Decreased toll-like receptor 4 and CD11b/CD18 expression on peripheral monocytes of hypertensive patients correlates with a lesser extent of endothelial damage: a preliminary study

Background: Low-grade chronic inflammation is recognized to contribute to the physiopathology of arterial hypertension. Therefore, this study aimed to assess the pro-inflammatory phenotype of peripheral monocytes of hypertensive patients by analyzing Toll-like receptor 4 (TLR4) and CD11b/CD18 surface expression. In the second part, the influence of phenotypic alterations of monocytes on the endothelial status reflected by circulating endothelial cells (CECs) was evaluated. Patients: The study included 60 patients with arterial hypertension, who were divided into two subgroups based on the disease severity according to the applicable criteria. The mild hypertension and resistant hypertension groups included 30 patients each. The control group consisted of 33 normotensive volunteers matched for age and sex. Results: Both in the entire group of patients and individual subgroups, reduced surface expression of TLR4 and CD11b/CD18 was found compared to normotensive volunteers. A reduced percentage of monocytes with the CD14+TLR4+ immunophenotype was correlated with a lower MFI level of CD18 and CD11b in the entire group of patients and after division only in the mild hypertension group. Reduced surface expression of TLR4 in hypertensive patients correlated with a lower number of CECs. This relationship was not observed in the resistant hypertension group; instead, an independent effect of reduced CD11b/CD18 expression on the reduction of CEC number was demonstrated. Conclusion: Our preliminary study showed for the first time that hypertension of varying severity is accompanied by phenotypic changes in monocytes, manifested by reduced surface expression of both TLR4 and CD11b/CD18. These phenotypic changes were associated with a reduced degree of endothelial injury. Our study opens a new, unexplored area of research on the protective features of peripheral monocytes in hypertension.

hypertension.The strong correlation between hypertension and popular biomarkers of inflammation observed in many clinical investigations indicates that abnormal immune system activation is involved in this disease [3][4][5].Initially, it has seemed that low-grade systemic inflammation is a consequence of hypertension, but recent reports suggest that this process can be a causative factor [6,7].Resistant hypertension is a clinically distinct subgroup defined by the failure to achieve blood pressure (BP) control on optimal dosing of at least three antihypertensive medications of different classes, including a diuretic [8].Previous studies have demonstrated that patients with resistant hypertension have higher arterial stiffness and impaired endothelial function compared with normotensive and mild-to-moderate individuals [9].Endothelial dysfunction is closely related to inflammation, accompanied by excessive adhesion of leukocytes to the vessel wall and the liberation of various endothelial damaging molecules, such as chemokines, reactive oxygen species, and metalloproteases.Endothelial damage is manifested by its vasoconstrictive, prothrombotic, and pro-inflammatory phenotype, which promotes the development of arterial hypertension [10,11].Our previous studies confirmed endothelial damage reflected by an increased number of circulating endothelial cells (CECs) in hypertension of various clinical severity [12].CECs represent mature endothelial cells detached from blood vessels due to multiple mechanisms, including apoptosis, mechanical injury, weakening of intracellular connection, and endothelial structure injury caused by cytokines/proteases [13,14].The number of CECs, therefore, reflects the condition of the endothelium and can be used to assess the extent of blood vessel injury.This work is a continuation of those previous studies in which we extended the earlier issue of endothelial injury by attempting to clarify the role of inflammatory cells in this process.
We focused on toll-like receptors (TLRs), a large family of innate immune receptors that may constitute an essential link in leukocyte-endothelial interactions [15][16][17].TLRs, broadly distributed in the immune cells, play a critical role in innate and adaptive immune responses.Among the 11 identified in humans so far, a unique role is assigned to the TLR4, whose increased expression has been shown in conditions associated with chronic low-grade inflammation, such as atherosclerosis, diabetes, and rheumatoid arthritis [18][19][20].TLR4 is crucial in activating monocytes and their adhesion to the vascular endothelium [21].Recent studies have shown that activation of TLR4 induces intracellular signaling cascades, resulting in surface expression of CD11b/CD18 integrin [21][22][23].The CD11b/CD18 integrin (also known as Mac-1) is a heterodimer of aM (CD11b) and b2 (CD18) subunits.The binding of CD11b/CD18 to intracellular adhesion molecule 1 (ICAM-1) expressed on blood vessels triggers monocyte adhesion and subsequent transmigration, which, if exaggerated, may promote endothelial injury [24,25].In addition, signaling from TLR4 activates the NK-kB pathway through several adaptor molecules and regulates cytokine expression, such as IL-6 and TNF-a [26,27].These factors facilitate the recruitment and attachment of circulating leukocytes to the vascular endothelium.
The exact role of monocytes and TLR4-dependent mechanisms described above is not fully understood in the pathogenesis of arterial hypertension.For this reason, the present study aimed to evaluate the expression of TLR4 and CD11b/CD18 on the surface of peripheral monocytes of patients with mild and resistant hypertension.In the second part, we analyzed how the observed monocyte phenotypic alterations affect endothelial status reflected by the number of CECs.

Patients
The study was performed in accordance with the principles of the Declaration of Helsinki, and the investigational protocol was approved by the Local Bioethical Committee of Poznan University of Medical Sciences (no. 163/17).The study was carried out in a group of hypertensive patients (38 men and 22 women) aged 21-73 (mean age 55.57AE 12.91) years who had been admitted to the Department of Hypertension at the University of Medical Sciences in Poznan.Written informed consent was obtained from all participants.All patients underwent laboratory, and physical examination, including BP measurements, performed after resting for at least 5 min in a seated position with back support, using a validated upper-arm BP monitor (Omron 705IT, Omron Healthcare Europe B.V. Netherlands).The arm on which the measurement was made was bent at the elbow, relaxed, and supported at the level of the heart.
On the basis of the detailed interview and a clinical examination, the patients were divided into two groups: patients with mild hypertension, including 20 men and 10 women (mean age 52.87 AE 13.55), and with resistant hypertension comprising 18 men and 12 women (mean age 58.27 AE 11.85).All patients in both groups were taking multiple antihypertensive therapy.Resistant arterial hypertension was recognized when, despite using at least three antihypertensive agents (including a diuretic) in maximum doses, it was impossible to achieve the target values of arterial BP lower than 140/90 mmHg.Patients were diagnosed with resistant hypertension after a 6-month protocol to exclude pseudoresistance (white-coat hypertension and poor medication adherence) by ambulatory BP monitoring (ABPM) and the Morisky-Green questionnaire.Mild hypertension group included patients with controlled BP using three or less antihypertensive drugs and those not yet controlled using two or less of these medications.The control group consisted of 33 normotensive blood donors of Regional Blood Centre in Poznan (25 men and 8 women), aged 27-61 (mean age: 41.87 AE 6.99), without any diagnosed diseases and not taking medications.The exclusion criteria for all study groups were as follows: secondary hypertension, myocardial infarction, and revascularization within 6 months before the study, stroke and transient ischemic attack (TIA) within 6 months before the study, congestive heart failure with grade III-IV according to New York Heart Association grading, chronic kidney disease (eGFR <30 ml/min), addiction to alcohol and psychotropic substances, smoking, active cancer, or diabetes.The demographics and clinical characteristics of the control and study participants are given in Table 1.

Sample collection
Blood samples were drawn in the early morning, from the arms of patients and healthy donors, in the recumbent

Laboratory analysis
Direct immunofluorescence using flow cytometry to assess the expression of TLR4 and CD11b/18 molecules on the surface of monocytes PBMCs were obtained from whole venous blood using Ficoll-Histopaque density gradient centrifugation, following the method described by B€ oyum [28].Briefly, 2.5 ml of blood was gently layered over 2 ml of Histopaque solution (Sigma-Aldrich, St. Louis, Missouri, USA), then centrifuged at 1300 rpm for 30 min.The white band of mononuclear cells was collected and washed three times using PBS through centrifugation at 950 rpm for 10 min.The PBMCs were then dispersed in the saline and labeled with a cocktail of directly conjugated pre-diluted mAbs provided by Becton Dickinson (Franklin Lakes, New Jersey, USA).Aliquots 1 Â 10 6 PBMC were labeled with 60 ml of APC-Cy TM 7 mouse anti-human CD45 mAb (catalog no.:557833), PerCP mouse anti-human CD14 (catalogno.:340585),PE mouse anti-human TLR4 mAb (catalog no.: 564215), APC mouse anti-human CD11b/Mac-1 mAb (catalog no.: 550019), FITC mouse anti-human CD18 mAb (catalog no: 555923).After incubation for 15 min at room temperature, antibodies-antigen connections were fixed, and residual erythrocytes were lysed with 500 ml lysing solution (Becton Dickinson, Franklin Lakes, New Jersey, USA, catalog no.:349202) for 10 min, followed by twice washing in 3 ml PBS and immediate flow cytometric analysis.Monocyte analyses were performed using forward and side scatter parameters combined with CD14-positive and CD45-positive stained cells.Single-parameter histograms were then generated to identify TLR4, CD11b, and CD18 expression on CD45 þ CD14 þ monocytes.Appropriately conjugated isotype controls were used to eliminate non-specific staining (Fig. 1).Electronic color compensation was used to exclude any overlapping emission spectra, and each directly conjugated mAb was separately analyzed to ensure that the appropriate fluorescent signals appeared in a single detector filter.When all parameters were set, 10.000 of the gated CD14 þ /CD45 þ events were acquired for analysis.The surface receptor expression was measured as the mean fluorescence intensity (MFI).
Reverse transcription and real-time qPCR for TLR4 gene expression in peripheral blood mononuclear cells Total RNA was extracted from PBMCs with Gene-MATRIX universal RNA purification kit, according to the manufacturer's instructions (EURX, Poland, catalog no.: E3598).The concentration of total RNA was detected using an ultraviolet spectrophotometer.cDNA was synthesized by random primer reverse transcription using an iScript advanced cDNA synthesis kit (Bio-Rad, Hercules, California, USA, catalog no.:1725038).The housekeeping gene TATA box binding protein (TBP) was used as an endogenous reference gene [29].Primer-BLAST was used for Budzyn et al.
detection to confirm the specificity of primer sequences.The primer sequences for the TLR-4 gene were as follows: 5'-GGGAGACACAGATGGCTGGGA-3' (forward) and 5'CAAG-GAGCATTGCCCAACAGGA-3' (reverse); the primer sequences for TBP were: 5'-GTGACCCAGCAGCATCACTGT-3' (forward) and 5'-AACCAGAAACCCTTGCGCTG -3' (reverse).Primer sequences for TLR4 and reference gene TBP were synthesized by the Institute of Biochemistry and Physics (Polish Academy of Sciences, Poland).Real-time qPCR was performed with Azure-Cielo Real-Time apparatus (Azure Biosystems, Dublin, California, US) using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, California, USA, catalog no.: 1725272).The PCR reaction was conducted with a total volume of 20 ml containing 10 ml of SsoAdvanced Universal SYBR Green Supermix., 2 ml of cDNA template, 1 ml of 10 mmol/l each forward and reverse primer, and 6 ml of RNase-free H 2 O.All qPCR was conducted at 95 o C for 30 s, then 40 cycles of 95 o C for 5 s and 60 o C for 30 s.
The specificity of the reaction was verified by melt curve analysis.Relative TLR4 mRNA concentrations were normalized to the corresponding TBP internal control and calculated using the 2 ÀDDCt method.

Cytokine assay
Serum TNF-a (assay sensitivity: 1 pg/ml) and IL-6 (assay sensitivity: 0.3 pg/ml) concentrations were determined using a commercially available PicoKine ELISA kit (Boster Bio, Pleasanton, California, USA, catalog no.: EK0525, EK0411), according to the manufacturer's instructions.Cytokine concentrations were calculated using the mean optical density of two wells and comparison with a standard curve.

Multicolor flow cytometry analysis for circulating endothelial cells
Multicolor flow cytometry analysis was performed according to the method published by Szpera-Goz ´dziewicz et al.

Statistical analysis
The statistical analysis was conducted using GraphPad Prism software 9.0 (GraphPad Software, San Diego, California, USA).The normality of quantitative variables was tested using the Kolmogorov-Smirnov or Shapiro-Wilk test.Any parameter not following the normal distribution was presented as a median and interquartile range and analyzed using the non-parametric Mann-Whitney test.Categorical data and proportions were compared using Chi-square or Fisher's exact test, as appropriate.Normally distributed, continuous variables were presented as a mean and standard deviation and analyzed using the Student's t-test.Multiple group comparisons were performed by one-way analysis of variance or the Kruskal-Wallis test.The Pearson or the Spearman correlation coefficient was used to test the strength of any association between different variables.In all cases, P value 0.05 or less was considered significant.The relationship between TLR4, CD11b, and CD18 surface expression and endothelial status was evaluated using multiple regression analysis.

Demographic and clinical characteristics of control and hypertensive groups
No difference in age was observed between the control and the mild hypertension and resistant hypertension groups (Table 1).Both groups with hypertension showed a statistically significantly higher value of BMI (Table 1).As expected, the highest office SBP and DBP were found in the resistant hypertension group compared with mild hypertension and control individuals (Table 1).Blood morphology showed a statistically significant increase in the WBC count in both groups of hypertensive patients, which was the highest in the resistant hypertension individuals.In both the mild hypertension and resistant hypertension groups, a significant increase in the NEUT was observed with a simultaneous decrease in the LYMPH compared to the control group (Table 1).The NLR increased significantly in both groups of hypertensive patients, reaching the highest value in the resistant hypertension group.Other parameters, such as MONO, PLT, RBC count, and HGB concentration, did not differ statistically in the groups of patients with hypertension compared to the control (Table 1).There was also no statistical change in the value of the PLR between mild hypertension and resistant hypertension and the control individuals (Table 1).A higher percentage of patients from the resistant hypertension group used calcium channel blockers (CCBs) and beta-blockers.Thirteen resistant hypertension patients and none from the mild hypertension group were prescribed mineralocorticoid receptor antagonists (MRAs) (Table 1).
The percentage of monocytes expressing TLR4 strongly correlated with the MFI level of CD18 and CD11b in all HP and after division only in the mild hypertension group (Table 2).In addition, a significant positive correlation between TLR4 MFI and CD18 MFI was found in mild hypertensive subjects (Table 2).No correlation between TLR4 expression measured as a percentage or MFI and CD11b/CD18 was observed in resistant hypertension group (Table 2).

The association between TLR4 and CD11b/CD18 surface expression on monocytes and endothelial status reflected by circulating endothelial cells
In the first step, univariate analysis was performed to determine the association between antigens surface expression and endothelial injury reflected by CECs in hypertensive patients.In the next step, multivariate linear regression analysis was performed to determine TLR4 and CD11b/CD18 as independent factors potentially    influencing endothelial status.Univariate analysis showed that TLR4 correlated positively with the number of CECs in all HP (Table 3).After division into appropriate subgroups, the strength of this correlation increased, but only in the mild hypertension group, while it was not observed in the resistant hypertension group (Table 3).However, this relationship was not maintained in all HP and in individual subgroups in the multivariate analysis adjusted for confounders including, the age, BMI, SBP and DBP, WBC, NEUT, LYMPH, MONO, and NLR index (Table 3).Only in the resistant hypertension group, there was a positive correlation between the expression of CD11b and CD18 and the number of CECs, which was found to be independent of other potential confounders (Table 3).

DISCUSSION
Many previous studies have demonstrated that inappropriate immune system activation is involved in the pathogenesis of hypertension [3][4][5].Nonetheless, the exact mechanisms triggered by inflammatory cells to promote vasoconstriction and, thus, increase in BP are still being revealed.One assumes endothelial damage caused by activated inflammatory cells via several mechanisms, including chemotaxis, enhanced adhesion to the vessel wall, and liberation of various cytokines and reactive oxygen species [10,11,31,32].While the participation of lymphoid-derived immune cells in the pathogenesis of hypertension has been well described in the literature [33][34][35], the mechanisms by which myeloid-derived innate immune cells contribute to BP elevation remain an active area of investigation.Monocytes, whose pro-inflammatory phenotype we tried to assess by analyzing the surface expression of TLR4, were the main interest of this study.TLR4 plays a pivotal role in orchestrating inflammation, mainly by regulating the expression of an array of pro-inflammatory cytokines [26,27].Results of some studies using the animal model have demonstrated that hypertension is accompanied by increased expression of TLR4 in non-immune cells across the vascular, renal, and nervous systems [36][37][38][39][40][41].Some others have indicated that silencing or inhibiting TLR4 using specific anti-TLR4 antibodies resulted in lower BP and improved cardiac function in hypertensive rats [36,40,42,43].These findings support the involvement of TLR4 in the development and progression of multiple endorgan damage in hypertension.However, peripheral tissue inflammation results from cross-talk between the non-immune cells and the immune system.Only activated leukocytes can adhere to the vascular endothelium and infiltrate various organs, causing unfavorable morphological and functional changes.It means that knowledge of the phenotypic and behavioral alterations within inflammatory cells will provide complete insight into these interactions underlying the development of hypertension and its complications.Although TLR4 has been shown to be involved in promoting the inflammatory response, its status in inflammatory cells in hypertension is poorly understood.The number of studies involving humans is limited, and those using animal models are unreliable due to the species and tissue specificity of TLR4 [44].Our study evaluating TLR4 expression in peripheral monocytes of hypertensive patients brings new knowledge about the possible role of the TLR4mediated inflammatory response of monocytes in the complex systemic inflammatory response in hypertension.
Contrary to expectations, we have shown that both the percentage of TLR4-expressing monocytes and the surface expression of TLR4 receptors are significantly reduced in hypertensive patients in both groups.We also measured TLR4 mRNA expression in PBMCs.TLR4 gene expression remains constant regardless of disease severity and does not change compared to healthy control.Since the presence of other cell subsets and the relative frequency of each subset can significantly affect the result, a conclusion about the expression of TLR4 mRNA in monocytes alone cannot be drawn.We can only conclude that the overall TLR4 gene expression is not significantly perturbated in PBMCs during hypertension.The study does not answer whether the observed decrease in TLR4 surface expression on monocytes is related to the downregulation of the TLR4 gene.Marketou et al. [45] found elevated TLR4 mRNA expression in peripheral monocytes of non-diabetic hypertensive patients.The authors' observations, however, do not exclude the reduced expression of TLR4 as a protein on the surface of monocytes, which was demonstrated in our study.It has been shown that the flow cytometry results for various TLRs are not always consistent with mRNA expression levels.Decreased levels of protein surface expression were often observed with unchanged or even increased corresponding transcript levels [46,47].It may result from a post-transcriptional regulatory mechanism, which involves downregulating the TLR4 membrane receptor through internalization or shedding [48][49][50][51].It remains to be clarified whether transcriptional or post-transcriptional mechanisms within monocytes are responsible for the observed changes in TLR4 protein level on the cell surface.
In our study, for the first time, the monocyte expression of CD11b/CD18 was assessed in parallel with the surface expression of TLR4 in hypertensive patients.This approach is justified by reports indicating TLR4-dependent synthesis of CD11b/CD18 [21][22][23].Lee et al. [21] demonstrated that activation of TLR4 resulted in increased expression of Mac-1 on monocytes in a concentration and time-dependent manner.Moreover, monocyte adhesion to HUVEC was significantly inhibited by pretreatment with anti-TLR4 antibody [21].It is therefore suggested that TLR4 signaling in monocytes may be a major factor contributing to the initiation of monocyte recruitment.In our study, we showed that in hypertension, the percentage of monocytes presenting CD11b and CD18 separately does not change, but the density of these molecules on their surface decreases significantly.As we were particularly interested in the relationship between TLR4 and CD11b/CD18 described in the literature, we also examined the percentage of monocytes co-expressing TLR4 and CD11b/CD18.The TLR4þCD11bþ and TLR4þCD18þ monocyte subsets decreased significantly regardless of the severity of hypertension.The relationship between the receptor and integrin was most evident in mild hypertension, where an increasing percentage of TLR4-presenting monocytes correlated with higher surface expression of both CD11b and CD18.These observations may indicate that TLR4-positive monocytes represent this subset with an increased amount of CD11b/CD18 on the surface.Therefore, if this subset of monocytes decreases, the overall expression of CD11b and CD18, as measured by MFI, also decreases, as we observed in hypertensive patients.The correlation between TLR4 levels and CD18 expression also observed in mild hypertension patients further demonstrates the interplay between these two processes.Although the expression pattern of TLR4, CD11b, and CD18 was similar in resistant hypertension, none of the above molecule relationships were reported.This finding may indicate that in this clinical form of hypertension, the dynamics and direction of changes in the expression of TLR4 and CD11b/CD18 are analogous but completely independent of each other.
The decrease in TLR4 expression and the accompanying depletion of CD11b/CD18 on the monocytes did not confirm a pro-inflammatory phenotype of these cells.This result was unexpected, so we tried to find its explanation based on the existing literature reports.The phenomenon of reduced expression of TLR4 was first described as a mechanism responsible for bacterial lipopolysaccharides (LPS) tolerance in monocytes previously challenged with LPS [52][53][54].LPSinduced internalization of TLR4 after immune stimulation acted as an essential immune-homeostatic response, preventing excessive activation of the inflammatory response and septic complications [54,55].Depletion in TLR4 receptors desensitizes cells to stimuli and effectively inhibits the proinflammatory signaling pathway.Further studies have shown that this negative control of TLR4 surface expression not only appears in microbial infections but may also be a universal mechanism triggered in response to increased inflammation of various etiologies.Reduced expression of TLR4 on peripheral monocytes was observed in autoimmunology diseases, acute pancreatitis, or chronic kidney diseases [56][57][58][59][60]. Krejsek et al. [61] observed TLR4 downregulation on monocytes of patients undergoing coronary artery bypass grafting, an invasive procedure associated with activating an inflammatory response.The concept of the crucial role of TLR4 in suppressing inflammation is supported by recent studies that have shown that TLR4 deficiency in monocytes favors their differentiation into an anti-inflammatory M2-like phenotype [62].
Inflammation is an indisputable fact in hypertension, which we also observed in our study as changes in leukocyte count, especially the neutrophil population, and a decrease in NLR value.However, an overactive immune system strives to regain homeostasis, hence the pro-inflammatory activity of some immune cells may be balanced by the anti-inflammatory behavior of others.Such a relationship has been discovered and described between neutrophils and monocytes, as the former induced an anti-inflammatory phenotype of monocytes [63][64][65][66].The reduced expression of TLR4 on the surface of monocytes observed in our study may therefore be a systemic mechanism to counteract exaggerated inflammation and an attempt to restore immune homeostasis.As a result, this process may lead to a decrease in the synthesis and secretion of pro-inflammatory cytokines by circulating monocytes.
In our studies, we assessed serum concentration of IL-6 and TNF-alpha, which are known to be partly under the TLR4 signaling control [67,68].Despite a slight upward trend, their level was comparable to that found in healthy volunteers.The results related to the concentration of interleukins in hypertensive patients are contradictory.Some authors show an increase in their level [69,70], others, like us, do not observe any changes [71][72][73].Dziez ˙a-Grudnik et al. [71] evaluated a panel of inflammatory markers including TNFalpha and IL-6 in patients with newly diagnosed hypertension and found no significant changes in any of them.They explained that it may be the result of insufficiently intense inflammation at this stage of disease development [71].Similarly, Vazquez-Oliva et al. [73] found no difference in IL-6 levels between normotensive and newly diagnosed hypertensive patients; however, after irbesartan treatment, IL-6 levels decreased significantly.This result of the authors indicates that the treatment process may have a significant impact on the level of inflammatory markers.
Therefore, when considering the reasons for the reduced expression of TLR4 and CD11b/CD18 on monocytes as well as unchanged cytokine levels, observed in our studies, the anti-inflammatory properties of antihypertensive drugs should also be taken into account.Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) are common drugs used to treat high BP.Both inhibit the action of angiotensin II (Ang II), a molecule that binds to its receptors expressed in a variety of organ systems thought to play key roles in BP homeostasis, including the heart, kidney, blood vessels, and adrenal glands [74].Apart from its classic role in the regulation of circulatory homeostasis, Ang II also exerts immunomodulatory effects.Accumulating evidence demonstrated that Ang II exhibits pro-inflammatory responses partly via upregulation of TLR4 expression and/or by stimulating TLR4dependent signaling pathways in various cell types.Ang IIinduced upregulation of TLR4 promotes functional consequences such as an increase in NF-kB activation and chemokine/cytokine expression during hypertension [75].A similar stimulating effect of angiotensin has been demonstrated for CD11b as a component of Mac-1 integrin [76,77].Therefore, it can be assumed that ACEIs and ARBs used in antihypertensive treatment in both analyzed groups of patients could reduce the monocyte expression of TLR4 and CD11b/CD18.This assumption may be confirmed by the results obtained in animal models, during which such an effect was observed after the use of various angiotensin receptor blockers [75].b-blockers were also included in the treatment regimen for both resistant hypertension and mild hypertension patients.They regulate BP via several mechanisms, including decreased renin and reduced cardiac output [78].In addition, b-blockers, by reducing the release of pro-inflammatory cytokines via inhibiting NF-kB signaling, are considered potential anti-inflammatory agents [79].Some authors indicate decreased surface expression of TLR4 on peripheral monocytes in hypertensive patients prescribed beta-blockers [80].Others have revealed that a treatment regimen similar to that used in our study, including combinations of ACEIs, ARBs, calcium antagonists, and diuretics, reduces TLR4 gene expression in monocytes [45].
The lack of differences in the TLR4 expression and other analyzed pro-inflammatory molecules between patients with resistant hypertension and mild hypertension was also surprising.There are very few comparisons in the literature between the different forms and clinical grades of hypertension, further complicating the explanation.In one study, authors showed that patients with non-controlled hypertension exhibit higher TLR4 monocyte expression than well-controlled [80].However, it is difficult to relate the results of our research to those presented by the authors.First, the authors do not precisely define the group with uncontrolled hypertension.It is known that uncontrolled hypertension is not synonymous with resistant hypertension [81].The former includes patients who lack BP control secondary to poor adherence and/or an inadequate treatment regimen, as well as those with true treatment resistance [82].In our study, we recruited patients only with true resistant hypertension based on the applicable clinical criteria.In addition, in the mild hypertension group in our study, there were few patients in the initial period of treatment with still uncontrolled hypertension.First of all, these differences may have contributed to the inconsistency of the results in these two studies.Finally, we figured out several reasons for the lack of differences in inflammatory molecules between the two analyzed clinical forms of hypertension.First, an inflammatory state may be similar in both clinical forms of hypertension, which is confirmed by other authors who did not show differences in some inflammatory mediators between resistant hypertension and mild hypertension groups [72,83,84].Second, it may be the effect of the applied antihypertensive treatment, which in patients with resistant hypertension is associated with the use of a wider spectrum of drugs in the maximum dose.In addition, patients with resistant hypertension were taking MRAs, which, like the previously described drugs, have anti-inflammatory effects [85].Taking high doses of several anti-inflammatory medications can equate an inflammatory state to the level seen in milder forms of hypertension.The last explanation assumes that the group with resistant hypertension could include patients with pseudoresistant hypertension despite the application of the recommended clinical criteria.Identifying true resistant hypertension remains challenging and many patients defined as resistant simply do not follow recommended lifestyle guidelines and/or do not take some (or all) of the prescribed medications [86].Chromatographic detection of antihypertensive drugs in the serum of patients seems to be the optimal method to screen for non-adherence to hypertension therapy [86].However, currently, this method is not routinely used and has not been implemented in the diagnosis of resistant hypertension in our study either.
The last aspect we analyzed concerned the influence of the observed phenotypic changes of monocytes on the endothelium status reflected by the CECs.In a previous study, we demonstrated that the number of CECs is significantly elevated in both clinical types of hypertension [12].This work showed that in mild hypertension patients, the observed decreased surface expression of TLR4 on monocytes is correlated with a reduced number of CECs and thus a better endothelial status.However, this association did not remain independent after adjusting for some confounding factors.This observation may indicate that the involvement of monocytes and TLR4-dependent signaling pathways in the process of vascular injury may be enhanced or suppressed by other factors, such as the patient's age, obesity, or other inflammatory cells.Interestingly, in resistant hypertension, such a relationship has not been demonstrated at all.This finding may indicate the lesser importance of TLR4 and TLR4-dependent mechanisms in vascular damage in this clinical form of hypertension.In contrast to mild hypertension in the resistant hypertension group, decreased expression of CD11b and CD18 was independently correlated with endothelial improvement.This observation proves that reduced monocyte adhesion, which is known to be largely regulated by CD11b/CD18, may have a protective effect on the vessel wall.Taken together, we can conclude that the endothelial status in these two clinical forms depends on the expression profile of the different molecules on monocytes.This may be evidence that different inflammatory processes and factors are involved in the development of individual clinical forms of hypertension and its complications.
We are aware of the limitations of our research.The number of patients is relatively small, which requires verification of our results on a larger population in further studies.The results obtained may have been influenced by the BP-lowering drugs taken by the patients.Therefore, the www.jhypertension.comaim of further research should be to assess the expression of TLR4 and CD11b/CD18 in patients with newly diagnosed hypertension to determine whether the observed decrease is a pharmacological effect or a natural compensatory mechanism of restoring immune homeostasis.At a later stage, it is also important to determine the impact of these phenotypic features on the functional behavior of the monocytes themselves and other cellular effectors of the immune response.

CONCLUSION
Our preliminary study showed for the first time that hypertension of varying severity is accompanied by phenotypic changes in monocytes, manifested by reduced surface expression of both TLR4 and CD11b/CD18.These phenotypic changes were associated with a reduced degree of endothelial injury.Our study opens a new, unexplored area of research on the protective features of peripheral monocytes in hypertension.Such knowledge will provide a broader insight into the inflammatory mechanisms involved in the pathogenesis of hypertension, and may also point to a new strategy involving the therapeutic manipulation of monocytes.

FIGURE 2
FIGURE 2 TLR4, CD11b, and CD18 expression on CD14 þ monocytes measured as a percentage or MFI in all HP and appropriate subgroups.The percentage of CD14 þ monocytes separately presenting TLR4, CD11b, and CD18 on their surface (a,b,c).Results are shown as the median and Min-Max of cells expressing a particular antigen and analyzed using the Kruskal-Wallis test with Dunn's multiple comparisons.TLR4, CD11b, and CD 18 expression density on CD14 þ monocytes (d,e,f).Results are presented as the median and Min-Max of MFI of a particular antigen and analyzed using the Kruskal-Wallis test with Dunn's multiple comparisons.All HP, all hypertensive patients; MH, mild hypertension; RH, resistant hypertension; Ns, nonsignificant.Ã P < 0.01; ÃÃ P < 0.001; ÃÃÃ P < 0.0001.

FIGURE 4 FIGURE 5
FIGURE 4 TLR4 mRNA relative expression in PBMCs of all HP and appropriate subgroups.Results are shown as the median and Min-Max of cells expressing a particular antigen and analyzed using the Kruskal-Wallis test with Dunn's multiple comparisons.All HP, all hypertensive patients; MH, mild hypertension; RH, resistant hypertension; Ns, nonsignificant.

TABLE 1 .
Demographic and clinical characteristics of control and hypertensive groups.
to-lymphocyte ratio; NS, not statistically significant; PLR, platelet/lymphocytes ratio; PLT, blood platelets; RBC, red blood cells; RH, resistant hypertension; WBC, white blood cells.a Results are shown as median and interquartile range and analyzed using Kruskal-Wallis test with Dunn's multiple comparisons.

TABLE 2 .
The relationship between TLR4 and CD11b/CD18 in all HP and hypertensive subgroups.

TABLE 3 .
Correlation between TLR4 and CD11b/CD18 surface expression on peripheral monocytes and endothelial status reflected by CECs.Results are shown as Spearman's rank correlation coefficient.Multiple regression analysis models for significant correlations from the univariate analysis, adjusted for age, BMI, systolic blood pressure, diastolic blood pressure, WBC, NEUT, LYMPH, MONO, and NLR.