Open AccessArticle

Turning the Tables: Loss of Adaptive Immunity Reverses Sex Differences in Tuberculosis

David Hertz

Lars Eggers

¹,

Linda von Borstel

¹,

Torsten Goldmann

^2,3

Hanna Lotter

⁴ and

Bianca E. Schneider

^1,*

Host Determinants in Lung Infections, Priority Research Area Infections, Research Center Borstel, Leibniz Lung Center, 23845 Borstel, Germany

Core Facility Histology, Research Center Borstel, Leibniz Lung Center, 23845 Borstel, Germany

Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), 22927 Großhansdorf, Germany

⁴

Department of Molecular Parasitology and Immunology, Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany

Author to whom correspondence should be addressed.

Immuno 2025, 5(1), 4; https://doi.org/10.3390/immuno5010004

Submission received: 6 November 2024 / Revised: 13 December 2024 / Accepted: 27 December 2024 / Published: 4 January 2025

(This article belongs to the Section Innate Immunity and Inflammation)

Download

Browse Figures

Versions Notes

Abstract

Sex-based differences in innate immunity may play a crucial role in susceptibility to and progression of tuberculosis (TB), a disease that disproportionately affects men. This study aimed to examine whether early host–pathogen interactions contribute to the heightened vulnerability of males to Mycobacterium tuberculosis (Mtb) infection. Using recombination activating gene 2 knockout (RAG2 KO) mice, which lack adaptive immunity, we were able to isolate and analyze innate immune responses to Mtb without the influence of T and B cells. Surprisingly, and in stark contrast to wild-type mice that reflect the male bias as observed in humans, female RAG2 KO mice were more susceptible to Mtb than their male counterparts. Increased lung CFU in females was accompanied by a significant rise in inflammation, indicated by elevated levels of inflammatory cytokines and chemokines, as well as a massive influx of neutrophils into the lungs. In contrast, male mice exhibited higher levels of IFN-γ and CCL5, along with a greater presence of NK cells in their lungs, suggesting that, in the absence of adaptive immunity, males benefit from a more robust NK cell response, potentially offering greater protection by better controlling inflammation and slowing disease progression.

Keywords:

RAG2 KO mice; Mycobacterium tuberculosis; sex differences

1. Introduction

Tuberculosis (TB) is the most prevalent bacterial infectious disease in humans. The causative agent, Mycobacterium tuberculosis (Mtb), is carried by an estimated 2–3 billion people globally and claims approximately 1.5 million lives each year [1]. Like many other infectious diseases, TB exhibits a strong male preponderance in disease development, with males being nearly twice as likely to have active TB compared to females. Historically, this difference has been attributed to behavioral factors such as higher rates of smoking and alcohol consumption among males [2]. However, recent research, including our own, has begun to shed light on biological reasons behind the differing risks of TB between males and females [3]. Particularly, studies using mouse models that isolate the impact of biological sex on TB susceptibility emphasize its importance, demonstrating that males experience a more rapid progression of TB and earlier death [4,5,6].

Increased male susceptibility to Mtb manifests in the chronic phase of the infection and is accompanied by marked differences in the organization of lesions in the lung [5,6]. While the underlying reasons for these differences in lesion formation remain to be elucidated, it has become clear that the early stages of infection are crucial in determining its final outcome [7]. Consequently, inherent functional differences among innate immune cells involved in early host–pathogen interaction could profoundly influence the overall outcome of the infection. Therefore, our aim was to investigate potential sex differences during early Mtb infection while excluding the influence of adaptive immunity. To do so, we studied recombination activating gene knockout (RAG2 KO) mice, which lack mature B and T lymphocytes [8]. Unexpectedly, while males are typically more susceptible to Mtb in immunocompetent mice, RAG2 KO mice revealed the opposite pattern. Female RAG2 KO mice infected with Mtb HN878 exhibited higher bacterial loads, more severe weight loss, and earlier mortality compared to their male counterparts. The inflammatory response in females was more pronounced, marked by neutrophil-dominated lung pathology. In contrast, the increased presence of NK cells and significantly higher IFN-γ levels in the lungs of males suggest that they benefit from a more robust NK cell response, which may play a role in regulating inflammation and slowing disease progression. These findings highlight significant sex-specific differences in the innate immune response to Mtb infection, with females, unexpectedly, showing increased susceptibility to severe infection and inflammation when adaptive immunity is absent.

2. Material and Methods

2.1. Ethics Statement

Animal experiments were in accordance with the German Animal Protection Law and approved by the Ethics Committee for Animal Experiments of the Ministry of Agriculture, Rural Areas, European Affairs and Consumer Protection of the State of Schleswig-Holstein.

2.2. Mice

Recombination Activating Gene 2 deficient (RAG2 KO) mice were bred under SPF conditions at the Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany, and transferred to the experimental animal facility at the RCB at least 5 weeks before the experiments for acclimatization. For infection experiments, male and female mice aged between 12 and 16 weeks were maintained under barrier conditions in the BSL 3 facility at the Research Center Borstel in individually ventilated cages.

2.3. Mtb Infection and Determination of Bacterial Load

Mtb HN878 was grown in Middlebrook 7H9 broth (BD Biosciences, Franklin Lakes, NJ, USA) supplemented with 10% v/v OADC (Oleic acid, Albumin, Dextrose, Catalase) enrichment medium (BD Biosciences, Franklin Lakes, USA), 0.2% v/v Glycerol, and 0.05% v/v Tween 80 to logarithmic growth phase (OD₆₀₀ 0.2–0.4) and aliquots were frozen at −80 °C. Viable cell number in thawed aliquots were determined by plating serial dilutions onto Middlebrook 7H11 agar plates supplemented with 10% v/v OADC and 0.5% v/v Glycerol followed by incubation at 37 °C for 3–4 weeks. For infection of experimental animals, Mtb stocks were diluted in sterile distilled water at a concentration providing an uptake of approximately 100–200 viable bacilli per lung. Infection was performed via the respiratory route by using an aerosol chamber (Glas-Col, Terre-Haute, IN, USA) as described previously [9]. The inoculum size was quantified 24 h after infection in the lungs, and bacterial loads in the lungs, mediastinal lymph nodes, spleen, and liver were evaluated at different time points after aerosol infection by determining colony forming units (CFU). Organs were removed aseptically, weighed, and homogenized in 0.05% v/v Tween 20 in PBS containing a proteinase inhibitor cocktail (Roche, Basel, Switzerland) prepared according to the manufacturer’s instructions for subsequent quantification of cytokines (see below). Tenfold serial dilutions of organ homogenates in sterile water/1% v/v Tween 80/1% w/v albumin were plated onto Middlebrook 7H11 agar plates supplemented with 10% v/v OADC and incubated at 37 °C for 3–4 weeks.

2.4. Clinical Score

Clinical score was used to indicate severity of disease progression. Animals were scored in terms of general behavior, activity, feeding habits, and weight gain or loss. Each of the criteria is assigned score points from 1 to 5 with 1 being the best and 5 the worst. The mean of the score points represents the overall score for an animal. Animals with severe symptoms (reaching a clinical score of ≥3.5; defined as moribund) were euthanized to avoid unnecessary suffering, and the time point was recorded as the end point of survival for that individual mouse.

2.5. Time Points of Organ Harvest

Organs were collected for downstream analysis at specific time points, as indicated in the figure legends: 13 or 21 days post-Mtb infection, a flexible (flex) time point, and from moribund animals. The flex time points were selected as follows: once a mouse reached a score of 3, a randomly chosen mouse of the opposite sex, pre-assigned before the experiment, was also harvested, regardless of its score. This allowed us to determine the bacterial burden and immunological and histopathological changes in relation to the disease stage and progression. The range of days for the flex time point was between days 24 and 27 post-infection.

2.6. Multiplex Cytokine Assay

The concentrations of various cytokines and chemokines in lung homogenates were determined by LEGENDplex^TM (Mouse Inflammation panel and Mouse Proinflammatory Chemokine Panel, BioLegend, San Diego, CA, USA) according to the manufacturer’s protocol. Data were acquired on the MACSQuant Analyzer 10 Flow Cytometer (Miltenyi, Bergisch Gladbach, Germany) and analyzed with the LEGENDplex™ Data Analysis Software Suite version 2024-06-15 (Biolegend, San Diego, CA, USA).

2.7. Histology

Superior lung lobes from infected mice were fixed with 4% w/v paraformaldehyde for 24 h, embedded in paraffin, and sectioned (4 μm). Tissue sections were stained with hematoxylin and eosin (HE) to assess overall granulomatous inflammation. Macrophages were detected by a polyclonal rabbit anti CD68 (Abcam, Cambridge, UK), neutrophils by monoclonal rat anti neutrophils (clone 7/4, Cedarlane Laboratories), and NK cells by a rabbit recombinant monoclonal anti NKR-P1C (EPR22990-31, Abcam), respectively, followed by secondary antibody (biotinylated goat anti-rabbit or biotinylated goat anti-rat; Dianova, Hamburg, Germany), amplification (avidin-HRP), and color reaction (DAB solution; Vectastain, Vector Laboratories, Inc., Newark, CA, USA). Slides were imaged with PhenoImager™ HT instrument (Akoya Biosciences, Marlborough, MA, USA). The affected lung area was quantified in relation to the total lung area, and the number of different cell subsets was calculated in relation to the total number of cells in the entire tissue using the QuPath software version 0.5.1 (freeware).

2.8. Statistical Analysis

All data were analyzed using GraphPad Prism 10 (GraphPad Software, La Jolla, CA, USA). Statistical tests are indicated in the individual figure legends. Correlation was determined by calculating Pearson’s coefficient using a two-tailed analysis. A correlation was taken into account as of r ≥ 0.60 (defined as strong correlation). Values of * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001 were considered significant.

3. Results

3.1. Female RAG2 KO Mice Are More Susceptible to Mtb HN878 Compared to Males

To test the ability of male and female mice to control HN878 infection in the absence of adaptive immunity, we infected RAG2 KO mice and monitored disease progression and survival according to a clinical score, as described in the Materials and Methods. Moreover, we determined the bacterial load in the lungs, mediastinal lymph nodes (LN), spleen, and liver after 13 days, 21 days, at a flex time point, and in moribund animals that had reached the clinical endpoint according to our clinical scoring (Figure 1A).

While there were no differences in bacterial burden between the sexes on day 13, females exhibited significantly higher bacterial loads in the lungs and LN 21 days post-infection (Figure 1B–E). As the disease progressed, evidenced by increased weight loss and clinical scores (Figure 1F,G), bacterial loads rose further, especially in the lungs and livers of females, and were significantly higher than in males (Figure 1B–E, flex). This aligns with the more pronounced weight loss and higher clinical scores observed in females compared to males (Figure 1F,G). In moribund animals that had reached the endpoint according to our clinical scoring, the CFU did not differ between the sexes, likely reflecting comparable disease states. Overall, diminished bacterial control and accelerated disease progression culminated in earlier mortality in females (Figure 1H). These findings contrast sharply with Mtb infection in immunocompetent C57BL/6 mice, where males are more susceptible than females [5,6].

3.2. Sex Differences in Cytokine and Chemokine Production in Response to Mtb in Lungs of RAG2 KO Mice

To examine the immunological environment in the lungs that was associated with differences in Mtb control in male and female RAG2 KO mice, we quantified the levels of inflammatory cytokines and chemokines. Figure 2 illustrates sex-based differences in immune mediator profiles at day 21 post-infection and the later “flex” phase (no significant differences between sexes were observed on day 13 post Mtb infection; Supplementary Figure S1). Panels A and B present radar plots of cytokine and chemokine levels, comparing male and female RAG2 KO mice. Females exhibited elevated levels of several key inflammatory markers, including TNF, IL-1α, IL-1β, IL-6, IL-17A, CCL2, CCL4, CCL20, and CXCL1, as shown in Figure 2C–K. These results suggest that female RAG2 KO mice have a generally heightened inflammatory response at both day 21 and during the flex phase, with statistically significant differences observed between sexes. However, CCL5 and IFN-γ stand out as notable exceptions to this trend. In contrast to the other immune mediators, both CCL5 and IFN-γ were significantly higher in males compared to females, particularly during the flex phase (Figure 2L,M).

These findings indicate marked differences between the sexes in the inflammatory response, driven by innate immunity. Since these cytokines and chemokines play a critical role in orchestrating immune cell recruitment, the data suggest that male and female RAG2 KO mice may exhibit distinct patterns of immune cell infiltration in the lung in response to Mtb infection.

3.3. Females Exhibit Neutrophil-Dominated Inflammation, While Males Have More NK Cells in the Lungs

To support the cytokine and chemokine profiles, HE staining and immunohistochemistry (IHC) were conducted to evaluate lung pathology and cellular infiltration in Mtb-infected male and female RAG2 KO mice. The analysis revealed significantly larger areas of lung inflammation in females compared to males, particularly during the flex phase (Figure 3A,B). This increased inflammation was associated with a higher abundance of CD68⁺ cells, which is a marker of macrophages and monocytes, in females than in males (Figure 3C,D). Additionally, female lesions exhibited a significant influx of neutrophils, as evidenced by staining for 7/4 (Figure 3E,F). The elevated presence of neutrophils in female lungs corresponds with the increased levels of CCL20 and CXCL1 observed in females (Figure 2J,K), both of which are known to play roles in neutrophil recruitment and activation. In contrast, NKR-P1C⁺ NK cells were more abundant in males than females (Figure 3G,H). NK cells are a crucial early source of IFN-γ in the lungs during Mtb infection [10]. Moreover, NK cell-derived IFN-γ was shown to regulate the pulmonary inflammatory response to Mtb in the absence of T lymphocytes by down-modulating neutrophil infiltration [11]. Consistent with this, NKR-P1C⁺ cells were positively correlated with IFN-γ levels and negatively with 7/4⁺ cells and lung inflammation in our study (Figure 3I–K). Conversely, the abundance of 7/4⁺ cells was weakly correlated with the area of lung inflammation, while the strongest positive correlation was observed between the number of 7/4⁺ neutrophils and the clinical score (Figure 3L,M). This underscores the importance of neutrophils in contributing to a detrimental clinical outcome [12,13,14].

In conclusion, the data suggest that in the absence of adaptive immunity, female mice exhibit a neutrophil-dominated lung pathology, whereas males show a higher abundance of NK cells that may mitigate inflammation through IFN-γ production.

4. Discussion

The initial immune response to Mtb, including the activation of innate immune cells and the subsequent inflammatory response, plays a crucial role in shaping the overall disease trajectory [7,15]. In this study, we aimed to investigate whether early events in Mtb infection might already indicate the higher susceptibility and impaired long-term control observed in males [5,6]. Our study reveals striking sex-specific differences in the innate immune response to Mtb infection in RAG2 KO mice, highlighting how the absence of adaptive immunity can lead to unexpected variations in disease susceptibility and progression. Contrary to what is observed in immunocompetent C57BL/6 mice, where males generally exhibit greater susceptibility to TB [5,6], female RAG2 KO mice demonstrated significantly poorer control over HN878 infection compared to males. This was evidenced by higher bacterial loads in the lungs, increased weight loss, and earlier mortality among females. The heightened susceptibility of female RAG2 KO mice was paralleled by an exaggerated inflammatory response, characterized by elevated levels of inflammatory cytokines and chemokines such as TNF, IL-1α, IL-1β, IL-6, IL-17A, CCL2, CCL20, and CXCL1. These factors are known to orchestrate immune cell recruitment and activation, and as a result, we observed a neutrophil-dominated lung pathology in females. This is in sharp contrast to males, who exhibited higher levels of NK cells and IFN-γ. In addition to its role in activating macrophages, IFN-γ has a regulatory role protecting from immunopathology by suppressing neutrophil recruitment [16,17]. NK cells are an important early source of IFN-γ in the Mtb-infected lung [18]. Studies in immunocompetent C57BL/6 mice suggest that NK cells do not play a direct role in regulating TB immunity since depleting NK cells did not affect bacterial burden [10]. However, NK cell-derived IFN-γ was indispensable for the survival of RAG2-deficient mice after Mtb infection where it regulated macrophage activation and limited neutrophil-driven immunopathology [11]. Consistent with this, the increased abundance of NKR-P1C⁺ NK cells in males in our study correlated with reduced neutrophil infiltration and less severe lung inflammation, suggesting a protective role of NK cells in modulating inflammation in a sex-specific manner. While we did not investigate macrophage activation in this study, the importance of IFN-γ for macrophage activation and enhancing their effector function against Mtb is well established [19,20,21]. Impaired macrophage activation due to lower IFN-γ production, particularly in the later phase of infection, could contribute to the reduced control of bacterial replication observed in female RAG2 KO mice. Therefore, future investigations should aim to dissect the exact roles of the different innate immune cell compartments and their contribution to the increased susceptibility of female RAG2 KO mice.

Sex differences in immune cell function may arise from variations in the expression of sex chromosome-encoded genes or the influence of sex steroid hormones [22]. Notably, sex differences in NK cells have been observed previously [23,24,25,26]. All studies consistently showed that the number of NK cells was higher in males than in females, but the data on their activation status were inconsistent. Patin and colleagues found a larger number of activated NK cells in men than in women [24]. Likewise, Huang and colleagues showed several pathways, such as the IFN-γ-mediated signaling pathway, and the positive regulation of immune effector processes to be up-regulated in male compared with female NK cells, which increased during aging [23]. In a mouse model of polycystic ovary syndrome, androgen-exposed mice displayed a higher frequency of NK-cells and elevated levels of IFN-γ, while a number of studies focusing on the effect of estrogen on NK cells have shown reduced cytotoxicity and IFN-γ production in response to estradiol stimulation [27,28,29]. In contrast, other studies reported enhanced NK cell activity as a result of androgen deprivation therapy, which is a standard treatment for prostate cancer due to the central role of androgens in carcinogenesis [30,31]. Finally, the study from Cheng found enhanced effector function in female compared to male NK cells [26]. These differences were primarily attributed to the X-linked epigenetic regulator UTX, encoded by the gene Kdm6a, which escapes X-inactivation [32]. UTX influenced the expression of gene loci crucial for NK cell fitness and effector functions and was expressed at significantly lower levels in both male mouse and human NK cells [26]. While the experimental studies were conducted on immunocompetent mice, potential sex differences in the NK cell compartment of RAG2 KO mice including potential hormonal differences between RAG2 KO compared to wild-type mice remain unexplored. However, recent research has demonstrated that NK cells in RAG2-deficient mice are hyperactive [33,34]. Both the number and frequency of NK cells were elevated in RAG2 KO mice compared to WT mice in peripheral blood, spleen, and bone marrow, with these cells also expressing higher levels of activation markers and IFN-γ. These hyperactive NK cells in RAG2-deficient mice showed enhanced antileukemia effect in several models of acute myeloid leukemia [33]. Our study suggests that the potent NK cell response prevents neutrophil-driven immunopathology in males but not in females. Currently the reasons for these sex differences remain unclear and deserve further evaluation.

5. Conclusions

Our findings reveal significant differences in disease outcomes following Mtb infection based on the presence or absence of adaptive immunity. In immunocompetent mice, males show greater susceptibility to Mtb infection [4,5,6], whereas in the absence of B and T cells, females exhibit heightened susceptibility. This shift underscores how adaptive immunity influences innate immunity and disease progression differently between sexes. Moreover, the pronounced neutrophil response in females and the potential regulatory role of NK cells in males underscore distinct innate mechanisms by which each sex responds to Mtb infection. Understanding these immune differences, and how innate and adaptive immunity interact in males and females, is crucial for designing interventions that address the unique immune profiles of each sex and enhance overall disease management.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/immuno5010004/s1, Figure S1. Radar chart of cytokines and chemokines measured in lung homogenates of female and male RAG2 KO mice at day 13 p.i . Concentrations of cytokines/chemokines are shown as pg/ml. Data is represented as mean of cytokine and chemokine concentration from 2 experiments (n = 10/group).

Author Contributions

B.E.S. and D.H. conceived and designed the experiments; D.H., L.E. and L.v.B. performed the experimental work; D.H. analyzed the data; T.G. and H.L. provided advice, reagents and mice; B.E.S. and D.H. wrote the paper. All authors revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

We would like to thank Silvia Maass for culturing Mtb; the staff of the animal facility at the Research Center Borstel for animal care; Christian Rosero and Jasmin Tiebach for their help with the immunohistochemistry. Data acquisition was performed on instruments in the core facility Fluorescence Cytometry at the Research Center Borstel.

Conflicts of Interest

The authors declare no competing interests.

References

WHO. Global Tuberculosis Report 2023; World Health Organization: Geneva, Switzerland, 2023. [Google Scholar]
Hertz, D.; Schneider, B. Sex differences in tuberculosis. Semin. Immunopathol. 2019, 41, 225–237. [Google Scholar] [CrossRef]
Gupta, M.; Srikrishna, G.; Klein, S.L.; Bishai, W.R. Genetic and hormonal mechanisms underlying sex-specific immune responses in tuberculosis. Trends Immunol. 2022, 43, 640–656. [Google Scholar] [CrossRef]
Bini, E.I.; Mata Espinosa, D.; Marquina Castillo, B.; Barrios Payan, J.; Colucci, D.; Cruz, A.F.; Zatarain, Z.L.; Alfonseca, E.; Pardo, M.R.; Bottasso, O.; et al. The influence of sex steroid hormones in the immunopathology of experimental pulmonary tuberculosis. PLoS ONE 2014, 9, e93831. [Google Scholar] [CrossRef] [PubMed]
Dibbern, J.; Eggers, L.; Schneider, B.E. Sex differences in the C57BL/6 model of Mycobacterium tuberculosis infection. Sci. Rep. 2017, 7, 10957. [Google Scholar] [CrossRef] [PubMed]
Hertz, D.; Dibbern, J.; Eggers, L.; von Borstel, L.; Schneider, B.E. Increased male susceptibility to Mycobacterium tuberculosis infection is associated with smaller B cell follicles in the lungs. Sci. Rep. 2020, 10, 5142. [Google Scholar] [CrossRef] [PubMed]
Cadena, A.M.; Flynn, J.L.; Fortune, S.M. The Importance of First Impressions: Early Events in Mycobacterium tuberculosis Infection Influence Outcome. mBio 2016, 7, e00342-16. [Google Scholar] [CrossRef]
Shinkai, Y.; Rathbun, G.; Lam, K.P.; Oltz, E.M.; Stewart, V.; Mendelsohn, M.; Charron, J.; Datta, M.; Young, F.; Stall, A.M.; et al. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 1992, 68, 855–867. [Google Scholar] [CrossRef]
Ring, S.; Eggers, L.; Behrends, J.; Wutkowski, A.; Schwudke, D.; Kroger, A.; Hierweger, A.M.; Holscher, C.; Gabriel, G.; Schneider, B.E. Blocking IL-10 receptor signaling ameliorates Mycobacterium tuberculosis infection during influenza-induced exacerbation. JCI Insight 2019, 4, e126533. [Google Scholar] [CrossRef]
Junqueira-Kipnis, A.P.; Kipnis, A.; Jamieson, A.; Juarrero, M.G.; Diefenbach, A.; Raulet, D.H.; Turner, J.; Orme, I.M. NK cells respond to pulmonary infection with Mycobacterium tuberculosis, but play a minimal role in protection. J. Immunol. 2003, 171, 6039–6045. [Google Scholar] [CrossRef]
Feng, C.G.; Kaviratne, M.; Rothfuchs, A.G.; Cheever, A.; Hieny, S.; Young, H.A.; Wynn, T.A.; Sher, A. NK cell-derived IFN-gamma differentially regulates innate resistance and neutrophil response in T cell-deficient hosts infected with Mycobacterium tuberculosis. J. Immunol. 2006, 177, 7086–7093. [Google Scholar] [CrossRef]
Muefong, C.N.; Sutherland, J.S. Neutrophils in Tuberculosis-Associated Inflammation and Lung Pathology. Front. Immunol. 2020, 11, 962. [Google Scholar] [CrossRef]
Eruslanov, E.B.; Lyadova, I.V.; Kondratieva, T.K.; Majorov, K.B.; Scheglov, I.V.; Orlova, M.O.; Apt, A.S. Neutrophil responses to Mycobacterium tuberculosis infection in genetically susceptible and resistant mice. Infect. Immun. 2005, 73, 1744–1753. [Google Scholar] [CrossRef] [PubMed]
Yeremeev, V.; Linge, I.; Kondratieva, T.; Apt, A. Neutrophils exacerbate tuberculosis infection in genetically susceptible mice. Tuberculosis 2015, 95, 447–451. [Google Scholar] [CrossRef] [PubMed]
Sankar, P.; Mishra, B.B. Early innate cell interactions with Mycobacterium tuberculosis in protection and pathology of tuberculosis. Front. Immunol. 2023, 14, 1260859. [Google Scholar] [CrossRef] [PubMed]
Desvignes, L.; Ernst, J.D. Interferon-gamma-responsive nonhematopoietic cells regulate the immune response to Mycobacterium tuberculosis. Immunity 2009, 31, 974–985. [Google Scholar] [CrossRef]
Nandi, B.; Behar, S.M. Regulation of neutrophils by interferon-gamma limits lung inflammation during tuberculosis infection. J. Exp. Med. 2011, 208, 2251–2262. [Google Scholar] [CrossRef]
Abebe, F. Immunological basis of early clearance of Mycobacterium tuberculosis infection: The role of natural killer cells. Clin. Exp. Immunol. 2021, 204, 32–40. [Google Scholar] [CrossRef]
Ahmad, F.; Rani, A.; Alam, A.; Zarin, S.; Pandey, S.; Singh, H.; Hasnain, S.E.; Ehtesham, N.Z. Macrophage: A Cell With Many Faces and Functions in Tuberculosis. Front. Immunol. 2022, 13, 747799. [Google Scholar] [CrossRef] [PubMed]
Flynn, J.L.; Chan, J.; Triebold, K.J.; Dalton, D.K.; Stewart, T.A.; Bloom, B.R. An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection. J. Exp. Med. 1993, 178, 2249–2254. [Google Scholar] [CrossRef] [PubMed]
Herbst, S.; Schaible, U.E.; Schneider, B.E. Interferon gamma activated macrophages kill mycobacteria by nitric oxide induced apoptosis. PLoS ONE 2011, 6, e19105. [Google Scholar] [CrossRef]
Klein, S.L.; Flanagan, K.L. Sex differences in immune responses. Nat. Rev. Immunol. 2016, 16, 626–638. [Google Scholar] [CrossRef]
Huang, Z.; Chen, B.; Liu, X.; Li, H.; Xie, L.; Gao, Y.; Duan, R.; Li, Z.; Zhang, J.; Zheng, Y.; et al. Effects of sex and aging on the immune cell landscape as assessed by single-cell transcriptomic analysis. Proc. Natl. Acad. Sci. USA 2021, 118, e2023216118. [Google Scholar] [CrossRef] [PubMed]
Patin, E.; Hasan, M.; Bergstedt, J.; Rouilly, V.; Libri, V.; Urrutia, A.; Alanio, C.; Scepanovic, P.; Hammer, C.; Jonsson, F.; et al. Natural variation in the parameters of innate immune cells is preferentially driven by genetic factors. Nat. Immunol. 2018, 19, 302–314. [Google Scholar] [CrossRef]
Menees, K.B.; Earls, R.H.; Chung, J.; Jernigan, J.; Filipov, N.M.; Carpenter, J.M.; Lee, J.K. Sex- and age-dependent alterations of splenic immune cell profile and NK cell phenotypes and function in C57BL/6J mice. Immun. Ageing 2021, 18, 3. [Google Scholar] [CrossRef]
Cheng, M.I.; Li, J.H.; Riggan, L.; Chen, B.; Tafti, R.Y.; Chin, S.; Ma, F.; Pellegrini, M.; Hrncir, H.; Arnold, A.P.; et al. The X-linked epigenetic regulator UTX controls NK cell-intrinsic sex differences. Nat. Immunol. 2023, 24, 780–791. [Google Scholar] [CrossRef] [PubMed]
Hao, S.; Li, P.; Zhao, J.; Hu, Y.; Hou, Y. 17beta-estradiol suppresses cytotoxicity and proliferative capacity of murine splenic NK1.1+ cells. Cell. Mol. Immunol. 2008, 5, 357–364. [Google Scholar] [CrossRef]
Stopinska-Gluszak, U.; Waligora, J.; Grzela, T.; Gluszak, M.; Jozwiak, J.; Radomski, D.; Roszkowski, P.I.; Malejczyk, J. Effect of estrogen/progesterone hormone replacement therapy on natural killer cell cytotoxicity and immunoregulatory cytokine release by peripheral blood mononuclear cells of postmenopausal women. J. Reprod. Immunol. 2006, 69, 65–75. [Google Scholar] [CrossRef]
Jiang, X.; Orr, B.A.; Kranz, D.M.; Shapiro, D.J. Estrogen induction of the granzyme B inhibitor, proteinase inhibitor 9, protects cells against apoptosis mediated by cytotoxic T lymphocytes and natural killer cells. Endocrinology 2006, 147, 1419–1426. [Google Scholar] [CrossRef] [PubMed]
Liu, Q.; You, B.; Meng, J.; Huang, C.P.; Dong, G.; Wang, R.; Chou, F.; Gao, S.; Chang, C.; Yeh, S.; et al. Targeting the androgen receptor to enhance NK cell killing efficacy in bladder cancer by modulating ADAR2/circ_0001005/PD-L1 signaling. Cancer Gene Ther. 2022, 29, 1988–2000. [Google Scholar] [CrossRef] [PubMed]
Shi, L.; Lin, H.; Li, G.; Sun, Y.; Shen, J.; Xu, J.; Lin, C.; Yeh, S.; Cai, X.; Chang, C. Cisplatin enhances NK cells immunotherapy efficacy to suppress HCC progression via altering the androgen receptor (AR)-ULBP2 signals. Cancer Lett. 2016, 373, 45–56. [Google Scholar] [CrossRef]
Berletch, J.B.; Ma, W.; Yang, F.; Shendure, J.; Noble, W.S.; Disteche, C.M.; Deng, X. Escape from X inactivation varies in mouse tissues. PLoS Genet. 2015, 11, e1005079. [Google Scholar] [CrossRef]
Sugimoto, E.; Li, J.; Hayashi, Y.; Iida, K.; Asada, S.; Fukushima, T.; Tamura, M.; Shikata, S.; Zhang, W.; Yamamoto, K.; et al. Hyperactive Natural Killer cells in Rag2 knockout mice inhibit the development of acute myeloid leukemia. Commun. Biol. 2023, 6, 1294. [Google Scholar] [CrossRef] [PubMed]
Karo, J.M.; Schatz, D.G.; Sun, J.C. The RAG recombinase dictates functional heterogeneity and cellular fitness in natural killer cells. Cell 2014, 159, 94–107. [Google Scholar] [CrossRef] [PubMed]

Figure 1. Increased susceptibility of female RAG2 KO mice to Mtb infection. (A) Experimental setup. Female and male RAG2 KO mice were aerosol-infected with Mtb HN878 and organs were collected at the indicated time points. Flex time point is defined as the time point where a mouse reached a score of 3. At the same time, a randomly selected mouse of the opposite sex, which had been assigned before the experiment, was also taken, regardless of its score. CFU of female and male RAG2 KO mice at indicated time points in (B) lung (day 1 n = 8; days 13 and 21 n = 10; flex n = 7; moribund n = 12 (f) or 10 (m)), (C) mediastinal LN (day 13 n = 8; day 21 n = 10 (f) or 9 (m); flex n = 7 (f) or 5 (m); moribund n = 12 (f) or 9(m)), (D) spleen (day 13 n = 10; day 21 n = 9 (f) or 10 (m); flex n = 7; moribund n = 12 (f) or 10 (m)) and (E) liver (day 13 n = 10 (f) or 9 (m); day 21 n = 10; flex n = 7; moribund n = 12 (f) or 10 (m)). Body weight change (F), clinical score (G), and survival (H) of female and male RAG2 KO mice. (B–E) Each data point represents one mouse from two experiments. (F,G) Each data point represents one mouse from one representative experiment out of two (n = 3–15). (H) n = 10 mice per group of one experiment. Statistical analysis was performed by Welch’s t-test (B–E), 2way ANOVA followed by Tukey’s multiple comparisons test (F,G) or log rank test (H).

Figure 2. Inflammatory responses in the lungs of Mtb-infected RAG2 KO mice. Female and male RAG2 KO mice were aerosol infected with Mtb HN878 and lungs were collected at the indicated time points. Radar chart of cytokines and chemokines measured in lung homogenates of female and male RAG2 KO mice at day 21 p.i. (A) and flexible time point p.i. (B). Concentrations of cytokines/chemokines are shown as pg/ml. (C–M) Selected cytokines and chemokines measured in lung homogenates of female and male RAG2 KO mice at indicated time points. (A,B) Data are represented as mean of cytokine and chemokine concentration from 2 experiments ((A); n = 10/group) or 1 experiment ((B); n = 7/group). (C–M) Each data point represents one mouse from two experiments (d21; n = 10/group) or one experiment (flex; n = 7/group). Statistical analysis was performed by 2way ANOVA followed by Tukey’s multiple comparisons test.

Figure 3. Increased NK cell numbers in male RAG2 KO mice associated with higher IFN-y responses and reduced neutrophil influx. Female and male RAG2 KO mice were aerosol-infected with Mtb HN878. Lungs were collected at indicated time points and PFA-fixed paraffin-embedded tissue sections were stained with HE (A) or antibodies to detect (C) macrophages (CD68⁺), (E) neutrophils (7/4⁺), or (G) NK cells (NKR-P1C⁺; red arrows). (A,C,E,G) Representative micrographs from one female and male RAG2 KO mouse from the flexible time point p.i. are shown. Bar = 1 mm (A,C,E) or 50 µm (G). (B,D,F,H) Quantitative analysis of the area of lung inflammation and respective immune cells as shown in (A,C,E,G). Correlation of NKR-P1C DAB⁺ cells with IFN-γ level (I), 7/4 DAB⁺ cells (J), and area of lung inflammation (K) as well as correlation of 7/4 DAB⁺ cells with area of lung inflammation (L) and clinical score (M) at the flexible time point p.i. (B,D,F,H) Each data point represents one mouse from one representative experiment out of two (d21; n = 5/group) or one experiment (flex; n = 6–7/group). Statistical analysis was performed by 2-way ANOVA followed by Tukey’s multiple comparisons test. (I–M) Each data point represents one mouse from one experiment (flex; n = 13–14). Correlation was calculated using Pearson correlation.

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

© 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Share and Cite

MDPI and ACS Style

Hertz, D.; Eggers, L.; von Borstel, L.; Goldmann, T.; Lotter, H.; Schneider, B.E. Turning the Tables: Loss of Adaptive Immunity Reverses Sex Differences in Tuberculosis. Immuno 2025, 5, 4. https://doi.org/10.3390/immuno5010004

AMA Style

Hertz D, Eggers L, von Borstel L, Goldmann T, Lotter H, Schneider BE. Turning the Tables: Loss of Adaptive Immunity Reverses Sex Differences in Tuberculosis. Immuno. 2025; 5(1):4. https://doi.org/10.3390/immuno5010004

Chicago/Turabian Style

Hertz, David, Lars Eggers, Linda von Borstel, Torsten Goldmann, Hanna Lotter, and Bianca E. Schneider. 2025. "Turning the Tables: Loss of Adaptive Immunity Reverses Sex Differences in Tuberculosis" Immuno 5, no. 1: 4. https://doi.org/10.3390/immuno5010004

APA Style

Hertz, D., Eggers, L., von Borstel, L., Goldmann, T., Lotter, H., & Schneider, B. E. (2025). Turning the Tables: Loss of Adaptive Immunity Reverses Sex Differences in Tuberculosis. Immuno, 5(1), 4. https://doi.org/10.3390/immuno5010004

Article Menu