The Participation of Prolactin in the Immunopathology of Experimental Pulmonary Tuberculosis

Abstract

Introduction

Prolactin and its receptor are widely recognized for their roles in lactogenesis and galactopoiesis. However, they have also gained attention for their role in immune pathophysiology, acting as mediators of both innate and acquired immune responses and potentially playing a significant role in chronic infectious diseases such as tuberculosis.

Methods

In this study, we examined the kinetics of prolactin and its receptor during the progression of experimental pulmonary tuberculosis in BALB/c mice infected intratracheally with a high-dose Mycobacterium tuberculosis (M. tuberculosis) reference strain, H37Rv. Groups of infected mice were euthanized at various time points, and their lungs were collected to quantify gene expression of prolactin and its receptor by RT-PCR, as well as to assess cellular expression by immunohistochemistry. In vitro infection experiments with alveolar macrophages and type II pneumocyte cell lines were conducted to evaluate the effects of prolactin on bacteriolysis and the expression of various innate immune response factors. After 2 months of infection, BALB/c mice with progressive pulmonary tuberculosis were treated with ovine PRL, and the effects were assessed by measuring bacillary loads and the extent of tissue damage (pneumonia).

Results

In vitro studies showed that prolactin increases bacterial phagocytosis in alveolar macrophages and significantly enhances the production of reactive oxygen and nitrogen species, which reduces bacillary loads. In type II pneumocytes, prolactin, under certain conditions, induces higher, though not statistically significant, expression of cathelicidin and surfactant proteins A and D. In vivo studies revealed that, during early infection (within the first week), there was high expression of prolactin and its receptor in the lungs. Immunostaining for prolactin and its receptor was observed at the early stage of infection, mainly in alveolar epithelial cells, macrophages, and lymphocytes. During the second week of infection, immunostaining showed the organization of macrophages and lymphocytes within granulomas and in the inflammatory infiltrate around blood vessels and airways, with macrophages showing the strongest staining. Over the course of progressive disease, after 1 month of infection, pulmonary prolactin production decreased, and some macrophages and lymphocytes showed minimal prolactin immunostaining, although high prolactin receptor expression persisted; 4 months later, prolactin immunostaining was observed in the bronchial epithelium. Administration of prolactin via intraperitoneal injection 2 months after infection resulted in a significant reduction in bacillary loads after 2 months of treatment, along with increased but not statistically significant expression of TNFα and IFNγ.

Conclusions

Prolactin and its receptors appear to contribute to immune activation during pulmonary tuberculosis, particularly in the early stages of infection. Prolactin administration during late active disease reactivates immune protection. Although we do not demonstrate the mechanisms underlying these observations, these results could serve as a basis for expanding future experimental research.

Introduction

Worldwide, M. tuberculosis is the infectious bacterial disease with the highest morbidity and mortality rates. According to the 2025 WHO tuberculosis (TB) report, approximately 25% of the world population is latently M. tuberculosis infected, and 10.8 million new active TB cases were reported, with 1.2 million deaths [1]. TB treatment continues to be challenging because it requires long and complex chemotherapeutic regimens that often produce adverse effects, reduce patient adherence, and contribute to the emergence of multidrug-resistant strains [1]. These limitations underscore the need for new adjuvant therapeutic approaches to shorten treatment duration, with immunoendocrine therapy as an interesting area to explore.

Prolactin (PRL) is a peptide hormone that performs at least 300 physiological functions, making it one of the most pleiotropic hormones of the pituitary gland [2]. Although it is best known for its roles in lactogenesis and galactopoiesis, it also participates in osmoregulation, metabolism, immune response, inflammation, and even in the repair of the central nervous system [3].

While PRL is primarily secreted by the anterior pituitary, its gene is regulated by two independent promoters – a central and a peripheral one – allowing tissue-specific expression depending on microenvironmental conditions [4]. PRL is also produced by lymphocytes, macrophages, fibroblasts, neurons, adipocytes, and endothelial cells [5]. Post-translational modifications generate multiple PRL isoforms (ranging from 11 to 40/60 kDa), each capable of inducing distinct biological effects depending on the receptor it binds [6]. The prolactin receptor (PR) is not completely specific to PRL as it can also bind growth hormone and placental lactogen [3]. Activation of this receptor triggers diverse intracellular signaling pathways, including STAT, MAPK, c-src, and Fyn kinase cascades, which account for the hormone’s pleiotropic biological actions [7]. The classical PR belongs to the type I cytokine receptor family and is widely expressed in mammary glands, gonads, liver, kidneys, brain, heart, pituitary, uterus, skeletal muscle, skin, and immune cells, including T lymphocytes, B lymphocytes, and macrophages. Moreover, immune cells in the thymus, bone marrow, and spleen can produce PRL mRNA and secrete bioactive isoforms similar to those of pituitary origin, including variants ranging from 11 to 80 kDa and a 25-kDa glycosylated form [8].

PRL exerts immunoregulatory functions by increasing the activity of transcription factors such as IRF-1 and NF-kB [8], enhancing the production of proinflammatory mediators, including cytokines, chemokines, and nitric oxide [9]. Heavy PRL isoforms can activate the JAK2/STAT1 signaling pathway, intensifying the secretion of proinflammatory cytokines [8, 9]. The activities of PRL on the immune cells are related, among other mechanisms, to mitogenic activity and prevention of programmed cell death, as well as to the production of interleukins and antibodies [8, 9].

In the context of mycobacterial infection, in vitro studies have shown that culture filtrate proteins from Mycobacterium bovis (M. bovis) induce PRL expression and overexpression of its short isoforms [10], especially in myeloid cells [11]. Both isoforms of PRL have been associated with antimicrobial mechanisms, including activation of the inducible nitric oxide synthase pathway. These findings suggest that PRL may contribute to host defense against M. bovis; however, no published studies have informed about the role of PRL during infection with M. tuberculosis, and there is no evidence currently published that links hyperprolactinemia or hypoprolactinemia to susceptibility to TB in humans.

Given this knowledge gap, the present study aimed to determine the contribution of PRL and its receptor during M. tuberculosis infection. Using a model of progressive pulmonary TB in BALB/c mice, we studied the expression of PRL and PR in the lungs over the course of infection and evaluated whether exogenous PRL administration affects bacterial burden and tissue damage in advanced disease stages. In addition, we assessed the in vitro effects of PRL on bacteriolysis and the production of immune mediators in infected and uninfected alveolar macrophages and alveolar epithelial type II cells.

Methods

Experimental Model of Progressive Pulmonary Tuberculosis

We used a well-characterized murine model of progressive pulmonary TB induced by intratracheal (it) infection [12]. Briefly, virulent M. tuberculosis reference strain H37Rv was cultured in Middlebrook 7H9 broth supplemented with oleic acid, albumin, dextrose, catalase enrichment. After 21 days of culture at 37°C, when mycobacteria were in log phase, bacilli were harvested and adjusted to 2.5 × 10⁵ live bacilli in 100 μL of phosphate-buffered saline (PBS) and maintained at −70°C until used. Pathogen-free male BALB/c mice, 6–8 weeks old, and 22 grams of weight on average, were anesthetized with sevoflurane (Abbott Laboratories) and 100 μL of PBS, and 2.5 × 10⁵ viable bacteria were inoculated intratracheally using a stainless-steel cannula. Forty animals were infected, and 24 were used as a noninfected control group; all of them were maintained in cages fitted with micro-isolators in a level 3 biosafety facility.

Following infection, a group of 5 mice (n = 5) was euthanized under anesthesia with pentobarbital at days 1, 3, 7, 14, 21, 28, 60, and 120 post-infection; at the same time points, three noninfected mice were euthanized and used as controls. Three right lungs were immediately frozen by immersion in liquid nitrogen and used to determine bacilli burden by colony-forming units (CFUs) quantification. The same procedure was performed on three left lungs used for molecular biology determinations, while the remaining lungs were perfused with 100% ethanol and immersed in the same fixative for 24 h to perform histopathological analysis. For the control group, the left lungs were processed for molecular biology and the right ones for histology analysis. Two independent experiments were performed.

Pulmonary Bacillary Loads Determination

Frozen lungs were disrupted using ceramic beads in tubes containing 1 mL of PBS with 0.05% Tween. Four dilutions of each homogenate were spread onto duplicate plates containing 7H11 Middlebrook agar enriched with oleic acid, albumin, dextrose, catalase. Plates were incubated for 21 days, after which the number of CFU was counted. Data points are the means of three animals.

Determination of PRL and PR Gene Expression during the Course of Pulmonary Tuberculosis

Lungs were placed in 2 mL of RPMI-1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) containing 0.5 mg/mL collagenase type 2 (Worthington, Lakewood, NJ, USA) and incubated for 1 h at 37°C. The lungs were then homogenized, and mRNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. RNA was treated with one unit of DNase (Invitrogen Life Technologies) per microgram of RNA. The quality and quantity of RNA were evaluated by spectrophotometry (260 nm and A260/280 ratio, NanoDrop-1000; Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized using the Omniscript RT kit (Qiagen, Hilden, Germany), oligo dT (Promega Corporation, Madison, WI, USA), and 5 µg of RNA. The cDNAs were amplified with Taq DNA polymerase Hot Start (Qiagen, Germantown, MD, USA). Real-time PCR was performed using the PCR system 7500 (Applied Biosystems, Bedford, MA, USA). Overall, 100 ng of cDNA was used with the Quantitect SYBR Green MasterMix kit (Qiagen, Hilden, Germany) and sense and antisense primers (50 pmol of each). The production of a single PCR product and the expected amplicon size were confirmed by electrophoresis of the conventional PCR product. Each PCR cycle included negative controls. Specific primers were designed using Primer Express software (Applied Biosystems) for PRL: (forward primer 5′-CCC​AGA​AAG​CAG​GGA​CAC​TC-3′, reverse primer 5′-CAG​TCA​CCA​GCG​GAA​CAG​AT-3 [94 base pairs expected amplicon size]) and PR (forward primer 5′-CCA​TCT​TTG​AGG​TCA​CAG​GAC​T-3′, reverse primer 5′-GCA​TGA​TCA​GGG​GAC​CTT​CTT-3′ [93 base pairs expected amplicon size]). Conditions used were initial denaturation at 95°C for 15 min, followed by 40 cycles at 95°C for 20 s, 60°C or 58°C for 20 s, and 72°C for 34 s. The number of copies of PR and PRL mRNA transcripts was related to one million copies of the housekeeping gene RLPO mRNA. Data were reported as mean +/− standard deviation (sd) of 3 different mice for each experimental group.

Histology and IHC for PR and PRL Cellular Detection

Parasagittal sections were taken through the hilum, dehydrated, embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin for histological and automated morphometric studies. The same paraffin-embedded tissues were used for immunohistochemistry; overall, 4 μm sections were obtained on Kling-On HIER slides (Biocare Medical, Lake Concord, CA, USA). For dewaxing, sections were placed at 60°C for 20 min and immersed in xylene for 5 min. The slides were then immersed five times in xylene alcohol (1:1), absolute alcohol, 96% alcohol, and distilled water. Once hydrated, antigen retrieval was performed by placing the slides in citrate buffer (pH 9) at high temperature in a pressure cooker. Endogenous peroxidase was blocked with H2O2-MeOH (9:1) for 20 min, followed by washes with PBS Tween 20 (0.05%). Tissue sections were covered with 2% background sniper (Biocare Medical) dissolved in PBS and incubated for 30 min in a humid chamber, then incubated for 24 h at room temperature in a humid chamber with the following antibodies: biotinylated rabbit polyclonal anti-Prolactin E−9 (sc-48383, Santa Cruz Biotechnology Inc.) and rabbit polyclonal anti-Prolactin Receptor D4A9 (#13552, Cell Signaling Technology), diluted at 1:100 and 1:250, respectively. Subsequently, slides incubated with anti-PR antibodies were washed and incubated with goat anti-rabbit antibodies labeled with horseradish peroxidase for 1 h at room temperature, while anti-PRL samples were incubated for 1 h with the VECTASTAIN Elite ABC HRP Kit (PK-6106). After extensive washes, horseradish peroxidase was revealed with 100 µL of the PolyDetector Liquid DAB HRP Brown kit (Bio SB, USA). Slides were washed, counterstained with hematoxylin, and mounted with a synthetic resin.

Study of the Effect of PRL in vitro in Alveolar Macrophages and Pneumocytes Type II

Murine alveolar macrophages (MH-S CRL-2019-ATCC) and pneumocytes type II (A549 cell line ATCC^® CCL-185™) were used to determine the effect of PRL on bacterial phagocytosis and bacteriolysis, as well as in alveolar macrophages the production of reactive oxygen species (ROS) and nitric oxide (NO) and in pneumocytes type II the production of surfactant proteins and the antimicrobial peptide cathelicidin (LL-37). For the maintenance of both cell lines, RPMI medium (Caisson Labs, USA, Cat. RPL03) and 10% fetal bovine serum (Gibco, USA) were used; once infected, RPMI alone was used. MEM medium without phenol red (Gibco 2766569) was utilized for ROS/NO determination. Infections were performed with the M. tuberculosis H37Rv reference strain at a 1:5 MOI. PBS + antibiotic-antifungal (MP 1670049) was used as the washing solution [13]. For the bacterial phagocytosis assay, 2.5 × 10⁴ MH-S cells were seeded per well in a Chamber Slide (Nunc™ 154,526 Lab-Tek^® II) and incubated for 48 h at 37°C/5% CO₂. Four conditions were studied: (1) control without PRL, (2) PRL added post-infection, (3) during infection, and (4) pre-infection. Recombinant PRL 0.008 μg/μL (1445-PL-050, R&D Systems) was added per well, then fixed with methanol for 10 min, and stained with Ziehl-Neelsen. In each condition, 300 cells were counted, quantifying the number of infected cells and intracellular bacteria to determine their percentages. Pneumocytes type II were infected and the bacteriolysis was determined in 12-well plates (2.5 × 10⁴ cells per well) in RPMI medium (Caisson Labs, USA, Cat. RPL03) supplemented with 10% fetal bovine serum (Gibco, USA). There were two groups: the control and the group treated with PRL; the bacterial killing was determined at one and 21 h of incubation by CFU quantification as described above.

To study the effect of PRL on the gene expression of surfactant proteins (SP) and LL-37 in infected and uninfected type II pneumocytes, cells were lysed in Buffer RLT Plus (Qiagen, Hilden, Germany) supplemented with 10% β-mercaptoethanol to preserve RNA integrity. RNA was extracted using the RNeasy^® Mini Kit (Qiagen, Germantown, MD, USA) following the manufacturer’s protocol. The concentration and purity of the extracted RNA were determined by measuring the A260/A280 ratio using an Epoch Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). For complementary DNA (cDNA) synthesis, 100 ng of total RNA was reverse-transcribed using the Omniscript^® Reverse Transcription Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. The gene expression of SP-A, SP-D, and LL-37 was evaluated by reverse transcription quantitative PCR (RT-qPCR). Reactions were performed as previously described using a 7500 Real-Time PCR System (Applied Biosystems Inc., Foster City, CA, USA) and the QuantiTect SYBR^® Green PCR Master Mix Kit (Qiagen, Hilden, Germany). The sequences of specific human primers for LL-37 were forward: 5′-CCA​AAG​GAA​TGG​CCA​CTC​CC-3′, reverse: 5′-CAC​ACA​CTA​GGA​CTC​TGT​CC-3′; for SP-A: forward: 5′-CTG​TGT​GTG​GCC​TGG​AGA​C-3′, reverse: 5′-GGC​TGC​CAT​CAA​GAT​GAG​GT-3′; for SP-D: forward: 5′-GCT​CCT​GGG​GCC​TAA​CAA​AA-3′, reverse: 5′-TGG​GCA​TTG​TTC​TGT​GGG​AG-3′. Cycling conditions were initial denaturation at 95°C for 15 min, followed by 40 cycles of 95°C for 20 s, 60°C for 20 s, and 72°C for 35 s. All reactions were performed in duplicate, including a negative control lacking the cDNA template. The mRNA encoding RPLP0 was used as an internal reference gene to normalize gene expression. Results are presented as relative expression levels compared to the control group (uninfected cells), using the ΔΔCt method.

PRL Administration to Tuberculous Mice during Late Active Disease

To study the effect of PRL on the course of late progressive disease, mice after 2 months of M. tuberculosis infection were treated with ovine PRL (RayBiotech, cat.: 228-11313-3), 10 µg administered by intraperitoneal route each day for 2 months. This concentration was selected based on previous published studies [14]. To evaluate the PRL effect on the course of the infection, groups of five animals were euthanized after 1 and 2 months of this treatment, right lungs were used to determine the bacterial load, and left lungs were used for tissue damage quantification (extension of pneumonia) by automated histomorphometry and quantification of the gene expression of the protective cytokines TNFα and IFNγ by RT-PCR. Two independent experiments were performed, each experiment with 5 mice per group (online suppl. material; for all online suppl. material, see https://doi.org/10.1159/000552692).

Statistical Analysis

Data were analyzed using Prism 8 (GraphPad Software In, USA). Results are expressed as mean ± SD. Statistical significance was determined by Student’s two-tailed t test for comparisons between two groups, or two-way ANOVA followed by Sidak’s multiple comparisons test for experiments with multiple variables. Survival curves were analyzed using the log-rank (Mantel-Cox) test. A p < 0.05 was considered statistically significant.

Results

Kinetics of Gene Expression of PRL and PR during the Course of Experimental Pulmonary Tuberculosis

BALB/c mice were infected with a high dose of M. tuberculosis H37Rv that produces progressive pulmonary TB, as demonstrated by a progressive increase in bacillary load (Fig. 1a) [12]. In the early phase of this model, there is increased expression of Th1 cytokines (IFN-γ, IL-12) and TNF-α, which permits transient control of bacillary growth. Granulomas appear at day 14, and immunological activity peaks on day 21. One month after infection, the expression of Th1 cytokines and TNF-α decreases in coexistence with progressive pneumonia. Extensive pneumonia and high bacillary burden cause the animal’s death [12]. The progressive increase of bacilli loads confirms our model of progressive pulmonary TB (Fig. 1a). Quantification of PRL gene expression by RT-PCR in infected lungs showed high expression after 1 day of infection, followed by a sharp decrease at days three and seven. Two weeks after infection, the peak of PRL expression was observed, followed by a pronounced decline at 1 and 2 months, but at 4 months of infection, PRL expression showed a mild increase (Fig. 1b). Noninfected mice did not show PRL gene expression.

Fig. 1.

Kinetics of prolactin (PRL) gene expression during pulmonary tuberculosis. a Progressive increase in bacillary loads in the lungs of mice infected with M. tuberculosis at different post-infection time points. b PRL gene transcription determined by RT-PCR throughout the course of infection. Data are shown as mean ± SD (n = 5 mice per time point). c–f Representative micrographs show PRL detection by immunohistochemistry. c No PRL is detected in the lungs of control, non-infected mice. d In contrast, alveolar epithelial cells display strong PRL immunostaining after one day of M. tuberculosis infection. e After two weeks of infection, granulomas contain some lymphocytes and macrophages with positive PRL immunostaining. f After four months of infection, certain lymphocytes and activated macrophages—large cells with abundant, compact cytoplasm (arrowheads) in pneumonic areas—as well as bronchial epithelial cells (arrow), exhibit PRL immunoreactivity, while foamy macrophages are negative (asterisks).

Gene expression of PR was highly expressed in the lungs at day one of infection, showing a decrease after three and 7 days, but it increased again after 2 weeks of infection, when granulomas started their formation and reached their peak at day 21 (Fig. 2a), when in this model is seen the maximal level of immune protection [12, 15]. During late infection, after 2 and 4 months of infection, similar PR expression to that on day one was seen (Fig. 2a). Noninfected mice did not show PR gene expression.

Fig. 2.

Kinetics of gene and protein expression of prolactin receptor (PR) during pulmonary tuberculosis. a PR mRNA expression measured by PCR (n = 5 mice). Data are shown as mean ± SD. b–e Representative photomicrographs of PR detection by IHC. b No PR detection in the lungs of control noninfected mice. c After 21 days of infection, numerous lymphocytes and macrophages are present in granulomas, and lymphocytes from mediastinal lymph (d) show PR immunostaining. e In contrast, few lymphocytes in the pneumonic areas after 120 days of infection exhibit PR immunoreactivity (all photomicrographs are at 40× magnification, except for 2D, which is at 20×).

Cellular Sources of PRL and PR Determined by Immunohistochemistry

The lungs from control noninfected mice did not show PRL immunostaining (Fig. 1c), while mice after 1 day of infection showed intense PRL immunoreactivity in the alveolar epithelium (Fig. 1d). This immunostaining pattern was also observed after three and 7 days but to a lesser degree. After 2 weeks of infection, granulomas started their formation and showed some lymphocytes and macrophages with strong PRL immunostaining (Fig. 1e). At 1 and 2 months of infection, some lymphocytes and macrophages located in granulomas, and in the inflammatory infiltrate around airways, blood vessels, and pneumonic patches, showed PRL immunostaining; a similar pattern of immunostained cells distribution was seen at day 120, but at this late time point the bronchial epithelium also showed immunostaining (Fig. 1f). In the pneumonic patches, macrophages with morphological characteristics of activated cells, large cells with abundant and compact cytoplasm, also some lymphocytes showed PRL immunostaining, while foamy macrophages were negative.

PR was not detected in control healthy lungs (Fig. 2b). In contrast, after 21 days of infection, numerous lymphocytes and macrophages in granulomas showed immunostaining (Fig. 2c), as well as many lymphocytes in the mediastinal lymph nodes (Fig. 2d). At day 120 of infection, only small groups of lymphocytes in pneumonic patches showed positive immunostaining (Fig. 2e).

Effect of PRL on Infected and Noninfected Alveolar Macrophages

In vitro studies showed that after 1 h of incubation, 3% of macrophages contained 1 or 2 phagocytosed bacilli in their cytoplasm. The incubation with PRL before exposing macrophages to mycobacteria increased the percentage of infected cells fourfold, and nearly 50% of these cells contained 5 bacilli per macrophage (Fig. 3a). PRL incubation during and after 1 h of macrophage infection also increased the number of infected cells and phagocytosed bacilli but in a lesser proportion (Fig. 3a). After 7 days of incubation with PRL, infected macrophages showed significantly higher elimination of phagocytosed mycobacteria than control infected cells without PRL (Fig. 3b). PRL induced a significant increase in intracellular ROS production in uninfected and infected macrophages (Fig. 3c). Furthermore, NO production increased significantly at 24 h, with higher levels in infected cells (Fig. 3d).

Fig. 3.

Effect of PRL in vitro on alveolar macrophages (cell line MHS). a Percentage of infected cells and the number of phagocytosed bacilli per cell after 1 h of incubation with M. tuberculosis in the presence of PRL before, during, and after bacterial exposure. Preincubation with PRL is the most effective method to induce phagocytosis. b Determination of intracellular live bacilli in macrophages infected after 1 h and 7 days, with or without PRL. There is significant intracellular bacteriolysis after 7 days of incubation with PRL. c Determination of superoxide and NO (d) in infected and noninfected alveolar macrophages incubated with PRL (two-way ANOVA with Sidak multiple comparisons; data are expressed as mean ± SD. bp < 0.05. cp < 0.001. dp < 0.01, p < 0.001). Two independent experiments were performed with similar results.

Effect of PRL on Infected and Noninfected Pneumocytes

As shown previously, the alveolar epithelium showed strong PRL immunostaining after 1 day of intratracheal M. tuberculosis infection, suggesting that these cells, particularly pneumocytes type II, could participate in the innate immune response against mycobacterial infection. Surfactant and antimicrobial peptides are significant immune factors produced by type II pneumocytes, and PRL may be involved in their production. Although it was not statistically significant, our in vitro results showed high production of SP-A by noninfected pneumocytes type II after 1 h of PRL incubation, without significant change in M. tuberculosis-infected cells at the same time (Fig. 4a); but after 21 h of PRL incubation, infected pneumocytes showed the highest expression of SP-A. PRL also induced a higher, but not significant, expression of SP-D in noninfected pneumocytes after 1 h of incubation, and M. tuberculosis-infected cells at the same time showed a higher, but not significant, expression induced by PRL. After 21 h of incubation, PRL induced much higher SP-D expression in noninfected pneumocytes than in infected cells (Fig. 4b). These results show important dispersion despite being performed in controlled in vitro conditions, and only mRNA was measured. Thus, further experiments are needed to confirm PRL’s induction effect on the production of these immune reactants.

Fig. 4.

Effect of PRL in vitro on pneumocytes type II (cell line A549). a Gene transcription of surfactant protein A, surfactant protein D (b), and human cathelicidin LL-37 (c) in the indicated experimental conditions after 1 and 21 h of incubation with PRL. d Intracellular bacilli load, determined by CFUs in pneumocytes type II after one and 21 h of infection, with or without PRL (data are expressed as mean ± SD; two-way ANOVA with Sidak multiple comparisons. d p < 0.05).

Although it was not statistically significant, PRL also induced expression of the human antimicrobial peptide cathelicidin (LL-37) after 1 h of incubation in noninfected pneumocytes, but M. tuberculosis infection alone induced the highest expression at the same time point (Fig. 4c). Interestingly, infected cells incubated with PRL showed lower expression than infected pneumocytes (Fig. 4c). Finally, PRL induced a significant decrease in intracellular live bacteria after 21 h of incubation in infected pneumocytes (Fig. 4d). Thus, despite phagocytosis being higher, final CFU counts were lower, and no clear role for SP-A, SP-D, or cathelicidin, as inferred from their transcription levels, was found.

Effect of PRL Administration in Mice with Late and Active Pulmonary Tuberculosis

Mice were treated with PRL after 2 months of M. tuberculosis infection, when late-stage, active pulmonary TB is present, characterized by a high bacillary load, extensive pneumonia, and low PRL production. After 1 month of treatment, there were no differences between treated and untreated mice, but after 2 months, the treated group showed a nonsignificantly higher survival (Fig. 5a), a significantly lower bacillary count (Fig. 5b), a nonsignificant decrease in the extent of pneumonic areas (Fig. 5c), and higher but not significant expression of TNFα and IFNγ (Fig. 5d).

Fig. 5.

Effect of ovine PRL treatment in mice with late progressive disease. a Survival curve of mice treated with PRL. Statistical significance was evaluated using the log-rank (Mantel-Cox) test (p = 0.6357). b Pulmonary bacillary load determined by CFUs; there is a significant reduction after 2 months of treatment. c Surface area affected by pneumonia assessed by automated morphometry. d Gene expression of TNFα and IFNγ determined by RT-PCR (data are expressed as mean ± SD. a log-rank (Mantel-Cox) test. b–d Two-way ANOVA with Sidak multiple comparisons. bp < 0.01).

Discussion

PRL is a hormone known to modulate immune responses, especially in autoimmune diseases [16]. Its role in infectious diseases is less clear, with most studies showing protective effects in protozoal infections. Research on PRL in bacterial infections is limited, particularly for mycobacterial infections, which have been studied only in vitro using M. bovis-infected macrophages [10, 11, 17].

In this study, we used a pulmonary TB model in BALB/c mice to assess PRL and PR expression during infection and to evaluate the effects of PRL on alveolar macrophages and type II pneumocytes. Our results show that PRL is highly produced in infected lungs from day one, primarily by alveolar epithelial cells, followed by lymphocytes and macrophages. In vitro, we found that PRL apparently stimulates type II pneumocytes to express innate immune molecules, including surfactant proteins A (SP-A) and D (SP-D), as well as LL-37. Although these in vitro results show important dispersion, only mRNA was measured. Thus, further experiments should be done in the future to confirm the PRL induction in the production of these important molecules that could mediate the control of infection, considering that SP-A and SP-D play a significant role in innate pulmonary defense [18], acting as pattern recognition molecules, enhancing phagocytosis and inhibiting bacterial growth in the lung [18, 19], including M. tuberculosis [20–24]. Our in vitro results also suggest that PRL could induce the production of LL-37, an antimicrobial peptide effective against M. tuberculosis in type II pneumocytes [25]. We observed that PRL increased LL-37 production in uninfected alveolar pneumocytes, but infection alone induced greater LL-37 production, suggesting that mycobacteria may possess a mechanism that reduces PRL’s effectiveness in producing this antimicrobial peptide.

PRL has been shown to induce macrophage phagocytosis [17]. But few studies have linked this activity to intracellular pathogens, such as M. tuberculosis. Our in vitro findings show that alveolar macrophages exposed to PRL before, during, and after infection exhibited increased phagocytosis, with PRL incubation before infection being the most effective condition. PRL also enhanced intracellular killing of bacilli by alveolar macrophages, likely through ROS- and NO-mediated mechanisms, as both were produced by infected and uninfected macrophages incubated with PRL. Similarly, PRL induced NO production in noninfected cells at 6 and 24 h post-infection. These results suggest that PRL locally in the lung promotes activation of innate immunity by stimulating alveolar macrophages and pneumocytes.

PRL can regulate both cellular and humoral components of the acquired immune system, acting in a dose-dependent manner to stimulate or suppress immunity [26, 27]. PRL, alone or in combination with pro-inflammatory molecules such as LPS, increases NF-κB and IRF-1 activity in macrophages and lymphocytes, leading to the secretion of TNF-α, IL-1β, IL-12, and IFN-γ, thereby promoting a protective Th-1 response against intracellular bacterial infections such as TB [35]. These in vitro findings match our in vivo results. During the first 2 weeks of infection, the lungs showed a progressive increase in macrophages and lymphocytes that were positively immunostained for PRL and PR, along with increased PRL gene expression, which peaked at day 14, coinciding with the beginning of granuloma formation composed of Th-1 cells and classically activated macrophages in this model [12, 15]. PR gene transcription reached its maximum 1 week later, on day 21, when granulomas were fully developed, and the protective immune response was at its peak [12, 15]. After 3 weeks, numerous lymphocytes and macrophages expressing PRL and PR were observed not only in granulomas but also within the inflammatory infiltrates of the alveolar-capillary interstitium and around blood vessels. Lymphocytes in mediastinal lymph nodes also showed strong immunostaining, particularly for PR, suggesting these cells could play a role in local autocrine and paracrine immune-endocrine responses that temporarily control bacillary growth.

After 1 month of infection, marking the onset of progressive pulmonary disease, PRL gene expression in the lungs declined, and only small groups of macrophages and lymphocytes in pneumonic regions maintained PRL and PR expression, as detected by immunostaining. On day 120, it was also detected in some lymphocytes from the pneumonic areas; however, PRL expression in the lungs increased again. The bronchial epithelium exhibited strong PRL immunostaining, whereas only a few lymphocytes and macrophages in the pneumonic areas remained positive. At this late stage, extensive pneumonic areas coexist with high bacillary burden and the emergence of anti-inflammatory responses, including Th-2 cytokines and T-regulatory cells [12, 28], which antagonize the protective Th-1 response by reducing IFN-γ and TNF-α production. Probably, the low PRL production during this stage could contribute to weakened host defense.

Given the stimulatory effects of PRL on Th-1 lymphocytes and activated macrophages [29], we hypothesized that PRL treatment during late active disease might restore Th-1 response activity, thereby improving bacterial elimination. Our results showed that in mice infected for 2 months and treated with PRL for 2 months, there was a significant reduction in bacillary loads compared with untreated controls, with a nonsignificant increase in IFN-γ and TNF-α.

In conclusion, PRL is produced by airway epithelial cells, macrophages, and lymphocytes, which likely contribute to the temporal control of M. tuberculosis growth by promoting innate defense mechanisms. PRL also enhances cell-mediated immunity, and its administration late in the disease course could reactivate protective immune responses.

Statement of Ethics

The Internal Committee for the Care and Use of Laboratory Animals (CICUAL) of the National Institute of Medical Sciences and Nutrition in Mexico approved the animal work in accordance with the Mexican regulatory guidelines for Animal Care and Experimentation, NOM-062-ZOO-1999 (Permit Document Code: CICUAL-PAT-1915-18-20-1).

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This study was supported by the Consejo Nacional de Ciencia y Tecnología (CONACyT IFC 2015-1, Project No. 115). GQ-B is a medical resident in the pathological anatomy course, part of the Programa Nacional de Residencias Médicas at the Universidad Nacional Autónoma de México (UNAM), and received a fellowship from the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán. The funder had no role in the design, data collection, data analysis, or reporting of this study.

Author Contributions

G.Q.-B., D.M.-E., and R.H.-P. contributed to the background work and conceived the experiments. G.Q.-B., D.M.-E., and D.C.-B. performed, organized, and analyzed the results. D.M.-E., B.T.-V., and J.B.-P. contributed to the design and supervised experimental work. G.Q.-B. and R.H.-P. wrote the manuscript. R.H.-P. provided the funds.

Funding Statement

This study was supported by the Consejo Nacional de Ciencia y Tecnología (CONACyT IFC 2015-1, Project No. 115). GQ-B is a medical resident in the pathological anatomy course, part of the Programa Nacional de Residencias Médicas at the Universidad Nacional Autónoma de México (UNAM), and received a fellowship from the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán. The funder had no role in the design, data collection, data analysis, or reporting of this study.

Data Availability Statement

The data that support the findings of this study are not publicly available for privacy reasons but are available from the corresponding author.