IgA nephropathy (IgAN) is one of the most frequent types of primary glomerulonephritis and a principal cause of end-stage renal disease worldwide. The clinical manifestations of IgAN include repeated episodes of gross or microscopic hematuria, proteinuria, hypertension, loss of glomerular filtration function, and pathological changes featuring mesangial deposition of galactose-deficient IgA1 (Gd-IgA1)-containing immune complexes. Although 30–40% of patients develop end-stage renal disease within 20–30 years of diagnosis, current therapies such as blood pressure control or non-specific immune inflammation inhibition cannot impede the malignancy of IgAN, partly due to the incomplete understanding of the complicated pathogenesis.

Apart from the attack of Gd-IgA1 on mesangial cells, infiltration and activation of immune cells also provoke progressive renal lesions of IgAN. For instance, the cytokines derived from circulating CD4⁺ T cells in IgAN patients were able to trigger over-secretion of Gd-IgA1 by B cells (Yamada et al., 2010; Ruszkowski et al., 2019). In addition, glomerular CD68⁺ cells of monocyte–macrophage lineage were recognized as marker of endocapillary hypercellularity and indicator of chronic tubulointerstitial damage in IgAN (Soares et al., 2019). More importantly, probing the immune landscape of IgAN by single-cell transcriptomics revealed significantly higher proportions of macrophages and CD8⁺ T cells in the early stage of IgAN (Zheng et al., 2020). Accordingly, evaluating the immune cell status, as well as the altered gene expression patterns associated with immune cell infiltration, is important for yielding insights into the pathogenic process of IgAN.

The Cell-type Identification By Estimating Relative Subsets Of known RNA Transcripts (CIBERSORT) deconvolution algorithm method (https://cibersort.stanford.edu/) has been successfully developed to distinguish and assess the relative fraction of 22 immune cell phenotypes from bulk tissue gene expression profiles obtained by RNA-sequencing. This deconvolution technique can provide crucial information about the variation of immune cells that arise at different disease stages throughout the onset, development, and treatment, thus allowing earlier and more accurate diagnosis of comorbidities and prediction of therapeutic response.

In the present study, we for the first time found that M2 macrophage was apparently increased in both glomerular and tubulointerstitial compartments of IgAN by bioinformatic approaches. In addition, 21 differentially expressed genes (DEGs) (1 upregulated and 20 downregulated) were identified as the commonly shared DEGs between glomerular and tubulointerstitial compartments in IgA patients. Correlation analysis found that hemoglobin subunit beta (HBB) gene was positively associated with infiltrating M2 macrophage in the glomerular compartment and resting CD4⁺ memory T cells in the tubulointerstitial compartment of IgAN patients. Most importantly, FBJ murine osteosarcoma viral oncogene homolog B (FOSB) gene deficiency was identified as the dominant factor in promoting M2 macrophage recruitment and infiltration in the glomerular compartment of IgAN, and it may be a useful biomarker and therapeutic target in diagnosis and prognosis for IgAN patients.

In the current study, we studied both glomerular and tubulointerstitial mRNA expression profiles by computational analysis and present novel observations of immune filtration involvement in the IgAN patients. Our current study revealed clear separation between the IgAN patients and healthy living controls by the distinct landscape of infiltrating immune cells, highlighting the role of infiltrating immune cells as one of the driving forces in the pathogenesis of IgAN. Specifically, our findings identified significantly increased proportions of M1 and M2 macrophages in the glomerular compartment as well as significantly high fractions of M2 macrophage and resting CD4⁺ memory T cells in the tubulointerstitial compartment from IgAN patients compared to control healthy subjects. Moreover, to explore the similarities and differences between glomerular and tubulointerstitial compartments, 21 DEGs were recognized as the commonly shared regulators between these two entities in the pathogenesis of IgAN, among which the HBB gene was positively associated with M2 macrophage infiltration in the glomerular compartment and resting CD4⁺ memory T cells infiltration in the tubulointerstitial compartment of IgAN patients. Most importantly, we confirmed the negative association of FOSB gene in the activation of M2 macrophage in the glomerular compartment of IgAN patients. Overall, our findings suggest a potentially important role of infiltrating immune cells, particularly M2 macrophages, in the pathophysiology of IgAN-associated renal damage, in which FOSB and HBB genes may provide important roles in this immune response.

The positive correlation between macrophages counts per glomerulus and the number of glomerular crescents in IgAN patients (Arima et al., 1991; Ikezumi et al., 2011; Silva et al., 2012; Xie et al., 2021) suggests an important role of the mononuclear phagocytes in the pathogenesis of mesangial hypercellularity and irreversible glomerular damage. Macrophages have been divided into classically activated M1 types that are characterized by a pro-inflammatory phenotype and alternatively activated M2 types that display regulatory functions in tissue repair and remodeling. In a study of 204 patients with IgAN, the presence of CD68⁺ (a marker of pan-macrophage or M1 type) macrophages was positively correlated with the following: Serum creatinine at the time of biopsy, proteinuria, progression of renal disease, and a worse disease outcome (Myllymaki et al., 2007; Soares et al., 2019). In line with this previous evidence, our current study demonstrates an obvious higher proportion of M1 macrophage in the glomerular compartment, strengthening the key role of M1 macrophage in the development of IgAN.

In the meantime, we observed increased M2 macrophage fractions in both the compartments of glomerular and tubulointerstitial, although there is still controversy regarding the long-term influence of M2 macrophage in the pathogenesis of IgAN. CD163⁺ (a marker of M2 type) M2 macrophages play a central role in hemoglobin clearance and limit oxidative heme toxicity, which are significant prognostic factors for an incomplete recovery of renal function in IgAN patients with macro-hematuria-associated acute kidney injury, warfarin coagulopathy, and paroxysmal nocturnal hemoglobinuria (Martin Cleary et al., 2010; Ballarin et al., 2011; Gutierrez et al., 2012). Despite the renoprotective role, emerging evidence suggests a distinct role of M2 macrophage to promote the development of IgAN. For example, the CD163⁺ M2 macrophages were found to be higher in both glomerular and tubulointerstitial compartments of kidney tissue from new-onset IgAN patients or IgAN patients with crescents (Ikezumi et al., 2010; Ikezumi et al., 2011; Silva et al., 2012; Han et al., 2013; Kim et al., 2015), and the glomerular M2 macrophage counts were positively correlated with percentage of crescents and fibrotic lesion (Ikezumi et al., 2011; Li et al., 2015; Zhao et al., 2015). Taken together, our observations, in accordance with previous evidence, precipitate the conclusion that M2 macrophages are positioned as potential effectors of glomerular and tubulointerstitial injury in IgAN and may be potential target for therapeutic intervention.

CD4⁺ T cells play critical in orchestrating B-cell responses (Yamada et al., 2010), and cytokines produced by subpopulations of CD4⁺ T cells can dramatically impact B-cell responses in IgAN pathogenesis and correlate with its clinical severity (Ruszkowski et al., 2019). Importantly, resting CD4⁺ memory T cells have been shown to be superior helpers for precipitating B-cell antibody production in comparison with naïve CD4⁺ T cells, and this is most readily derived from the capability of memory cells to more quickly attain a helper-competent activation state than naive cells (McKinstry et al., 2010). Our study agrees with these previous findings to show the apparently increased proportion of resting CD4⁺ memory T cells in the tubulointerstitial compartment of IgAN. Therefore, we presume that the unique functional qualities of resting CD4⁺ memory T cells have the strength to elicit a secondary response in diverse approaches via its continuum.

Further efforts are urgently needed in numerous areas if the clinically feasible imbalance of mononuclear phagocytes and T-cell subpopulations are highlighted as promising therapeutic targets and biomarkers for the treatment and monitoring of disease to succeed. Notably, the diagnostic role of mononuclear phagocytes and specific T cells have not been confirmed in studies of adequate sample size, different patient populations, and in patients with other forms of glomerulonephritis as controls (Yamada et al., 2010; Ruszkowski et al., 2019; Soares et al., 2019; Zheng et al., 2020). As a tool for risk stratification and disease monitoring, data on the association between mononuclear phagocytes, various T-cell subsets and the degree of histological damage, and the rate of decline in renal function as well as renal end points are limited. In addition, no data exist on the serial monitoring of these immune cell subpopulation changes at different phases of IgAN. Accordingly, future studies to validate their roles in risk stratification and monitoring of patients with IgAN, preferably in different stages of the disease and using several different populations of patients, are required before advancements can be made.

The KEGG enrichment analysis mainly reflect transcripts associated with some classic signaling pathways associated with immune cell activation and inflammation during the pathogenesis of IgAN, such as PPAR signaling pathway in the glomerular compartment but IL-17, tumor necrosis factor (TNF), and the Forkhead box O (FoxO) signaling pathways in the tubulointerstitial compartment. The PPAR signaling pathway has long been recognized as an important transcriptional activator in macrophage polarization toward the M2 phenotype (Chang et al., 2015), which to some extent is consistent with our current finding of significantly increased M2 macrophage in glomeruli. Similarly, IL-17, TNF, and FoxO signaling pathways are able to regulate the frequency of effector and/or memory CD4⁺ T cells by providing proliferative and survival signals either directly to the T cells or to the antigen-presenting cells with which they interact (Nembrini et al., 2009; Chen et al., 2016; Delpoux et al., 2021), which is in line with our current findings of apparently elevated resting CD4⁺ memory T cells in tubulointerstitial.

Few studies have pinpointed the commonly shared gene-expressing changes occurring in the glomerular and tubulointerstitial compartments using a sensitive transcriptomic profiling approach. Our study for the first time revealed that 21 DEGs were shared in both glomerular and interstitial compartments, including 1 upregulated gene (HBB) and 20 downregulated genes. These commonly shared DEGs indicated that these two different entities share some general mechanisms. However, in contrast to the positive association between HBB gene and CD8⁺ T-cell activation in the glomerular compartment, a negative association between HBB gene and CD8⁺ T-cell activation was observed in the tubulointerstitial entity. This discrepancy indicates that these two entities also differ from each other in the underlying pathophysiology. Our study further found that HBB gene was positively associated with M2 macrophage activation in the glomerular compartment and CD4⁺ memory T-cell activation in the tubulointerstitial compartment. The HBB gene provides instructions for generating a protein called beta-globin that is a subunit of a larger protein called hemoglobin. Although how the HBB gene expression in nonerythroid cells affected cellular physiology remains largely undetermined, the capability of hemoglobin in maintaining oxygen homeostasis and mediating inflammatory response in some other cell types precipitates one hypothesis that the binding of soluble hemoglobin to a yet unidentified hemoglobin receptor expressed in infiltrating immune cells (Saha et al., 2014). Such a receptor could facilitate the internalization of HBB into the target immune cells to elicit a series of subsequent immune response in IgAN. The elucidation of the mechanism underlying the activity of hemoglobin in exaggerating the immune response and boosting the development of IgAN has to be elucidated yet.

FBJ murine osteosarcoma viral oncogene homolog B (FOSB) is a protein that is encoded by the FOSB gene localized on chromosome 19q13 and composed of four exons in humans. FOSB gene is a member of the Fos family of genes that functions as transcription factors by forming the transcription factor complex active protein 1 (AP-1). AP-1 acts as a regulator of cell homeostasis and mediates gene expression in response to a variety of environmental and physiological factors such as cytokines, oxidative stress, and other cell stressors. It was confirmed in various experimental systems that renal inflammation, fibrosis, and podocyte function are deteriorated by elevating AP-1 complex composition (Mezzano et al., 2001; Le et al., 2005), indicating a potential regulatory effect of FOSB gene in the development of IgAN. Indeed, several clinical studies were consistent with our current study to identify the downregulated expression of FOSB in the kidney from IgAN patients, cultured podocytes, or tubule cells exposed to polymeric IgA extracted and purified from the serum of IgAN patients (Miraji et al., 2019; Zhang et al., 2019; Liao et al., 2020; Park et al., 2020). More importantly, FOSB gene overexpression in cultured podocyte or tubule cells was able to reduce the secretion of pro-inflammatory mediators (Liao et al., 2020). Special noteworthy is that our current study revealed not only an obvious deficiency of FOSB gene in the glomerular compartment of IgAN but also a close relation between the glomerular FOSB deficiency and M2 macrophage infiltration. It thus seems reasonable to speculate that the decreased FOSB gene expression in glomeruli might be tightly involved in the development of IgAN, possibly thorough a boosting influence on M2 macrophage activation (Ranghino et al., 2017).

Altogether, abnormal expression of effector molecules and disordered activation and differentiation of immune cells synergistically stimulates local inflammation and immune response, causing renal tissue damage and pathological repair in IgAN. Aiming at all aspects of immune mechanism involvement, it is helpful to develop novel and promising clinical early-diagnosis and prognostic indicators as well as ideal specific therapeutic targets in translational medicine. Notably, in the current study, the identification of mononuclear phagocytes and specific T cells involved in the occurrence and development of IgAN highlights the imbalance of these immune cell subsets as new therapeutic targets and biomarkers for the treatment and monitoring of the disease.

To the best of our knowledge, no study has analyzed the immune cell transcriptome within the renal microenvironments of IgAN and associated molecular patterns through a combination of all the currently available datasets; the original studies derived from all the selected datasets (Reich et al., 2010; Berthier et al., 2012; Liu et al., 2017) have not performed any microarray analysis regarding the immune cell states, either. Therefore, we employed a cross-dataset integration strategy of defining the landscape of the immune cells and associated molecular patterns from human renal biopsies of IgAN. Our current study for the first time revealed that the diversity in the immune cell landscape could clearly separate IgAN patients from healthy living controls, and enhanced M2 macrophage infiltration possibly plays an important role in boosting the pathogenesis of IgAN in both glomerular and tubulointerstitial compartments. In specific, glomerular elevation of HBB gene and deficiency of FOSB gene were found to be closely associated with M2 macrophage infiltration, suggesting the potential role of these two DEGs in keeping the differentiation toward a M2-skewed polarization phenotype, which consequently triggers the immune response and the renal damage of IgAN. These findings may be useful to develop M2 macrophage-targeted therapy for IgAN treatment in the future.