Background
Suboptimal immune recovery occurs in up to 40% of HIV-infected individuals receiving long-term Highly Active Antiretroviral Therapy (HAART) in sub-Saharan Africa (SSA)[
1]–[
3]. The exact mechanisms for suboptimal immune recovery are not fully established, although the phenomenon has been associated with low nadir CD4 count at HAART initiation, irreversible fibrosis of the reticulo-endothelial system during advanced HIV disease, persistent T-cell activation and immune exhaustion, among other factors[
2,
4,
5]. There is limited data on how HIV-associated dysfunction of the innate immune system influences immune recovery, in particular Natural Killer (NK) cells that are known to participate in the initiation and development of adaptive immune responses. NK cells also participate in host innate responses to viral and intra-cytoplasmic bacterial infections[
6‐
8], and may have a role in immune recovery among HAART-treated HIV-infected adults. HIV-associated NK cell dysfunction has been reported in association with severity of HIV disease[
9] and the impaired immune responses associated with HIV/AIDS[
10,
11]. In addition, increased NK cell activation and degranulation have been associated with Immune Reconstitution Inflammatory Syndrome (IRIS) and TB/HIV co-infections[
12,
13], which contribute to HIV-associated morbidity and mortality during HAART[
14]–[
16]. There is a need to understand the role of innate immune dysfunction in post-HAART immune recovery, to inform therapeutic advances to optimize HIV treatment outcomes. This paper explores the association of NK cells with immune recovery during suppressive HAART in an African HIV treatment cohort.
The role of the innate immune system in HIV immune-pathogenesis has been explored with particular focus on NK cell subsets, function and expression of receptors[
11,
17]–[
19]. Three distinct subsets of NK cells are recognized in human peripheral blood; CD56
bri, CD56
dim and CD56
neg; categorized according to the expression of NK cell lineage markers CD56 and CD16[
20,
21]. CD56
bri are pre-dominantly cytokine producing cells and CD56
dim are mainly cytotoxic[
22]. NK cell function is directed by a complex repertoire of activating and inhibitory natural cytotoxicity receptors (NCRs), such as NKp46, NKp30 and NKp44, as well as NKG2D, CD16, 2B4 and NKp80[
22]. During HIV infection, NK cells are directly infected[
23] and the distribution of NK cell subsets is altered[
20]; with an expansion of CD56
neg among viremic patients[
20]. In addition, HIV causes up-regulation of inhibitory natural killer receptors (iNKRs) leading to impairment of NK cell lysis of virally-infected cells[
11]. Antiretroviral therapy reverses the effects of HIV infection on NK cells; however, there is no consensus on the degree to which suppression of HIV replication restores NK cell function[
10]. We hypothesized that the distribution and function of NK cell subsets differs among individuals with poor versus excellent CD4+ T-cell recovery during antiretroviral therapy. This study describes the profiles of NK cell subsets and their expression of activating receptors, NKG2D and cytotoxicity receptor NKp46, among individuals with poor CD4 T-cell reconstitution relative to individuals with excellent CD4 T-cell count reconstitution after four years of suppressive HAART. Our results highlight the need for studies to further understand the short and long-term recovery of the innate immune system including NK cell function among African HAART-treated HIV-infected patients.
Discussion
This study compared proportions of NK cell subsets as well as expression of activating receptor NKG2D and cytotoxicity receptor NKp46 among adults with ‘suboptimal’ and ‘super-optimal’ immune recovery despite four years of suppressive HAART. We found that CD56
dim was the largest NK cell subset among HAART-treated adults, irrespective of immune recovery status. Our data is consistent with previous reports that CD56
dim is the largest population of NK cells in peripheral blood and the main cytotoxic NK cells participating in Antibody-dependent cell-mediated cytotoxicity (ADCC); followed by CD56
neg and CD56
bri NK cells. Whereas the functionally defective CD56-CD16+ (CD56
neg) population of NK cells expands in viremic versus aviremic patients[
11] and is associated with poor cytotoxic function[
20], this subset of NK cells was lower among suboptimal responders relative to super-optimal responders. Given that our study participants had received suppressive HAART for four years, our data suggests that HIV-associated expansion of the dysfunctional CD56
neg population was no longer significant. These results mirror previous reports that initiating HAART during acute HIV infection prevented further decline in NK cell subsets and improved NK cell function[
25]. It is therefore likely that initiating HAART earlier in HIV disease when the immune systems are still robust, as recommended in the 2013 WHO guidelines[
26], may result into faster recovery of HIV-associated NK cell dysfunction; among other benefits.
The CD56
++CD16
- (CD56
bri) subset, functionally cytokine producers, was higher among ‘suboptimal’ responders relative to ‘super-optimal’ responders. The high numbers of cytokine producing NK cells among ‘suboptimal’ responders may be reflective of the persistently high levels of immune activation that were previously documented among suboptimal responders in our cohort[
4]. Immune activation has been associated with high production of inflammatory cytokines and increased turn-over of T-cells, B lymphocytes, NK cells and accessory cells[
27,
28]. Immune activation in the first few years of HAART-mediated viral suppression predicted long-term CD4+ T-cell recovery after 15 years of antiretroviral therapy. It is likely that pre-HAART immune activation, not only predicts mortality during HAART[
29], but also predicts suboptimal immune recovery including suboptimal reversal of the HIV-associated NK cell dysfunction. We therefore postulate that controlling immune activation among HIV-infected individuals may also stabilize the NK cytokine producing cells, modulate their immune function and subsequently optimize short and long-term immune recovery during antiretroviral therapy.
Expression of NKG2D and NKp46 receptors by NK cells was comparable among suboptimal and super-optimal responders. NK activating receptors correlate with the NK effector function[
30,
31], so it is likely that NK effector function is comparable among suboptimal and super-optimal responders. We could attribute this result to suppressive HAART that has provided partial immune recovery during the first four years. It has been previously shown that HAART modulates NKG2D receptor expression among HIV-infected viremic individuals[
10,
13,
21]. Increased NKp46 expression on NK cells was previously shown to correlate with HIV-1 disease severity among HIV-infected children[
9], although its role in immune recovery is not yet well understood. Although we did not perform NK functional assays, previous data shows that abnormal expression of NK activating and inhibitory receptors was associated with impaired cytolytic function[
11]. In addition, reduced surface expression of the NK cytotoxicity receptor, NKp46 was associated with poor cytolytic function during viremic HIV disease[
21]. Given that NKG2D and NKp46 expression was similar in the ‘suboptimal’ and ‘super-optimal’ immune responders, it is likely that these receptors are not involved in the mechanisms that lead to poor CD4+ T-cell reconstitution in HIV-infected adults receiving HAART unless functional assays reveal significant differences. In addition, NK cell activation is controlled by a dynamic balance between complementary and antagonistic pathways[
31,
32]. We did not evaluate NK cell surface inhibitory receptors that antagonize activating pathways through protein tyrosine phosphatases (PTPs), therefore our data is not conclusive on the NK cell activation status in the study population.
Implications of the study
Our results imply that after four years of suppressive antiretroviral therapy, the HIV-associated NK cell dysfunction was only partially restored, with a predominant CD56
dim (CD56 + CD16-/+) population and a high CD56
bri (CD56
++CD16
-) NK cell population among suboptimal responders. The high CD56
bri, functionally cytokine producers, among suboptimal responders may be reflective of the persistently high levels of immune activation previously described in the same cohort[
4]. We postulate that earlier initiation of HAART and control of immune activation could contribute to faster and more comprehensive recovery of the immune system. It is also important to note the trend shown that specific T-cell subsets recover faster, while other subsets require longer periods of suppressive HAART. Given the significant cytokine producing function of CD56
bri NK cells, it might be worthwhile to further investigate NK cell dysfunction among individuals that initiate HAART at CD4 < 500 cells before severe damage of the immune system, as well as potential interventions to enhance comprehensive immune recovery. In addition, increased NK cell degranulation capacity was significantly associated with Immune Reconstitution Inflammatory Syndrome (IRIS) among HIV/TB co-infected individuals in Cambodia, with activating receptor expression higher among IRIS patients relative to non-IRIS patients[
17]. Similarly NK cell activation was shown to distinguish Mycobacterium tuberculosis-mediated IRIS from chronic HIV and HIV/TB co-infection[
12]. Thus, further examination of the mechanisms of NK cell dysfunction, co-infections and suboptimal recovery is critical for suboptimal immune responders that remain at risk of life-threatening opportunistic infections[
1,
24].
Limitations
We did not perform NK cell function assays due to logistical limitations. In addition, this study was limited to the extremes of immune recovery (suboptimal and super-optimal immune responders), and did not include average responders. Our results, however, highlight the need for NK function assays to conclusively ascertain defects in NK cell effector function that might be relevant to immune responses to viral and bacterial infections among HAART-treated HIV-infected adults.
Acknowledgements
The authors thank the Infectious Diseases Institute (IDI) research cohort and staff for accepting to participate in this study. We acknowledge the IDI translational laboratory and the Immunology Laboratory, at Makerere University College of health Sciences where the laboratory assays were conducted. The work was supported by a Wellcome Trust Uganda Post-doctoral Fellowship in Infection and Immunity held by Damalie Nakanjako, funded by a Wellcome Trust Strategic Award, grant number 084344.
Competing interests
The authors declare that they have no competing interests.
Authors’ contribution
LB, DN, HMK and JO made substantial contribution to the conception, design and interpretation of the data. LB, DN, MJ, RN, SK and PNS made substantial contribution to the data collection, flow cytometry assays and data analysis. DN, MRK, AK and AK contributed substantially to the clinical cohort from which the samples were collected. LB and DN drafted the manuscript. All authors reviewed the manuscript and approved the final version for publication.