for the impaired CD4 recovery among cases in this study.
We thank Dorthe Petersen from the Department of Infectious Diseases, Hvidovre Hospital, Bente Baadegaard and Lene Pors Jensen from the Department of Infectious Diseases, Rigshospitalet, for their excellent work with logistics and provision of information to the patients. Marie Noan Bruun is thanked for outstanding laboratory assistance.
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