Impact of Sex and Gender on Autoimmune Lung Disease: Opportunities for Future Research: NHLBI Working Group Report

Understanding the roles of sex and gender in autoimmune lung diseases is central to the advancement of science in this field. Although it is well known that most autoimmune diseases disproportionately affect women of childbearing age (1), the mechanism underlying this disparity remains poorly understood. Female mammals are largely underrepresented in basic research, thereby undermining efforts to fully elucidate the impact of biological sex on autoimmune lung diseases.

There are also many examples of sex and gender bias in the context of human research, resulting in untoward health consequences. For instance, the NIH’s Baltimore Longitudinal Study of Aging (2), commencing in 1958, enrolled 75,000 men. Women were excluded for the first 20 years of the study; thus, the study failed to address age-related issues concerning women, such as osteoporosis and menopause. Women were also excluded from early-phase clinical trials before 1994 out of concern for safety (3). Thus, it is not surprising that 8 out of 10 prescription drugs withdrawn from the U.S. market from 1997 to 2001 were withdrawn because of health risks identified in women (4).

Recognizing the inherent role played by sex and gender in autoimmune lung diseases (5), the NHLBI partnered with the National Institute of Arthritis and Musculoskeletal and Skin Diseases and the Office of Research on Women’s Health to organize a 2-day virtual sex- and gender-related autoimmune lung diseases workshop in June 2021. Participants with diverse areas of expertise convened to consider the impact of sex and gender on research conducted across various autoimmune lung diseases. The goals of the workshop were to discriminate the influences of biological sex, operating on a molecular level (e.g., regulated by genes, hormones), and gender (a social construct based on environmental, cultural and behavioral factors and choices that affect a person’s self-identity) (6) on experimental methods and results in the context of autoimmune and inflammatory lung diseases.

Sex- and Gender-related Factors Affecting Lung Anatomy and Physiology

Sex differences in lung growth and development are apparent in the prenatal period (7). Beginning at birth and extending into adulthood, females have smaller lungs with fewer respiratory bronchioles than males (6). In addition to anatomical differences, cyclical variations in sex hormones during the menstrual cycle affects airway behavior, manifesting as increased inflammatory airway responses during certain phases of the menstrual cycle (8).

Beyond the influence of biological sex, gender-related variations in environmental exposures across various cultures can adversely affect the airways and lung parenchyma (9). In addition, self-reported breathlessness and sputum production can vary by gender (10). Interestingly, patient gender influences the physicians’ perception of chronic airway disease: it is reported that men are more often diagnosed with emphysema, whereas women are more often diagnosed with asthma and chronic bronchitis (11).

Sex and Gender Differences in Autoimmune Lung Diseases

The prevalence of various autoimmune diseases with lung involvement varies by sex, with the majority occurring more commonly in females. In certain conditions, age is an important moderating factor in disease prevalence. For example, the incidence and prevalence of sarcoidosis is more common in females, particularly among Black populations, with a greater female predominance noted after age 50 (12, 13). In other conditions, sex differences in disease prevalence exist across the lifespan. For example, in pulmonary arterial hypertension (PAH), females comprise the majority of cases, particularly among patients with connective tissue disease (CTD)-associated PAH (14). Rheumatoid arthritis (RA) and systemic sclerosis (SSc) also afflict females more than males; however, male sex is one of the risk factors for the development of interstitial lung disease (ILD) in both of these conditions (15, 16).

Understanding the biological basis for the female predominance of autoimmune diseases that affect the lungs may provide important insight in disease pathogenesis (17). For example, the observation that sex differences in the prevalence of certain autoimmune lung diseases diminishes in older individuals (i.e., postmenopausal females), suggests that hormones and hormone receptors likely play a key role in propagating these diseases, as discussed further below.

In contrast to general trends toward exclusion of females, given the aforementioned sex differences in disease prevalence, women represent the majority of patients engaged in clinical research in many forms of ILD. For instance, in the three largest clinical trials for SSc-ILD, the percentage of individuals identifying as women ranged from 70% to 75% (18–20). Similarly, in the multicenter PHAROS (Pulmonary Hypertension Assessment and Recognition of Outcomes in Scleroderma) registry, which prospectively follows subjects with SSc at high risk for or with incident pulmonary hypertension, 87% are women (21). Although the paucity of men in these studies reflects disease prevalence, it limits our ability to adequately understand the relationship between sex and disease course, as well as treatment response. For example, in therapeutic trials for SSc-ILD, men randomized to placebo had more rapid progression (worsening) of ILD than women (22). Although this may reflect a more severe SSc-ILD phenotype among men, it could also result from selection bias (i.e., men with a more severe phenotype). Thus, there are challenges in understanding the impact of sex and/or gender on pathobiology, disease manifestations, and outcomes when there is underrepresentation of a given sex and/or gender in studies.

Relatively simple solutions could remedy sex imbalance in research. For one, programs designed to educate our biomedical workforce and trainees on the importance of examining sex and gender across the research spectrum are needed (23). In addition, researchers are inevitably influenced by government-based funding agencies prioritizing sex and gender considerations (24). Additional measures to ensure women are equally enrolled in clinical research studies on conditions affecting both genders include partnering with advocacy groups in the community that interface with underrepresented and/or marginalized patients and incorporating sex and gender variables into basic science studies. In addition, future studies could explore how autoantibody profiles and genetic factors contribute to sex disparities as discussed further below. This will promote greater scientific rigor in autoimmune diseases.

Sex, Gender, and the Pathobiology of Autoimmune Lung Diseases

The influence of sex and gender are evident in all phases of research, including cell physiology, metabolism, and immune responses. These factors should be considered in the design of research, data collection, analysis, and interpretation (Figure 1) to address research pitfalls that could undermine research progress in the field of autoimmune ILD and related disorders.

Figure 1.

Optimizing research designs to incorporate sex- and gender-related variables in studies of autoimmune lung diseases.

Genetic and Epigenetic Factors

Sex-linked genetic and epigenetic mechanisms strongly influence differential immune responses in men and women. For example, the X chromosome, encoding a high-density of immune-related genes (e.g., TLR 7/8. IRAK1, NEMO, and others) is a significant risk factor for autoimmune susceptibility for systemic lupus erythematosus and Sjogren’s syndrome. Dosage compensation of X-linked genes between the sexes is achieved during early embryonic development by expression of the long noncoding RNA Xist (X-inactive specific transcript), which is expressed from the inactive X chromosome and functions in cis. Xist RNA recruits a variety of repressive protein complexes that convert an active X into a heterochromatic and transcriptionally silent inactive X chromosome. However, approximately 15–20% of human and 1–3% of mouse X-linked genes escape X-chromosome inactivation, and partial reactivation of the inactive X chromosome is thought to promote lymphocyte deregulation, contributing toward the female predominance of autoimmune diseases, such as systemic lupus erythematosus (25), and other autoimmune diseases (26). In particular, the literature indicates that autoimmune diseases are more often associated with an escape from Xist inactivation within the X chromosome, especially in B cells (25). This gene is usually not evaluated in genome wide association studies because of this complexity, further limiting our understanding of the contribution of the X chromosome in disease risk.

Sex Hormones

Specific to the lung, sex hormones such as estradiol (E2) can elicit responses in different cell types, including airway smooth muscle cells, vascular endothelial cells, and bronchial epithelial cells (27). Sex hormones affect both innate and acquired immunity throughout the body (28). In particular, female gonadotropic hormones promote signaling via STAT3, leading to T-cell–mediated fibrosis, and E2 promotes deposition of extracellular matrix proteins by human fibroblasts in human tissues (29, 30). Examples of these diseases, in which E2 promotes fibrosis, include CTD-associated ILD (31), such as SSc (32), and sarcoidosis (33). The sources of E2 include the reproductive system, but E2 is also produced locally in lung tissues that express aromatase, the enzyme converting testosterone to estrogen and estrone.

Animal models shed additional light on the role of estrogen in ILD. The most commonly used model of lung fibrosis in rodents is the bleomycin-induced lung fibrosis murine model. Bleomycin is known to increase the expression of different profibrotic factors and pathways. However, in male mice, it also increases concentrations of ERα (estrogen receptor α) while decreasing concentrations of ERβ in male mice, paralleling concentrations of these receptors in lung tissues of patients with idiopathic pulmonary fibrosis (IPF) (34). Furthermore, the key players in lung fibrosis such as IGF-I (insulin-like growth factor I) and EGF (epidermal growth factor) can increase ERα independent of E2 (34), suggesting that ligand-independent regulation of the estrogen receptors can promote fibrosis in IPF.

Sex Hormone Metabolites

The role of sex hormones and their metabolites are evident based on research in PAH. In the setting of PAH, E2 increases pulmonary artery smooth muscle cell proliferation and aromatase expression. In fact, concentrations of aromatase are elevated in pulmonary arteries of patients with PAH, with concentrations in tissues from women being higher than those derived from men (35). Men and postmenopausal women with PAH have higher circulating concentrations of E2 than matched controls, and higher E2 concentrations are associated with more severe PAH (36, 37). Genetic variants in the genes encoding cytochrome P4501B1 (converting estrogens to 2OHE [metabolites 2-hydroxyestrone] and 16α-OHE1 [16α-hydroxysterone]) or aromatase (converting DHEA [dehydroepiandrosterone] to estrogen) increases the risk of PAH penetrance in women (but not in men) who carry a BMPR2bone morphogenetic protein receptor type 2 (BMPR2) mutation (38), the major cause of heritable PAH. These studies exemplify the complexities of female hormone responses, as indicated by studies showing that some E2 and estrone metabolites can exhibit antimitogenic and antiinflammatory effects, whereas others are promitogenic and inflammatory. For example, 16α-OHE1 promotes a PAH phenotype, whereas another metabolite, 2-ME (2-methoxyestradiol), may be protective.

As noted above, higher concentrations of E2 in women promote PAH (35). In contrast, the use of ERα agonists rescues the pulmonary hypertension phenotype in rodent models via increasing the endothelial-cell and cardioprotective peptide apelin, suggesting that the Estradiol/ERα/BMPR2/apelin axis might be cardioprotective in the right ventricle and endothelial cell compartment in PAH (39). In support of protection by E2 among those with established PAH, testosterone was found to worsen right ventricle function, whereas DHEA was protective (35). The observed increased susceptibility to PAH and paradoxical improved outcomes among women with PAH due to the effects of estrogen has been referred to as the “estrogen paradox”. These findings emphasize the importance of examining the role of sex hormones in the appropriate context, tissue, and cell type and suggest that sex hormone effects may differ based on these factors.

Mitochondrial DNA

Emerging roles for extracellular vesicles in a variety of disease processes have been described. In the setting of ILD, circulating mitochondrial DNA (mtDNA) predicted worse outcomes in patients with IPF and SSc-associated ILD. Mitochondrial DNA increased concentrations of α-smooth muscle actin in lung fibroblasts, thus activating them into myofibroblasts (40). Elevated concentrations of plasma mtDNA were observed in men compared with women and were found to predict disease progression in SSc-ILD (40) and IPF (41).

Gaps and Opportunities for Research on Sex and Gender in Autoimmune Lung Diseases

On the basis of the examples provided above, a comprehensive study of sex and gender in autoimmune lung diseases is not only essential for preserving the integrity of science but will undoubtedly lead to new discoveries. We now have at our disposal exceptional tools and technological resources to address sex and gender disparities; incorporating sex and gender roles in both preclinical and clinical research study design (Figure 1) is important, especially in studies of autoimmune lung diseases.

Preclinical Research

The effects of sex and gender are quite complicated, especially in the context of diseases in which mechanisms are complex and dynamic, such as ILD and PAH. The development of more advanced disease models promises to accelerate the rate of discovery and provide for preclinical testing of novel therapeutics. This includes in vitro models representing complex, dynamic disease-specific immune cell interactions (e.g., during granuloma formation) or when insights into tissue-specific disease mechanisms are investigated in human organoids, lung on a chip, and humanized animal models (Table 1). Animal modeling of diseases should include both sexes, and the same applies to investigations of organoid models derived from human pluripotent stem cells, which are often derived from female embryos as opposed to true gender imbalance attributed to the source of embryonic cell lines (42). These studies should also focus on the contribution of chromosomes. It is appropriate to consider other confounding factors such as hormonal concentrations, early-life exposures, age, diet, and sources of bedding in study design, analysis, and interpretation. For example, the antifibrotic effects of the hormone relaxin may vary based on sex (43) and estrogen concentrations (44). Disclosing information about animal sex, striving to balance representation of males and females, and documenting estrous cycle phases and related biological effects are other key elements to consider when reporting results. Estrous cycle and variable hormonal concentrations may affect outcomes, and it is also important to recognize that the rodent estrous cycle is 4–5 days compared with 28 days in humans.

Table 1.

Summary Statements Developed by Workshop Participants to Consider in Autoimmune Lung Disease Research to Incorporate the Study of Sex and Gender

Goals	Strategies for achieving these goals
Promote sex inclusivity	• Include both sexes in animal studies • Disclose and consider estrous cycle phase • Include designation of sex in in vitro methods section
Evaluate confounding factors that may affect the hormonal milieu	• Measure concentrations of hormones, hormone metabolites • Characterize hormone receptor isoforms and activity levels • Assess early life exposures • Consider diet and sources of bedding in study design, analysis, and interpretation • Distinguish between exogenous and endogenous hormones
Develop innovative tissue-culture–based models	• Create human organoids as models of disease • Examine sex differences in therapeutic responses in human tissue-based models, such as explants, tissue punches, and precision tissue sections
Model different stages of lung diseases	• Identify both early-stage biological mediators and late-stage mediators of disease • Examine sex differences in biological mediators across the disease
Participant selection	• Recruit patients from diverse backgrounds • Utilize social media and advanced marketing techniques to reach patients who may not otherwise participate in clinical research • Ensure that study documents do not include gender-biased language
Study conception and planning	• Collaborate with disease-specific non-profit organizations to understand sex and gender differences in disease experience from the patient perspective • Invite patients of all backgrounds to participate in the development of patient-reported outcomes for autoimmune lung diseases
Data analysis	• In smaller randomized studies, consider stratification by sex or gender to ensure balanced participation • Take into account the sex-specific age incidence to maximize statistical power • Consider the risk of false-negative results in studies underpowered to examine sex differences; use data from these smaller studies to contribute to meta-analyses, generate new hypotheses, and inform sample size calculations for future studies • Prespecify tests of sex differences to minimize the risk of type I error; clearly state when tests of sex differences are performed as post hoc exploratory analyses
Reporting of results and interpretation	• Report sex and gender differences in original research manuscripts, especially clinical trials where investigational therapies may have differential effects (both efficacy and safety) for men and women • Report results of sex and gender analyses in a standardized fashion to allow for future aggregate analyses

Although animal models of disease can provide insight into disease mechanism, they have limitations, as most do not completely recapitulate human disease. Furthermore, outcomes of drug efficacy studies in rodents and mice are rarely reproducible in humans. Therefore, innovative tissue-culture–based models such as human organoids or tissues within specific organs may provide a more viable alternative that better predicts in vivo conditions. Human, sex-specific, tissue-based models offer several advantages over animal models including tractability, throughput, and cost. Such models offer promise for developing and testing host-directed therapies for human lung diseases and for evaluating sex-related impact on diverse lung diseases.

Modeling the full spectrum of autoimmune lung disease, from early disease onset to end-stage lung disease, is central to understanding the evolution of these conditions. Because males with ILD typically have a more progressive ILD phenotype regardless of etiology (e.g., RA, SSc, or IPF), with the potential exception of sarcoidosis, research is needed to understand how the pathobiology of early ILD (when the disease may be treatable) differs in males versus females. Outcomes of this research may reveal important sex-dependent therapeutic targets and eventually guide precision medicine efforts in this field.

Lastly, preclinical research studies should consider designs that 1) distinguish between exogenous and endogenous hormones, as well as exposures to environmental factors such as endocrine disruptors (e.g., phytoestrogens in animal chow or phenol red in culture media); 2) assess the concentrations and functions of the various hormonal metabolites; 3) assess the activation state of a hormone-regulated axis; 4) account for the timing and dosage of hormones; and 5) take into account the rate of metabolism of hormones and the rate of conversion via enzymes such as aromatase. Preclinical studies should also consider integration of multiomic technologies and focus on how sex exerts differential effects across multiple omics layers, including microRNAs (34). Coursework at the pre- and postdoctoral level on topics related to understanding how sex hormones affect both animal models and human disease is instrumental to learning how to properly examine sex differences in research studies while controlling for potentially confounding variables.

Clinical Research

Given that autoimmune lung diseases are relatively rare, new clinical studies may want to consider designs that ensure the effective use of small samples and subgroup analyses. Because many autoimmune diseases disproportionately affect females, progressive recruitment efforts are needed to facilitate representative enrollment of all sexes and genders in balanced proportions (Table E1 in the online supplement). These recruitment efforts could include targeting patients both within and outside of academic centers, using social media to cast a wider recruitment net and developing research networks to facilitate multicenter collaborations (Table 1).

Efforts to encourage participation of individuals who identify as transgender or gender diverse should be considered. Study recruitment flyers typically solicit female and/or male participants, marginalizing those who do not identify themselves as gender binary. Moreover, a simple change to the language of a consent form may encourage broader participation (“this study seeks to enroll patients all of races and genders, including transgender and gender-diverse individuals”).

Similarly, clinical investigators should consider the recruitment of individuals across the lifespan (e.g., pre-female or -male menopause and post-female or -male menopause), in addition to individuals from diverse racial and socioeconomic backgrounds. The aforementioned factors may affect the relationship between sex and gender and the clinical outcomes of interest. In addition, observational studies may consider environmental exposures (e.g., exogenous sex hormone exposure and nutrition) and health behaviors (e.g., meditation, exercise, sleep hygiene, and adiposity) that might alter the hormonal milieu, thereby influencing patient-reported outcomes (43, 44). In this regard, patient-reported outcome metrics, such as dyspnea scores that are historically based on disease in men, could be modified based on balanced inclusion of women.

Clinical study designs that include a plan for an analysis of how both sex and gender affect the course of the disease, biomarker predictability, treatment response, as well as safety and toxicity (45) may lead to new discovery. Standardized reporting of sex differences may also facilitate the conduct of future aggregate analyses. Stratified analysis to determine how the observed differences affect patient health, personalized care. and clinical decision-making may also be very beneficial. Sex and gender differences in pain perception and reporting are particularly important to consider given that pain plays important roles in assessment of disease activity in many autoimmune diseases. Reporting of differences in outcomes by sex and gender in original research manuscripts, even if the study was not powered to detect significant differences between groups, is critical. Moreover, the international research community promotes methods for reporting in a standardized fashion so that aggregate analyses (e.g., meta-analyses) of specific patient subgroups can be performed at later stages (46, 47).

Finally, as direct patient interactions often inspire our research endeavors, efforts to train physician scientists to recognize the unique impacts of sex and gender on autoimmune lung disease may be valuable. Longitudinal curricula on humanism and doctoring can help physicians at all stages to understand their own gender biases, and often this awareness alone can cultivate greater empathy and improved clinical reasoning skills. Specialized courses in women’s health and LGBTQ care may further motivate trainees to pursue sex- and gender-based research in their field of interest.

Conclusions and Future Directions

Participants of the sex- and gender-related autoimmune lung diseases workshop presented compelling evidence that both sex and gender uniquely affect molecular and cellular processes, disease susceptibility, clinical manifestations, and responses to therapy in patients with autoimmune ILD. The workshop participants unanimously agreed that conducting sex- and gender-informed research increases scientific rigor, facilitates new discovery, and may lead to improved patient care through the application of precision medicine. The workshop results summarized herein provide an overview of gaps and opportunities related to sex- and gender-informed preclinical and clinical research on autoimmune lung diseases, and the key role of the next generation of scientists to carry this research forward. Ultimately, illuminating fundamental differences in disease pathogenesis based on both sex and gender will accelerate our understanding of disease pathogenesis and treatment, to improve the care of patients with ILD.