New Players in the Same Old Game: Disturbance of Group 2 Innate Lymphoid Cells in HIV-1 and Mycobacterium leprae Co-infected Patients

Abstract Leprosy control is achieved through a fine-tuning of TH1 and TH2 immune response pattern balance. Given the increasing epidemiological overlay of HIV and M. leprae infections, immune response in co-infected patients consists in an important contemporary issue. Here we describe for the first time the innate lymphoid cells compartment in peripheral blood of leprosy and HIV/M. leprae co-infected patients, and show that co-infection increases group 2 innate lymphoid whilst decreasing group 1 innate lymphoid cells frequencies and function.


Introduction
Leprosy is a chronic infectious disease caused by the intracellular bacillus Mycobacterium leprae. Although usually asymptomatic, some subjects develop the clinical form of disease, exhibiting skin and peripheral nerves lesions [1]. In 2013, the Brazilian Healthy Department, registered 31.988 new cases of leprosy, with 84% of cure [2]. However, Menezes et al., describe an increasing number of HIV-1/M. leprae co-infected patient admitted in a center of leprosy treatment in Rio de Janeiro state [3]. In a referral HIV and leprosy center in Amazonas state, Talhari et al., observed in 13-year follow-up study higher leprosy prevalence among HIV-positive individuals [4].
Normally, clinical forms of the disease can be divided under the light of T H 1/T H 2 paradigm; being tuberculoid, or paucibacillary, leprosy linked to a T H 1 response pattern, and lepromatous, or multibacillary, leprosy linked to a T H 2 response profile. Lately, T H 17 cells were shown to play a role in disease pathogenesis, especially in lesion formation and M. leprae control [5]. Moreover, it was suggested that T H 17 cells contribute to pathogen clearance, when T H 1 or T H 2 polarization is ineffective [6].
As in other chronic and latent infections, the control of pathogen load is highly dependent on the immunological fitness of the host. Therefore, immunosuppressive factors contribute to disruption of host-pathogen homeostasis, and may account to aggravation of disease and enhancement of M. leprae transmission [7]. HIV-1 infection is the most common acquired immunodeficiency. In HIV-1/Mycobacterium tuberculosis co-infection, the suppression of immune system caused by HIV was shown to accelerate disease progression [8]. Likewise, in HIV-1/M. leprae co-infected patients our group observed an imbalance towards T H 2 responses, with lower CD4:CD8 ratios and higher IL-4 levels when compared to healthy controls [9]. Furthermore, patients co-infected with HIV-1 and M. leprae exhibited lower frequency of plasmacytoid dendritic cells (pDCs) and Natural Killer T (NKT) cells when compared to healthy controls [9,10], indicating that innate immunity cells are also affected during co-infection.
In recent years, innate lymphoid cells (ILCs) have emerged as important effector cells of innate immunity, responsible for bridging innate and adaptive responses. Recently, ILCs have been divided in three major groups, regarding their effector function and cytokine production profile [11]. These groups correlate well with T helper lymphocyte subsets. Hence, Group 1 ILCs (here referred to as ILC1) are characterized by the production of "T H 1" cytokines, such as IFN-γ and TNF-α. Natural Killer (NK) cells are comprised in this group, and the ontogeny of other IFN-γ-producing ILCs is still controversial [11,12]. Group 2 ILCs (ILC2) is an important innate source of "T H 2" cytokines, such as IL-4, IL-5 and IL-13. These cells respond to IL-25 and IL-33 and are associated with protection in helminthes infection and allergic asthma [11,12]. Finally, Group 3 ILCs (here referred to as ILC3) is composed of the well known Lymphoid-tissue inducer (LTi) cells and NCR + and colitogenic NCRnon-LTi cells. These cells produce IL-17A and/or IL-22 and are involved in different bacterial infections [11,12].
There are only a few studies regarding the effects of HIV or SIV infection on ILCs subsets. In two macaque studies, SIV infection led to a disturbance of IL-17-producing ILC3 cells [13,14]. However, in HIV-infected patients IL-22-producing ILC3 cells were still fully functional [15]. Interestingly, there are no studies on ILCs in leprosy and, consequently, in HIV/M. leprae co-infection. Thus, in the present study we evaluated the frequency and function of all three ILCs subsets in the peripheral blood of M. leprae and HIV mono-infected patients and HIV/M. leprae co-infected patients.

Subjects
Leprosy patients were treated according to World Health Organization Guidelines [16], and co-infected patients were treated with the appropriate multidrug therapy (MDT) for paucibacillary (PB) and multibacillary (MB) leprosy. The initial treatment for patients with HIV mono-and co-infection o was defined using modified criteria adopted by the Brazilian Ministry of Health at the time of sample collection that includes patients with a CD4 + T cell count of < 350 cells/μL or any AIDS-defining clinical condition [17]. These guidelines have been recently updated [18]. The HIV mono-infected and co-infected patients received highly active antiretroviral therapy (HAART) and multidrug therapy (MDT). Patients with immune reconstitution inflammatory syndrome, with leprosy reactions and under systemic corticosteroid and/or thalidomide were not included in the present study, to avoid potential interference in the immune parameters as described in a previous studies. The study subjects were divided into four groups: 16 healthy controls (Healthy) and 11 HIV seropositive patients (HIV), most of whom had CD4+ T cell counts of less than 400 cells/μL, 7 patients infected with M. leprae (Leprosy), and 10 co-infected patients with M. leprae and HIV co-infection (Dual), recruited at Leprosy Outpatient Clinics at both sites. In this the major presentations of leprosy were multibacillary form rather than paucibacillary form. For all analysis we grouped the leprosy monoinfected and co-infected patients (leprosy per se) (S1 Table).
Fluorescence voltages were determined using matched unstained cells. Compensation was carried out using CompBeads (BD Biosciences) single stained with all fluorochromes used in the experiments. Samples were acquired to reach at least 1,000,000 events.
Groups were compared using non-parametric models; data were reported with median and 25-75% interquartile range. p values were considered significant if below 0.05. Results are expressed in medians and interquartile ranges (IQR).

Results
Study subjects were divided into four distinct groups, 16 healthy controls (Healthy), 11 HIVinfected patients (HIV), 7 patients infected with M. leprae (Leprosy) and 10 co-infected patients with M. leprae and HIV co-infection (Dual). Demographic details can be found at S1 Table. The median age of all participants was 36 years and no difference in age distribution was found between groups. Most of the subjects were male (70.21%). All patients were properly treated with HAART and/or MDT therapy.
Additionally to ILC2 subset, we evaluated the frequencies of ILC1 and ILC3 populations on the peripheral blood of all subjects. ILC1 cells were defined as Lin -CD45 + CD56 + TNF-α + cells and ILC3 subset was defined as Lin -CD45 + CD56 + IL-17 + cells (S1 Fig and Fig 2A). Conversely to the increase of ILC2 cells frequencies in HIV/M. leprae co-infected patients, we found a decrease in ILC1 cells frequencies when compared to healthy controls (Fig 2B) (Fig 2C).

Discussion
To our knowledge, this is the first study to investigate and describe ILCs subsets in leprosy and HIV-1/M. leprae co-infected patients. Moreover, this is the first time that a complete profile of the three different ILCs subsets was carried out in HIV-1 infected patients, as the only study regarding this question focused on ILC3 cells [15]. One limitation of this study, however, is the relatively small sample size, which may have affected the ability to identify more subtle changes between groups. Nevertheless, we were able to identify major changes in ILCs compartment caused by HIV-1/M. leprae co-infection. To define the three major groups of ILCs we chose to use a functional criterion (i.e. cytokine secretion) in order to avoid misconception of ILCs subpopulations [12]. Consequently, when using the ILC1 term we are referring to both NK cells and T H 1 (i.e., TNF-α) cytokines-producing ILCs [11]. Similarly, here we used the ILC3 term to comprise LTi cells, NCR + and NCRcells, based on their ability to produce IL-17 [11]. Finally, ILC2 cells were defined both by their IL-4 and IL-13 secretory capacities and surface molecules, because this subset is composed by only one known population of cells [11].
Here we found that patients with HIV-1 and M. leprae co-infection showed an increase in ILC2 cells and a decrease in ILC1 cells frequencies in peripheral blood, when compared to healthy subjects. This data is in line with previous findings from our group, which showed a skewing in cellular immune response towards a T H 2 bias [9]. Thus, our data add new evidences to the hypothesis that HIV-1 in patients with an ongoing infection with M. leprae promotes a disturbance in T H 1 and T H 2 balance, sustaining a T H 2 environment. Even though not conclusive, our results indicate that HIV-1 infection is responsible for enhancing this T H 2 shift. However, it is not clear if this skewing in immune response is a viral escape mechanism or an attempt from the immune system to control the virus. Although higher in frequencies, ILC2 cells from HIV-1/M. leprae co-infected patients produce less IL-4 and IL-13, when compared to M. leprae mono-infected patients. Additionally, circulating ILC2 cells in HIV-1 mono- infected patients are present in similar frequencies and with comparable functional capacity of healthy subjects, suggesting that HIV-1 is not responsible for inducing a relevant T H 2 response, per se. Therefore, suggesting that HIV-1 infection impairs T H 2 response, through decrease of functional response, and the resulting increase in ILC2 frequencies is an attempt from the immune system to rescue this phenotype. Concurrently with the increase of circulating ILC2 frequencies it was observed a decrease of circulating ILC1 frequencies in co-infected patients when compared to their healthy counterparts, further supporting this notion of an unbalancing of T H 1/T H 2 responses. Of note, to better understand the immunological landscape in HIV-1/ M. leprae co-infection it would be crucial to dissect the immune response of ILCs and other immune cells located in the site of M. leprae infection (i.e. in the skin and nerves).
We found no differences in the frequencies of ILC3 subset from HIV-1/M. leprae coinfected patients when compared to healthy subjects and mono-infected patients, as opposed to previous findings that showed IL-17-producing ILCs cells depletion in SIV infected macaques [14]. Nonetheless, in the study led by Xu, cells were obtained from mucosal tissues. Moreover, these findings were obtained in an experimental macaque model [14]. Conversely, Fernandes and colleagues looked on IL-22-producing ILC3 cells population on mucosal surfaces of HIV-1 infected individual and found no differences in their frequencies when compared to healthy controls [15]. Although functionally different ILC3 subsets, the conditions in this study are the most comparable to ours, adding strength to our findings that HIV-1 does not alter ILC3 cells population.
Finally, in this study we added new information to the growing body of data that shows that ILCs have effector functions that parallels T helper cells subsets during infection. Additionally, our main finding that HIV-1 infection has a major effect on ILC2 cells demonstrate that these cells might have a role during viral infections, and not only on asthma and helminthes infections [11].