A Decade of Pathogenesis Advances in Non-Type 2 Inflammatory Endotypes in Chronic Rhinosinusitis: 2012–2022

Chronic rhinosinusitis (CRS) is a complex inflammatory disease characterized by localized inflammation of the upper airway and sinuses lasting at least 12 weeks and leading to a significant decline in quality of life [1]. About 10% of the world’s population is afflicted by the disease, resulting in reduced quality of life and productivity [2]. According to the results of endoscopic and sinus computed tomography examinations, CRS is usually divided into two main phenotypes [3], namely, CRS with nasal polyps (CRSwNP) [4] and CRS without nasal polyps (CRSsNP) [5]. CRSsNP is usually attributed to the mechanical obstruction of sinus drainage channels caused by secondary inflammation and infection [6]. CRSwNP is considered to be a diffuse eosinophilic mucosal inflammatory response process [7]. In addition, other phenotypes include aspirin-exacerbated respiratory disease [8, 9], infectious CRS [10, 11], allergic fungal rhinosinusitis [12], cystic fibrosis [13, 14], etc. However, the traditional phenotype-based classification method severely underestimates the complex cellular and molecular biological mechanisms [15, 16].

Currently, the mechanisms driving different phenotypes in CRS are unknown [17]. The purpose of this review was to summarize the latest evidence on the pathogenesis of non-type 2 inflammation in CRS, extract the main points of representative literature works in the past 10 years, reorganize the main research directions on the pathogenesis of non-type 2 inflammation, and update the relevant information on the heterogeneity. The participation of microvascular or microlymphangiogenesis in inflammatory cell recruitment, tissue remodeling, and inflammatory response in non-type 2 CRS is emphasized. Their potential contributions to the pathogenesis of non-type 2 inflammation in CRS are speculated. Hopefully, the study of nasal mucosal epithelium and subepithelial tissue will form a complete closed loop. The novelty and contribution of this review are to sort out the research direction of the pathogenesis of non-type 2 inflammation in CRS in the past 10 years and guide the balance of research on the nasal epithelium and subepithelial tissue. The potential roles of immune mechanisms within microvessels and lymphatic vessels in the pathogenesis of non-type 2 inflammation in CRS are predicted. A detailed explanation of the abbreviations used in this article can be found in the online supplementary material at https://doi.org/10.1159/000532067.

Different groups have proposed the possible pathogenesis of CRS from multiple perspectives in the past 10 years. They elaborated on the possible pathogenesis of CRS from the perspectives of genetics [18‒20] and immunology [21, 22]. Most of existing studies focus on the nasal mucosal epithelium [23‒25] and ignore the influence of immune function in blood vessels and lymphatic vessels in subepithelial tissue.

Existing studies on the pathogenesis of non-type 2 inflammation in CRS need to be improved in at least the following aspects. First, the research directions are scattered. It is hoped that a clear structure can be sorted out by the review. Second, current researches focus on the epithelium of the nasal mucosa [23, 24, 26] and ignore the impact of the immune regulation mechanism of microvessels and microlymphatic vessels. Currently, studies on microvascular and microlymphangiogenesis have been matured in the fields of tumor, trauma, transplantation, and immunology, which undoubtedly provide new ideas for the exploration of the pathogenesis of non-type 2 inflammation in CRS.

Phenotypes are usually classified according to clinically observed features [6]. CRSwNP and CRSsNP appear different from each other endoscopically and have long been thought to be pathophysiologically distinct [27]. It has been reported that approximately one-third of CRS suffer from CRSwNP [28], whereas the occurrence of CRSsNP is higher than that of CRSwNP [29]. Stevens et al. [30] reported the correlation between the endotypes and the clinical phenotypes. CRS patients with mixed type 2 and type 3 endotypes often have clinical manifestations shared by both type 2 and type 3 inflammatory endotypes. It is speculated that the clinical manifestations are directly related to the endotypes of inflammation in CRS.

In order to better understand the pathogenesis of CRS [31], researchers have introduced the concept of endotypes [32, 33]. Endotypes refer to pathophysiological mechanisms that can be identified by specific biomarkers [34, 35]. Endotypes are classified based on histological features such as trends in neutrophils, eosinophils, and cytokines. Biomarkers for endotype identification in CRS can be determined directly from biological tissues of patients [36]. Commonly used biological tissues include peripheral blood, turbinate tissue, and sinus mucosa tissue [37]. The sinus mucosa is the most commonly used tissue as the site of disease occurrence [38‒40].

Currently, CRS mainly includes two endotypes, namely, type 2 and non-type 2 endotypes. Type 2 endotype usually receives more attention from researchers due to refractory nature. Non-type 2 endotype often receives less attention [41]. The complicating matter is that a certain endotype may be associated with multiple clinical phenotypes, and endotypes may contribute to the heterogeneity of phenotypes in CRS. Inflammation in CRSsNP and CRSwNP has been reported to be highly heterogeneous [42]. Each phenotype can exhibit a type 2 inflammatory endotype and a non-type 2 inflammatory endotype [43].

The concept of endotype has been paid attention by researchers after it was proposed. Tomassen et al. [44] pioneered the definition of endotype in CRS and distinguished 10 endotypes by cluster analysis of biomarkers. Bachert et al. [21] proposed to differentiate endotypes in CRS based on the pattern of inflammatory cells, specifically T cells and innate lymphoid cells (ILCs), which control major cytokines and chemokines, leading to the accumulation of eosinophils and neutrophils. Therefore, the publication of the above work opened the prelude to the study of endotypes. Liao et al. [45] used cluster analysis to identify endotypes in CRS and divided them into 7 clusters. Cluster 1 is comparable to typical eosinophilic CRSwNP (eCRSwNP). Most of them are refractory CRS with severe clinical symptoms and high recurrence rate. Clusters 3 and 6 are characterized by severe or moderate neutrophil inflammation, respectively, as well as elevated interleukin 8 (IL-8) levels and a high proportion of refractory CRS.

The 2020 European Position Paper on Rhinosinusitis and Nasal Polyps (EPOS) guidelines recommend the evaluation and treatment of primary CRS inflammatory disease in the context of type 2 or non-type 2 endotype [1]. Studies have found that for most patients, the main difference is between type 2 and non-type 2 inflammation [46]. Two major endotypes can be found in the CRSwNP and CRSsNP phenotypes [47]. The non-type 2 classification includes all other unknown factors regarding inflammation and may change over time [43]. There are also literature works that divide non-type 2 inflammatory endotypes into type 1 and type 3 endotypes [48].

The research scope of the pathophysiology of CRS includes microbial flora [7, 49], environment [1], genetic factors [20], and immune responses [50]. Research on the pathophysiology of CRS has always been a hotspot in this field. Bachert et al. [21] reported four effective methods and pointed out the direction for revealing the pathology of CRS. The first is to analyze surgically collected patient tissues, such as sinus, nasal polyp (NP) tissue, and blood samples. The second is to establish animal models. The third is to establish a model that simulates the situation in the human body, such as explant culture of sinus tissue and NPs. The fourth is to observe the therapeutic response to biological products, such as anti-IgE and anti-cytokine therapy [51]. Lam et al. [52] reported six hypotheses on the pathogenesis of CRS, namely, the fungal hypothesis [24], the superantigen hypothesis, the biofilm hypothesis, the microbiome hypothesis [53], the eicosanoid hypothesis [54], and the immune barrier hypothesis [55]. By combing literature works in the past 10 years, it can be seen that the research on the pathophysiology of CRS is mainly based on the above six hypotheses. We will review the relevant research progress on the pathogenesis of non-type 2 inflammation in CRS in the past 10 years.

Non-type 2 endotype in CRS can be subdivided into type 1 and type 3/type 17 endotypes according to inflammatory cytokines [56, 57], as shown in Figure 1 [6, 21, 42].

Type 1 inflammation is commonly seen with microorganisms [49, 58] and viruses [11, 59]. It is characterized by preferential expression of interferon-γ (IFN-γ) produced by Th1 cells, natural killer cells (NKs), and type 1 innate lymphoid cells (ILC1s) [6]. Th1 cells activate macrophages to produce IFN-γ which is a hallmark marker of type 1 inflammation. IFN-γ induces apoptosis in nasal epithelial cells and stimulates neutrophil activity [60]. Type 1 inflammation may contribute to disease chronicity and is generally more common in the CRSsNP phenotype [61, 62].

IL-6, IL-8, and tumor necrosis factor (TNF) have been shown to stimulate the production of IFN-γ, which further promotes type 1 immune responses [43]. Bequignon et al. [63] found that wound closure of injured epithelium in CRSwNP patients was significantly faster under IL-6 stimulation. It is suggested that IL-6 can induce epithelial cell proliferation and promote NP formation. Therefore, it is speculated that IL-6 may be an important cytokine in the physiopathology of NPs.

Type 1 inflammation has been reported in Asian patients or second-generation Asian CRS patients in the USA [64]. Klingler et al. [47] found that type 1 CRSsNP is characterized by the accumulation of Th1 cells, CD8+ cytotoxic T cells, and NKs. Type 1 CRSsNP is associated with viral infection and NK cell-mediated cytotoxicity. Wang et al. [65] found that type 1 endotype is the main endotype of CRSsNP in Asia. It is unclear why Asian patients are more likely to have type 1 inflammation in CRSwNP compared with patients from Western countries. It has not been determined whether genetic factors play key roles [19, 66].

Type 3 inflammation is a response to bacteria and fungi [67] that triggers epithelial cells to produce osteopontin, which stimulates DCs to induce Th17 cell differentiation. Type 3 inflammation is characterized by the production of the T helper type 3(Th3) cytokines IL-17 and IL-22 by Th17 cells and ILC3 [68]. It results in neutrophil recruitment, activation, and proliferation after activation of the type 3 immune responses [69]. On one hand, neutrophils have the innate immune ability to phagocytize microorganisms and produce antibacterial products. On the other hand, the inflammatory factors released by them can aggravate tissue damage and inflammatory response [70]. Wang et al. [71] found that elastase secreted by neutrophils is the main activator of IL-36γ. IL-36γ promotes the expression of IL-17A in neutrophils, which in turn upregulates the expression of IL-36γ in nasal epithelial cells and leads to a vicious cycle of persistent neutrophil inflammation in CRS. Kim et al. [72] found that anti-IL-33 treatment in a mouse model of CRS reduced the thickness of edematous mucosa, subepithelial deposition of collagen, and neutrophil infiltration, but not eosinophil infiltration. It is speculated that IL-33 may become a key mediator of the pathogenesis of CRSwNP by recruiting neutrophils [70]. Klingler et al. [47] found that type 3 CRSsNP was characterized by the accumulation of Th17 cells, B cells, DCs, M1 macrophages (type 1 macrophages), and neutrophils. It is speculated that type 3 CRSsNP is related to bacterial infection. Li et al. [73] found that the Th17/Treg (T regulatory) ratio and IL-6 levels were significantly elevated in both eCRSwNP and non-eCRSwNP. It is speculated that IL-6 may regulate the function of Th17, Treg cells and the Th17/Treg ratio. It plays a role in the pathogenesis of CRSwNP. In order to facilitate sorting out the key information, the trend of changes in Th1, Th2, and Th3 cytokines in CRS is summarized in Table 1 [45, 71, 73‒77].

The complicating matter is that the prevalence of non-type 2 inflammation varies with geographically, environmental, genetic factors and lifestyles [78‒80]. Both type 2 and non-type 2 (type 1 and/or type 3) endotypes are present in Asian patients [2, 81].

Studies found that type 1 and type 3 endotypes have been demonstrated in parts of Asia, Europe, and the USA. The difference is that type 1, type 3, or their mixed endotypes are common in CRSsNP in Asia [78]. Wang et al. [78] demonstrated that type 1 and mixed type 1/3 endotypes were predominant in CRSsNP patients in Beijing, China. Type 1, type 2, or type 3 marker was not elevated in patients in Chengdu, China. This suggests that there are also significant geographical differences within a country and inflammatory endotypes are more controlled by environment than by genetic factors. In general, CRSsNP is classified as type 1 inflammation based on the elevation of IFN-γ. It is a characteristic marker of type 1 inflammation [42]. However, recent studies found that elevated IFN-γ in CRSsNP could not be confirmed [65]. It is speculated that the inflammation may be transferred from type 1 to type 2. Therefore, the inflammatory endotypes in CRSsNP are highly heterogeneous, and the occurrence of each endotype varies from region to region, which undoubtedly increases the difficulty of studying non-type 2 inflammatory endotype.

Asian CRSwNP patients especially in China, Korea, and Japan are characterized by Th1 and Th17 inflammation. They may have mixed type 1 or type 3 inflammation characteristics [82]. Compared with the inflammation observed in NPs of American or European patients, the neutrophils were more common [31]. Similarly, neutrophil inflammation may also play a role in the pathogenesis of Western NPs [83, 84]. Wang et al. [65] found that type 1, type 2, and type 3 inflammation showed diversity in Europe, Asia, and Oceania. Endoypes in CRSwNP are highly heterogeneous. The occurrence of each endotype varies geographically [65]. Studies have found that there are not only type 2 immune responses in CRSwNP but also non-type 2 immune responses [36]. Jiang et al. [85] found that eosinophilic inflammation was significantly enhanced in CRSwNP patients in central China over time. It may be related to increased T helper type 2 (Th2) responses and IgE production. Hulse et al. [86] found that second-generation Asian patients born in the USA had significantly fewer eosinophils in NP tissues than African-American and Hispanic patients living in the Greater Chicago area. It is suggested that genetic differences may play key roles in the development of NPs [66]. How do the environment [80] and genetics [19] influence the development of non-type 2 inflammation worthy of further study [87].

In addition, the study found that age may be one of the factors affecting the heterogeneity of CRS [88]. The function of innate and acquired immunity changes with age, which can be confirmed by observing the distribution of age-related immune cells in peripheral blood. The deterioration of immune function with age may affect the pathophysiological process of CRS [88]. Ryu et al. [89] reported differences in age-related cytokines change in CRS subtypes and control group. In the CRSsNP and non-eCRSwNP groups, type 2 cytokines were found to increase with age, and type 3 cytokines were found to decrease with age. In the eCRSwNP group, type 2 media were found to remain at high levels, and type 3 media increase with age. It is speculated that the age-related distribution of inflammatory mediators is different in CRS endotypes. In addition, the study suggests that smoking may be one of the factors influencing the age-related increase of inflammatory mediators in eCRSwNP. Kim et al. [90] reported a significant correlation between age and tissue eosinophils. They found that the proportion of non-eCRSwNP was significantly higher in younger patients than in older patients (79.2% vs. 56.6%). An association was hypothesized between age and non-type 2 inflammation in patients with CRSwNP. Understanding age-related immune regulation in CRS will be necessary to establish precision medicine in the future.

Recently, genomics and proteomics approaches have played increasing roles in identifying immune mechanism in non-type 2 inflammatory endotype [91]. Wang et al. [65] found that NPs from non-type 2 CRSwNP patients exhibited IFN-γ-induced genes (CXCL9, CXCL10, and CXCL11), IL-17A-induced genes (CXCL6), and neutrophil chemokines (IL-8, CXCL1, and CXCL6). It is speculated that the mixed type 1 and type 3 endotypes are the most common endotypes in non-type 2 CRSwNP in Beijing. It suggests that non-type 2 CRSwNP in Asia may predominantly exhibit a mixed type 1 and type 3 endotypes with neutropenia [65, 91]. Large-scale transcriptome studies are needed to further define immune mechanisms in type 1 and type 3 CRSwNP in the future.

It should be noted that sometimes the endotype does not belong to one of the above-mentioned type 1, type 2, or type 3. Nearly 25% of CRS patients have mixed endotypes. It is difficult to divide them into certain types of endotypes [92, 93]. Since inflammatory pathways can be related in multiple ways, it is understandable that CRS has crossed endotypes. For example, eCRSwNP may also have significant levels of neutrophils and non-type 2 cytokines [71]. Kato et al. [6] found that all three endotypes were elevated in patients with CRSsNP or CRSwNP. It indicates the existence of endotype overlap. It can be classified from the perspective of distinguishing which endotype is dominant.

In addition, sometimes the test results do not show visible elevations of type 1, type 2, or type 3 endotype markers in patients with CRSsNP. It cannot be classified under the current endotype classification system [6, 30]. Possible factors leading to this phenomenon include the several aspects. First, the unrecognized endotype markers of CRS patients may be slightly elevated, but the level of significant difference cannot be reached. Second, the patients may have type 1, type 2, or type 3 inflammation, but the researchers did not sample it. Furthermore, CRS patients may have unrecognized endotypes in addition to type 1, type 2, and type 3 [6]. It is required to understand the immune mechanisms and pathogenesis of unrecognized CRS in the future.

Research on microvascular permeability and microlymphangiogenesis has been successful in the fields of allergy, infection, and oncology [94‒98], which will provide inspiration for the study of the pathogenesis of non-type 2 inflammation in CRS. Currently, most studies on the pathogenesis of non-type 2 endotype focus on nasal mucosal epithelium [23, 25] and ignore the impact of immune mechanisms in blood vessels and lymphatic vessels in the subepithelial tissue.

Under normal physiological conditions, the main function of microvessels is to maintain blood flow, transport nutrients to tissues for material exchange [99], and transport inflammatory cells to effector organs or tissues. Increased vascular permeability is often observed under inflammatory conditions and is accompanied by increased expression of inflammatory genes [100]. Inflammatory cells and cytokines have effects on vascular permeability and angiogenesis. On the contrary, the absorption and permeability of blood vessel wall have influences on the transport and penetration of inflammatory cells during the inflammatory process. According to the published literature works, research on inflammation-driven microvascular dysfunction in CRS has focused on type 2 inflammation [101‒103]. It is typically characterized by the observation of a large number of eosinophilic infiltrates. In addition, some studies did not target specific endotypes in CRS [104‒106]. Possibly, no significant changes in inflammatory cytokines were observed.

Currently, inflammation-induced vascular dysfunction and its associated genes and signaling pathways have not been identified in non-type 2 endotype in CRS. Khurana et al. [105] found that vascular biomarkers and signaling pathways were significantly overexpressed in patients with CRS. It was speculated that vascular dysfunction occurred. These pathways are usually quiescent in adults. When stimulated by inflammation or injury, the migration of inflammatory leukocytes increases which releases more inflammatory cytokines. It leads to remodeling of vascular endothelial cells, initiating new angiogenesis, and regulating blood flow and inflammatory cells from the vascular space to infiltrate into the tissue. Vascular leakiness and dysfunction occur, which allow increased exchange of macromolecules. Another study by Khurana et al. [104] found a significant increase in the number of blood vessels in CRS patients compared to the control group. Luukkainen et al. [106] found that the average density of all vessels in the NP group was significantly lower than in the control group in the area of the highest vessel density. It was previously believed that the blood vessel density in polyps may be similar to that in normal tissues [107]. A larger sample size is needed to confirm in future research.

The public literature works on microvascular dysfunction of CRS driven by non-type 2 inflammation have not been retrieved. Similar studies are mainly published in the fields of asthma [95, 108], atherosclerosis [109], diabetes [110], diabetic retinopathy (DR) [111, 112], tumor [97, 98, 113], blood-brain barrier (BBB) [114, 115], hypertension [116‒118], and so on. Cytokines have been found to play a role in inflammation-driven vascular dysfunction [113]. Increased vascular risk is associated with elevated levels of non-type 2 inflammatory cytokines, such as INF-γ, TNF-α, IL-6, and IL-8 [119]. IFN-γ is thought to play a role in antiviral response and vascular dysfunction [119]. TNF-α is an important cytokine in injured blood vessels, which can regulate the expression of VEGF, fibroblast growth factor, adhesion molecules, etc. [119]. IL-6 has been found to be involved in the pathogenesis of specific vascular diseases [119]. IL-8 is produced by monocytes, lymphocytes, fibroblasts, endothelial cells, epithelial cells, etc. and is only released under inflammatory conditions [97]. Inflammation is mostly associated with pathological angiogenesis [100]. Angiogenesis can promote inflammation and the progression of vascular diseases [120]. Therefore, angiogenesis may be a therapeutic target for the treatment of various human diseases.

Angiogenesis is the main feature of airway remodeling in asthma research, which is mainly mediated by VEGF [108]. Akdis et al. [95] found that IL-17 plays a role in the progression of inflammation in patients with asthma. IL-17 is secreted by Th17 cells and is a proinflammatory cytokine. Th17 cells upregulate VEGF production in bronchial epithelial cells [108] and promote the development of angiogenesis. In addition, Kudo et al. [94] also found that IL-17 has a similar proinflammatory effect in airway hyperresponsiveness.

Vascular inflammation plays an important role in the occurrence and progression of atherosclerosis [109]. Xu et al. [96] found that endothelial cells are activated in response to injury, and they produce IL-8, chemokines, interferons, intercellular adhesion molecule-1, vascular adhesion molecule-1, and other inflammatory factors. Proinflammatory mediators such as TNF-α stimulate endothelial cells to secrete other proinflammatory cytokines, including IL-6. They regulate chronic vascular inflammatory responses [121].

Studies have found that vascular inflammation can promote atherosclerosis and vascular endothelial dysfunction in diabetes [110], resulting in a high incidence of cardiovascular disease in diabetic patients. Based on the elevated concentrations detected, inflammatory mediators such as IL-6 and vascular adhesion molecule-1 were implicated in this process [110]. Ma et al. [110] showed that exercise has a significant benefit in relieving vascular inflammation. Proper endurance exercise plays an anti-inflammatory role by inhibiting proinflammatory cytokines.

DR is one of the common complications of microangiopathy in diabetes [112]. Symptoms of retinal ischemia, osmosis, and angiogenesis are common in patients with the disease. Tsai et al. [111] found that the expression levels of proinflammatory factors IL-1β and INF-γ were significantly increased in DR patients, while the expression level of IL-6 was not affected. Both VEGF and VEGF-A are upregulated in patients with DR. It is speculated that proinflammatory factors and angiogenic factors are related to inflammation and angiogenesis [111]. They play key roles in the development of DR. One possible reason for the absence of IL-6 upregulation in DR patients is the inhibitory effect of IL-1β on IL-6 signaling.

Inflammation in tumor angiogenesis is mainly related to tumor-associated macrophage recruitment [113]. Various inflammatory mediators secreted by tumor-associated macrophage act on tumor endothelial cells [113]. Tumor endothelial cell upregulates the production of proinflammatory cytokines and chemokines, thereby regulating the activity of inflammatory cells, recruiting them to the site of inflammation, and promoting tumor angiogenesis. These inflammatory molecules include IL-1β, IL-6, IL-8, and TNF-α [98]. Matsushima et al. [97] found that IL-8 was involved in tumor-related angiogenesis. IL-8 interacts with CXCR2 receptors on endothelial cells to induce angiogenesis. Anti-IL-8 antibody combined with anti-VEGFR may synergistically inhibit tumor-related angiogenesis.

The BBB is an important regulator of material movement between blood and nerve tissue [113]. Recruitment of proinflammatory chemokines (CCL2, CCL5, and CXCL10) and proinflammatory mediators (IL-17 and IL-22) occurs during BBB injury in most neurological diseases [114]. Proinflammatory factors (IL-1β, IL-6, and TNF-α) promote the proliferation of endothelial cells in the BBB and enhance angiogenesis during neuroinflammation [115]. Increased VEGF-A stimulates BBB leakage, secretion of proinflammatory cytokines, and infiltration of leukocytes, which lead to neuroinflammation [115].

The link between endothelial dysfunction and inflammatory cytokine production has been demonstrated [116]. IL-17A plays an important role in endothelial dysfunction associated with hypertension [118]. IL-17A enhances the proinflammatory effects of other cytokines on endothelial cells, vascular smooth muscle cells, and macrophages. IL-17A stimulation increased the expression of proinflammatory genes in cultured mouse vascular smooth muscle cells compared to control mice [118]. Overexpression of IL-17A in mice leads to increased production of reactive oxygen species, which in turn triggers vascular dysfunction and perivascular fibrosis. Tanase et al. [116] showed that chronic inflammation leads to increased vascular permeability and thrombosis associated with persistent hypertension. The serum levels of IL-1β, IL-6, IL-8, IL-17, IL-23, TGF-β, TNF-α, and other proinflammatory cytokines in hypertensive patients increase the production of reactive oxygen species, leading to vascular endothelial dysfunction. In addition, studies have found that elevated serum IL-6 levels can lead to increased production of hepatitis markers such as C-reactive protein (CRP) and increase vascular permeability, apoptosis, and thrombosis [116, 117].

The findings suggest that controlling angiogenesis and VEGF-A holds promise for improving quality of life and longevity in patients with heart disease [122]. Braile et al. [122] reported the expression of VEGF-A in cardiomyocytes (CM) and the role of VEGF-A in cardiovascular diseases. VEGF can cause cell-cell contact disruption and increased permeability. CM is the source of inflammatory cytokines and angiogenic factors, and it secretes TNF-α, IFN-γ, IL-6, ANGPT1, ANGPT2, VEGF-A, etc. The results suggest that the lack of VEGF-A expression in CM may affect myocardial angiogenesis and lead to impaired cardiac function.

Inflammation-driven microvascular dysfunction plays an important role in the progression of COVID-19 [123‒125]. Chen et al. [123] showed that after the SARS-CoV-2 virus entered human cells by binding with ACE2, many critically ill patients developed cytokine storm [126] or cytokine release syndrome [127]. Serum levels of inflammatory factors such as IL-6, IL-1, IFN-γ and endothelial activating markers such as vasculopathy factor were elevated, leading to endodermatitis and microvascular thrombosis [124, 125].

Angiogenesis and inflammation are features of almost all chronic liver diseases [113]. Vascular capillaries and abnormal organization interfere with the interaction between endothelial and cells, thus activating the immune response and leading to inflammation and chronic liver disease [128]. Activated liver endothelial cells have proinflammatory effects and secrete a variety of cytokines and chemokines such as TNF-α, IL-1, IL-6, and CCL2 [129], which promote the development of inflammation. Inflammatory cells recruited in the damaged liver microenvironment secrete angiogenic factors to promote liver angiogenesis, aggravate inflammation through metabolism, and form a cycle between liver angiogenesis and inflammation [129].

In conclusion, studies on microvascular dysfunction triggered by inflammation have focused on vascular endothelial cells, while the role of the pericytes remains to be investigated. Pericytes provide functional support for endothelium, which play important roles in the functional maintenance of the blood-retinal barrier and BBB [130, 131]. Previously, pericyte loss in both the blood-retinal barrier and BBB has been reported [132]. Pericyte loss leads to increased vascular permeability and tissue damage in diabetes mellitus [133, 134]. The role of pericytes in microvascular dysfunction triggered by non-type 2 inflammation in CRS has not been reported and further studies are needed.

Lymphatic vessels are part of the immune organs and are participants in inflammation. They are responsible for transporting and reabsorbing inflammatory cells [135]. They are closely related to inflammatory response. Quantification of blood and lymphatic vessel density has previously been used as a prognostic indicator in cancer tumors, especially melanoma and head and neck cancer [136]. Recently, researchers have begun to pay attention to the relationship between CRS and lymphatic dysregulation [106]. Luukkainen et al. [106] found that the relative density of lymphatic vessels in CRSwNP was significantly lower than that in the control group, both in the area of the highest vascular density and throughout the tissue. It is speculated that low relative density of lymphatic vessels may be related to NPs. Chauhan et al. [135] showed that IL-17 promoted the growth of lymphatic vessels by inducing increased expression of VEGF-D in lymphatic angiogenesis.

As CRS is a heterogeneous disease, the sample size may have contributed to sampling bias. The existing research on blood vessel and lymphatic vessel dysfunction in CRS has great limitations at present. Further studies with a larger sample size are needed to clarify the influence of lymphatic vessel dysfunction on the pathogenesis of non-type 2 endotype in CRS.

CRS and COVID-19 are the most common causes of olfactory dysfunction [46]. However, there is little scientific information on the link between the two. Marin et al. [46] reported the impact of the COVID-19 pandemic on olfactory function in patients with CRS. They found that type 2 eosinophilic inflammation may decrease the expression of ACE2 in sinus epithelial cells. They speculate that eosinophilic inflammation in CRS may play a protective role against olfactory dysfunction caused by SARS-CoV-2 infection. In addition, they found that increased ACE2 expression was associated with the expression of type 1 inflammatory cytokine IFN-γ in non-eCRSwNP patients. They suggest that non-eCRSwNP may increase susceptibility to SARS-CoV-2 infection. Wang et al. [10] found that the expression of ACE2 was significantly increased in the nasal tissues of non-eCRSwNP patients. It was positively correlated with the expression of IFN-γ but negatively correlated with the expression of tissue-infiltrating eosinophils, IL-5 and IL-13. They found that TMPRSS2 expression was reduced in the nasal tissue of CRSwNP patients and was independent of the inflammatory endotypes of CRSwNP. Therefore, it is speculated that IFN-γ-regulated ACE2 expression is increased in nasal tissues of non-eCRSwNP patients and is positively correlated with type 1 inflammation. Xu et al. [92] found that SARS-CoV-2 utilizes ACE2 as a receptor for entry into cells and infection. In non-eCRSwNP tissues, ACE2 expression was further upregulated by IFN-γ. It is speculated that type 1 inflammation may increase susceptibility to COVID-19. However, there is currently no evidence that people with non-type 2 CRS are at higher risk of contracting severe COVID-19.

Marin et al. [137] reported that type 2 inflammation in tissues of CRSwNP patients may be related to the downregulation of ACE2/TMPRSS2 in the olfactory neuroepithelium. It may reduce the risk of further damage to the olfactory neuroepithelium and the entry of SARS-CoV-2 into the brain. Lee et al. [138] found that a prominent type 2 immune response caused by NPs may contribute to reducing SARS-CoV-2 infectiousness and COVID-19 severity compared to CRSsNP. Similar findings were reported by Jian et al. [139], who found that type 2 inflammation in the airway may have a protective effect against COVID-19 infection or its severity. It is pointed out that the expression and regulation of ACE2 receptor and TMPRSS2 protease are the key points of study.

Forster-Ruhrmann et al. [140] reported a reduction of disease in patients with severe CRSwNP treated with dupilumab during COVID-19 infection. It is speculated that continuous treatment with dupilumab may reduce local inflammation, improve nasal breathing, and ultimately control the symptoms of CRSwNP. Klimek et al. [141] reported several biologics targeting related cytokines or effector cells for the treatment of CRS during the COVID-19 pandemic. Dupilumab was shown to reduce IgE production, Th2 differentiation, and recruitment of eosinophils. Mepolizumab and reslizumab can induce apoptosis and reduce recruitment of eosinophils. Benralizumab may deplete tissue of eosinophils. Omalizumab can reduce IgE receptor expression and reduce mast cell mediator release.

At present, it is uncertain whether patients with non-type 2 CRS and targeted biologics for patients with non-type 2 CRS affect the risk of infection with SARS-CoV-2. In addition, the protective hypothesis of CRS against COVID-19 and the pathophysiological mechanism of non-type 2 CRS combined with COVID-19 remain to be further studied.

The guidelines recommend saline irrigation and intranasal corticosteroids as standard treatment and oral corticosteroids and antibiotics as adjuvant treatment [1]. When CRS patients do not respond to drug therapy, surgery is recommended [142]. However, the strategy does not take into account underlying pathophysiological mechanisms and does not respond optimally to the heterogeneity of CRS. Recently, precision medicine has received more and more attention [61, 143].

Glucocorticoids (GCs) have anti-inflammatory, immunosuppressive, and anti-allergic effects [1]. Oral and topical GCs are considered to be effective treatments for CRS and are widely used in most patients [61, 144]. It has been reported that the effective rate of GC therapy for CRS is 50%–80% [145]. Corticosteroids can effectively inhibit type 2 inflammation in CRS, including eosinophils, ILC2s, and TH2 cells. However, some patients with CRS may not respond to GCs [145]. Studies by Milara et al. [146] showed that approximately 30% of CRS patients responded poorly to GCs. Yan et al. [147], Lou et al. [143], and Wu et al. [148] showed that the effect of GC treatment depended on the concentration of eosinophils or neutrophils. Although systemic corticosteroids may relieve inflammatory symptoms, long-term use may produce adverse effects [1]. The benefits of GCs in the treatment of non-type 2 CRS need to be supported by more clinical data.

At present, microbial resistance to antibiotics is continuously increasing [145, 149]. Although oral antibiotics can alleviate acute respiratory symptoms, eliminating resistant bacteria remains a challenge [150]. There is conflicting evidence regarding the benefits of antibiotics as a form of treatment for CRS [151, 152], and recommendations for antibiotic use in CRS patients are controversial. Currently, the latest guidelines do not recommend the routine use of topical and intravenous antibiotics for the treatment of CRS [1, 151]. Actual evidence for the treatment of CRS with oral antibiotics remains scarce [145]. Carlton et al. [150] showed that topical antibiotic therapy is useful in the short term in CRS patients with refractory Staphylococcus aureus (S. aureus) infection. Although guidelines recommend against topical antibiotics, they are still used in clinical practice [150]. Miyake et al. [153] showed that local treatment has the potential to deliver higher drug concentrations to the disease site, reducing systemic side effects and antibiotic resistance. Miyake et al. [153] reviewed recent advances in topical drug therapy and compared the potential and shortcomings of topical drug therapy for CRS. Although the efficacy of local treatment varies, different local treatment strategies continue to emerge. It is speculated that local antibiotic therapy is a promising area.

Corticosteroids have a limited role in non-type 2 CRS, and treatment options often favor antibiotics and surgery [70]. In general, patients with CRSsNP are often treated with antibiotics [152]. The study found that patients with low tissue eosinophils benefited the most from antibiotic treatment, suggesting that patients with non-type 2 disease are most likely to benefit from the drugs [151, 152]. As the role of microbial infection in CRS patients is gradually explained, the rationality of antibiotic use will be reexamined [145]. Further validation of antimicrobial and anti-inflammatory effects of antibiotics on appropriate CRS patients is needed in the future to determine which patients are suitable for antibiotic treatment [145].

Sinus surgery is another effective way to treat CRS when medications fail to control symptoms [152]. Endoscopic sinus surgery for the treatment of CRS can achieve the purpose of relieving the obstruction of the nasal passage and removing inflammatory tissue [152]. Patel et al. [152] proposed three criteria for determining the rationality of surgical treatment. The first is a Lund-McKay computed tomography score of 1 or above. The second is adequate medication before surgery, including a course of antibiotics in patients with CRSsNP or a short course of oral corticosteroids in patients with CRSwNP. Third, the SNOT-22 score can reach 20 or above after drug treatment.

One of the advantages of surgery is that the diseased tissue of the patient can be collected [70] to obtain the most direct information about the disease, which can be used to predict subsequent treatment measures and treatment outcomes. The disadvantage is that the condition may be repeated after surgery. It has been reported that approximately 40% of patients with CRS develop recurrent disease 3∼5 years after surgery [70, 154]. In addition, patients who choose surgery also bear risks such as accidental injury and anesthesia [155]. Seys et al. [156] showed that multiple nasal endoscopic surgeries can reduce patients’ sense of smell. Because CRS is a heterogeneous disease, the goals and effects of surgery may vary. Sometimes, surgery and other treatment strategies can be combined. For example, in patients with type 2 inflammation at highest risk of recurrence, sinusitis incision and debridement of inflammatory tissue may be combined with high-dose topical corticosteroid irrigation [152]. Studies have found that surgery may affect the classification of endotypes [151]. Type 2 inflammatory mediators are reduced after surgery, and more than 50% of patients have endotype conversion [157].

Biotherapy is an important adjunct to the treatment of CRS [158] and is usually used in patients with refractory diseases and drug or surgical failures [151, 159]. Currently, the biologics used generally target patients with type 2 CRSwNP [158, 160]. The United States Food and Drug Administration (FDA) has approved dupilumab, omalizumab, and mepolizumab for the treatment of CRSwNP [41]. Biologic drugs mainly target downstream cytokines of type 2 inflammation, mainly targeting IgE, IL-4, IL-5, and IL-13 [152, 161]. The recruitment and survival of inflammatory cells are regulated by blocking the signaling of inflammatory mediators [160]. In phase III clinical trials, these biologics improved NP scores in patients with CRSwNP. They have positive effects on both NP size and outcome measures [41, 151].

Research on the mechanism and treatment of non-type 2 immune response lags far behind that of type 2 immune responses [162]. Currently, no biologics targeting non-type 2 factors have been used in CRS [163‒165]. The development of biologic therapies for non-type 2 inflammation seems to be more difficult than for type 2 inflammation [61]. Research on non-type 2 inflammation needs to be strengthened [61]. Similarly, type 2 asthma is well characterized, while there is a lack of adequate biomarkers and targeted treatment options for non-type 2 asthma [166, 167]. Targeting TH17 cell signaling may be an effective therapeutic option for patients with corticosteroid-insensitive asthma, including targeting TH17 cell-associated cytokines, cytokine receptors, and signaling pathways [168]. Brodalumab is a human, anti-IL-17RA antibody which may block IL-17A, IL-17F, and other IL-17 subtypes. Busse et al. [169] reported a randomized double-blind study of brodalumab targeting strategies blocking IL-17 receptor signaling in asthma treatment, which found no significant improvement in treatment. IL-6 is an important cytokine that induces TH17 cell differentiation [168]. Chen et al. [170] reported that blocking IL-6 signaling reduced TH2/TH17 cell response and improved symptoms in corticosteroid-insensitive asthmatic mice. Chu et al. [171] reported that anti-IL-6 antibodies reduced neutrophil and eosinophilic cytokines and chemokines in mice and reduced airway inflammation. Because TH17 cells are not homogenous [168], the possible consequences of blocking the TH17 cell pathway must be carefully evaluated to determine the risks. McDowell et al. [172] showed that IL-17 and IL-13 play mutually regulating roles in asthma, suggesting that targeting these pathways simultaneously may be more powerful for treatment. Choy et al. [173] showed that the expression of TH2 and TH17 induced gene signaling is mutually exclusive and reversely regulated in patients with asthma. Patients with a TH17 signature also had elevated levels of the type 2 biomarker. Therefore, they speculate that patients with type 2 asthma may shift between TH2 and Th17 molecular markers over time. Targeting both type 2 and type 3 cytokines in combination may be better than targeting one or the other alone.

The CXC chemokine 2 receptor (CXCR2) is a key mediator of neutrophil migration. Targeting CXCR2 may inhibit the progression of non-type 2 inflammatory mechanisms [174]. Nair et al. [175] and O‘Byrne et al. [176] reported that CXCR2 antagonists targeted the CXCR2 on neutrophils and blocked its activation through the chemokine IL-8. Although neutrophils were reduced in sputum and blood in patients with asthma, no clinical improvement was seen. Because the authors did not elaborate on possible factors that may have contributed to the deterioration of efficacy during the trial, the assessment of biologics may have deviated from their actual effect.

In addition to driving type 2 inflammation after release from epithelial cells, TSLP also plays a role in non-type 2 immune response [177]. TSLP blocking has been shown to be a promising approach for treating both type 2-driven and non-type 2-driven inflammation in asthma [177]. In particular, for patients with non-type 2 inflammation, treatment options are limited. Although the pathogenesis of the asthma is unknown, TSLP blocking has been shown to be effective in such patients [177].

For severe asthma patients with low type 2 inflammation, treatment targeting type 2 cytokines has no significant clinical effect [178]. It suggests that there is a gap between the means and the need to treat low type 2 inflammation and mixed type 2 and non-type 2 inflammation. Maun et al. [178] found that mast cell trypsin levels were correlated with asthma severity and that elevated trypsin in the airways of patients with severe asthma was independent of type 2 biomarker levels. For this type 2 inflammatory independent subgroup, they developed anti-trypsin antibodies as a therapeutic agent for severe asthma. The results indicate the potential role of neutralization of trypsin activity in the treatment of type 2 independent asthma.

The use of biologics in non-type 2 inflammatory diseases may interfere with COVID-19 vaccination response [179]. Anti-cytokine antibodies (e.g., anti-IL-6) may block the antiviral cellular response. Therefore, Jutel et al. [179] recommend a minimum 7-day gap between biotargeted non-type 2 immune responses and COVID-19 vaccination to determine the possible side effects of each other. Because of the differences between the effects of different biologics, it is worth considering how to select the best biologics [151].

Existing studies on the pathological mechanism of non-type 2 endotype in CRS have at least the following limitations [180]. First of all, there is a time limit. Most of the lesions have already occurred when sampling. It is difficult to determine the susceptibility genetic and environmental factors in the early stage of CRS [80]. It is impossible to obtain new information about the disease mechanism from the source. Second, how to select the most ideal anatomical site as the research object has not been specified yet. Inflammatory cytokines have been reported to be unevenly distributed throughout the sinus tissue, and cytokine levels may vary depending on the anatomical site of origin [92]. In addition, the control group used, including inferior turbinate, hook, or cribriform tissue, was also non-standardized in tissue sampling which makes comparison between studies challenging between different groups [37]. Finally, there is a lack of widely accepted animal models of CRS. Most studies have focused on examining nasal secretions, nasal mucosa, NPs, sinus secretions, sinus mucosa, and peripheral blood from human patients with CRSwNP or CRSsNP. Therefore, new perspectives are needed to understand the pathophysiology of non-type 2 endotype in CRS. Genomics and proteomics studies, which can examine the entire genetic information in an unbiased and independent manner [180], are effective ways to determine the pathogenesis of non-type 2 endotype in the future. In addition, further studies on the pathogenesis of non-type 2 endotype in CRS are needed at least in the following aspects [46, 48], as shown in Table 2 [46, 65, 78, 79, 103, 106, 181, 182].

Endotypes in CRS are mainly divided into type 2 and non-type 2 endotype. The mechanisms driving the pathogenesis of non-type 2 endotype in CRS are currently unknown. The occurrence of non-type 2 endotype in CRS varies with race, geography, environment, and lifestyle. Type 1, type 2, and type 3 and unrecognized endotypes in CRS promoted the study of CRS heterogeneity. The immune mechanisms of CRS with overlap and unrecognized endotypes remain to be further studied. In addition, as emerging aspects of the inflammatory response mechanism in CRS, lymphatic vessel density, vascular permeability, angiogenesis and their related genes and signaling pathways have not yet been identified. The impact of immune mechanisms in blood vessels and lymphatic vessels in the subepithelial tissue on CRS still needs to be confirmed in larger sample size studies. Finally, the pathophysiological mechanism of non-type 2 CRS combined with COVID-19 remains to be further studied. It is worth considering how to select the befitting biologics for CRS patients with non-type 2 endotype.

The authors have no conflicts of interest to declare.

This research was supported in part by the National Nature Science Foundation of China (81870701).

Na Cui, Xuewei Zhu, Chen Zhao, Cuida Meng, Jichao Sha, and Dongdong Zhu have contributed in writing and reviewing the final version of the manuscript.

Edited by: H.-U. Simon, Bern.