Overall Reduced Baseline Lymphocyte Subsets Closely Related to the Poor Prognosis and the Disease Severity in Patients With COVID-19 and Diabetes Mellitus

Introduction Dysregulated host immune response was common in COVID-19. In this study we aimed at the characteristics of lymphocyte subsets and its relationship with the disease progression in COVID-19 patients with or without DM. Methods The baseline peripheral lymphocyte and subsets were compared between 55 healthy cases (control group) and 95 conrmed cases with COVID-19 (COVID-19 group), and between COVID-19 patients with and without DM. Results The prevalence of DM in COVID-19 group was 20%, and severe cases had higher the prevalence of DM than non-severe cases (P=0.006), moreover signicantly poor prognosis and higher severe rate were found in those with DM relative to those without DM (P=0.001,0.003, respectively). In COVID-19 group overall and signicant reduced lymphocyte and subsets, especially B and T lymphocytes were found, especially in those with DM. Partial decreased lymphocyte subsets, age and DM closely related with the disease progression and the prognosis. These ndings provide a reference for clinicians that immunomodulatory treatment maybe disease and especially those with DM.


Methods
Objects A cross-sectional study with a sample size of 150 patients was conducted in the Public and Health Clinic Centre of Chengdu from January 16, 2020 to March 16, 2020. Among them, 95 patients with COVID-19 from the hospital isolation ward were assigned to the COVID-19 group (the source of the cases has been previously explained in the literature), [14] and 55 healthy people from medical examination clinic were assigned to the control group.The Ethics Committee of the Public and Health Clinic Centre of Chengdu approved the study and the ethical approval number was PJ-K2020-26-01. Written informed consent was waived by the Ethics Commission of the designated hospital because of emerging infectious diseases.
The clinical typing, disease diagnosis and cured criteria of COVID-19 were applied according to the seventh Trial Version of the Novel Coronavirus Pneumonia Diagnosis and Treatment Guidance. [7] The prognosis of COVID-19 inclued cured, unhealed and death. [7] The discharge criteria were as follows: normal body temperature over three days; obvious improvement of respiratory symptoms; obviously improved lung imaging; negative nucleic acid in two consecutive respiratory specimens at least 24hours interval. [7] The viral negative conversion time was the time from onset to the rst negative nucleic acid of meeting discharge criteria. [7] The DM diagnosis criteria were applied accorded to the corresponding guidelines. [18] The participants in the COVID-19 group were divided into the non-DM subgroup (patients without DM), the DM subgroup (patients with DM), respectively.
The participants in each subgroup were also divided into the non-severe subgroup (the clinical type belonged to light and common type) and the severe subgroup (the clinical type belonged to severe and critically illness type).

Collection of data
Demographic data, clinical data, lymphocyte and subsets of all 150 cases were collected, then established databases.
Researcher strictly control the accuracy, authenticity and completeness of data.

Statistical analyses
Statistical analysis was done through GraphPad Prism 8(GraphPad, CA, the USA). x±SD was expression for the measurement data, and ANOVA was used for a multi-group comparison with variance homogeneity and normal distribution data, and least signi cant difference (LSD) t test was used for further comparison between the two groups. While without variance homogeneity and normal distribution data independent sample Kruskal-Wallis H(K) test was used for a multi-group comparison, and Mann-Whitney U test was used for further comparison between the two groups. An independent-sample t-test were compared between two groups. Percentage or proportion was expression for enumeration data, and Chi-square test was used for comparison of this data. Spearman correlation analysis was used for two-factor correlation analysis, multiple stepwise regression was used for multi-factor correlation analysis. Statistically signi cant de ned as P<0.05.

Baseline conditions
Patients in the COVID-19 group were signi cantly younger than those in the control group, and those in the non-DM subgroup were younger than those in the DM subgroup (P=0.0097, 0.0022, respectively) ( Table 1). But there was no signi cant difference of age between the control group and the COVID-19 DM subgroup, of gender between the control group and the COVID-19 group, between the non-DM subgroup and the DM subgroup (all P 0.05) ( Table 1).
Patients in the non-severe non-DM subgroup was signi cantly younger than the other three subgroups (P=0.000), and similar age was found in the other three subgroups (P 0.05) ( Table 2). There was no signi cant difference of gender between four subgroups (P 0.05) ( Table 2).
The non-DM subgroup had lower HbA1c level and FPG level than the DM subgroup (all P=0.000) ( Table 3), moreover the non-severe non-DM subgroup had lower FPG level than the severe non-DM subgroup ( P=0.0336), while similar HbA1c level were found between severe subgroup and non-severe subgroup whether with or without DM (all P 0.05), and similar FPG level were found between severe DM subgroup and non-severe DM subgroup (P 0.05) ( Table 3).
The prevalence of DM and the severe rate in COVID-19 patients The prevalence of DM in the COVID-19 group was 20% (19/95), and those severe patients had higher the prevalence of DM than those non-severe patients (P=0.006), moreover the severe rate in the non-DM subgroup was lower than that in the DM subgroup (P=0.003), there were all signi cant differences.

The disease progression and prognosis of COVID-19 patients
In DM group the prognosis was worse and the severe rate was higher (P=0.001, 0.003, respectively), moreover the nonsevere DM subgroup and the severe DM subgroup all had longer the virus negative conversion time than those two non-DM subgroups, and the longest in-hospital time was found in the severe DM subgroup (P=0.000, 0.009, respectively) ( Table 4).

The relationship of lymphocyte subsets and DM with the disease progression and prognosis in COVID-19 patients
Through spearman correlation analysis, age and DM were positively correlated, while lymphocyte count level and percentage value, CD3+ count level and percentage value, CD3+CD4+ count level and percentage value, B(CD19+) count level and CD3+CD8+ count level were all negatively correlated with the disease severity (Table 5); simultaneously the factors positively correlated with the viral negative conversion time included age and DM (Table 5); furthermore the disease severity, the coronavirus negative conversion time, DM and age were positively correlated, but lymphocyte percentage value was negatively correlated with the prognosis (Table 5).
By multiple step wise regression analysis for the disease severity the indicating factors included CD3+CD4+ percentage value, lymphocyte percentage value, age and DM (Table 6); moreover for the virus negative conversion time those contained B(CD19+) percentage value and lymphocyte percentage value (Table 6); furthermore for the prognosis those consisted of the coronavirus negative conversion time, the disease severity and age (Table 6).

Discussion
In this study we found that the prevalence of DM in the COVID-19 group was 20% (19/95), and those severe patients had higher the prevalence of DM than those non-severe patients. As a comorbidities for COVID-19 patients, [8][9]20]the 20%(19/95) prevalence of DM in this cohort was consistent with one literature reported that 20%(8/41), [8]higher than other literature reported that 13% (13/99). [9] And the 36.67%(11/30) prevalence of DM in severe patients relative to 12.31%(8/65) in non-severe patients were not consistent with literature reported that no signi cant difference of DM prevalence was found between severe and non-severe patients. [13]The reasons may be that there were 8 newly diagnosed DM cases after admission in this cohort , of them 3 non-severe cases and 5 severe cases. This discovery prompt that closely monitoring plasma glucose, testing glycated hemoglobin and performing glucose tolerance test for COVID-19 patients are necessary in order to nd people with diabetes in time.
Further analysis we found that the severe rate in patients with COVID-19 and DM coexisting was lower than those Only COVID-9 and no DM, moreover DM was positively correlated with the viral negative conversion time and the disease severity, simultaneously it was an essential in uencing factor for the disease severity, these ndings was consistent with those literature reports that in patients with acute respiratory distress syndrome (SARS) and DM in 2003, the mortality rate ,the rate of check in the intensive care unit sand mechanical ventilation were 3.0~3.3 times of those no DM, [21]the intensive care units rate of patients withH1N1 in uenza and DM was 4.29 times of those no DM, [22]in 2014a high-risk factor of the Middle East Respiratory Syndrome coronavirus infection severe cases was also DM. [22] We also found that overall and signi cantly reduced lymphocyte and subsets existed in COVID-19 patients, especially those with DM. Moreover the severe rate was the highest in those with DM, the prognosis was worst, and lymphocyte and subsets especially CD4+, CD8+ T and B cells was the lowest in those severe cases with DM, these ndings was consistent with literature. [13] But in the literature there was no hierarchical analysis performed between COVID-19 patients with and without DM . [13] Further analysis we found that factors negatively correlated the disease severity were lymphocyte and subsets, negatively correlated the prognosis was lymphocyte. The indicating factors for the disease severity were lymphocyte percentage value, CD4+ percentage value, age and DM, moreover for the prognosis included the disease severity, age and the virus negative conversion time.
In this study we also found that severe patients without DM were signi cantly older than non-severe patients without DM, this was consistent with that a poor prognosis was found in elderly COVID-19 patients. [8][9]Study found that between old and young mice CD4 T cell subsets were markedly different, exhausted three cell subsets, cytotoxic, and activated regulatory T cells (a Tregs) rarely appeared in young mice, but with age that gradually accumulated. Extreme anti-in ammatory and pro-in ammatory phenotypes of cytotoxic CD4 T cells and a Tregs were most unexpected. [23] It was found that the relative frequency and total number of B cells will decrease with age. Plasma blasts, memory cell types and transitional B cells decrease in the older than 70 years group. [24] Lymphocytes and their subsets (including NK (CD56 +) cells, B (CD19 +) cells and T (CD3 +) cells) were mainly responsible for regulating host immunity. T cells played an important role in promoting or maintaining in ammation by producing in ammatory cytokines. [22,[25][26][27][28]A subtype of CD4+ effector T cells was activated Th1 cells, which triggered phagocyte-dependent in ammation and cellmediated immunity through the production of Interferon-γ (IFN-γ), interleukin 2 (IL-2) and tumor necrosis factor β (TNFβ). [22]In contrast, another subtype of CD4+ effector T cells, activated Th2 cells, modulated the antibody response by producing IL-13, IL-10, IL-9,IL-6, IL-5 and IL-4. [25]Viral infection played a major role in disease progression by inducing indirect host immune response and direct cytopathic effects. [13,26]A fast and well-coordinated innate host immune response was the rst line of defense against viral infections, but a dysregulated immune response could lead to excessive in ammation and even death. [13] Non-severe non-DM patients were younger than severe non-DM ones, and age was positively correlated with the prognosis, the viral negative conversion time and the disease severity, moreover age was an essential indicating factor for the prognosis and the disease severity, which was consistent with that the elderly had a poor prognosis. [8][9]However similar age was found between non-severe DM and severe DM patients, that is to say, age was not a factor in the disease severity for DM patients, which was inconsistent with those reports. [8][9]Type 2 diabetes mellitus (T2DM) is a systemic chronic low-grade in ammatory disease. The function of speci c T lymphocyte subsets (including T regulatory (Treg) cells) has changed, leading to the following hypothesis: partial in ammation exacerbate T2D autoimmunity. [29]T cells play an important role in promoting or maintaining insulin resistance and in ammation by inducing the production of pro-in ammatory cytokines in metabolic organs (such as pancreas, muscle, adipose tissue and liver). [28,[30][31][32] In adipose tissue the major in ammatory cells was macrophages. [ 28,[30][31][32]Proin ammatory M1 macrophages releasing proin ammatory cytokines such as IL-6, TNF-a and IL-1contribute to the local and systemic in ammation. [32]On the contrary, anti-in ammatory M2 macrophages secreting IL-10inhibit the activity of most proin ammatory cell types including M1 macrophages. [32]IL-10 by interacting with the p38/MAPK pathway suppress TNF-a. [32]Th1 cells producing TNF-a,IL-2,IFN-γpromote M1 polarization and enhance its pro-in ammatory functions. In contrast, Th2 cells skew the differentiation of macrophage towards M2 by producing anti-in ammatory IL-4, and IL-13. [28,[30][31][32] Therefore, Th1 and Th2 responses, which are closely related to M1/M2 polarization, may also have a critical role in T2DM. [ 28,[30][31][32] Previous research found that in COVID-19 patient higher expression of proin ammatory cytokines and chemokines, especially in the severe cases, the consumption of CD4+ and CD8+ T cells, and the decrease of regulatory T cells, might result in aggravated in ammatory responses, the production of cytokine storm and make damaged tissue worse. Maybe lower number of lymphocytes suggested a role for dysregulated immune responses in COVID-19 pathogenesis. [13][14]Our research discoveries suggested that the coexistence of viral infection and DM result in more dysregulated host immune responses thus worsen the already aggravated in ammatory process, more susceptible to bacterial infections, more severe organ damage and worse prognosis, simultaneously the coexistence of viral infection and DM can reduce or delay antibody production by decreasing B(CD19+) count level and percentage value, thereby delaying the removal of the virus, worsening prognosis.
In this work, we evaluated that the characteristics of lymphocyte subsets and its relationship with the disease progression in COVID-19 patients with or without DM, and found that the coronavirus disease 2019 (COVID-19) patients had overall reduced lymphocyte and subsets. Partial decreased lymphocyte subsets, age and DM as factors closely related with the disease severity, the viral negative conversion time and the prognosis. Our study demonstrated several novel information that the coexistence of viral infection and DM result in more dysregulated host immune responses thus worsen the already aggravated in ammatory process, more susceptible to bacterial infections, more severe organ damage and worse prognosis, simultaneously the coexistence of viral infection and DM can reduce or delay antibody production, thereby delaying the removal of the virus, worsen prognosis. Combination immunomodulatory therapy based on comprehensive treatment might improve prognosis of COVID-19 patients, especially those severe cases or with DM.
Our study had several limitations. It was a retrospective, single center and small sample study. Despite that, our study demonstrated several novel information that the coexistence of viral infection and DM result in more dysregulated host immune responses thus worsen the already aggravated in ammatory process, more susceptible to bacterial infections, more severe organ damage and worse prognosis, simultaneously the coexistence of viral infection and DM can reduce or delay antibody production, thereby delaying the removal of the virus, worsen prognosis.

Conclusions
The COVID-19patients had overall reduced lymphocyte and subsets and overall decreased lymphocyte subsets and DM maybe aggravated the prognosis by aggravating the disease severity and prolonging the viral negative conversion time. Combination immunomodulatory therapy based on comprehensive treatment might improve disease progression and prognosis of COVID-19 patients, especially those severe cases or with DM.

Declarations
Ethics approval and consent to participate The Ethics Committee of the Public and Health Clinic Centre of Chengdu approved the study and the ethical approval number was PJ-K2020-26-01. Written informed consent was waived by the Ethics Commission of the designated hospital because of emerging infectious diseases.

Consent for publication
All of participants understand that the information will be published without their child or ward's/their relative's (circle as appropriate) name attached, but that full anonymity cannot be guaranteed.All of participants understand that the text and any pictures or videos published in the article will be freely available on the internet and may be seen by the general public. The pictures, videosand text may also appear on other websites or in print, may be translated into other languages or used for commercial purposes.All of participants have been offered the opportunity to read the manuscript.

Availability of data and material
All data, models, or code generated or used during the study are available from the corresponding author by request: Dafeng Liu,E-mail:liudf312@126.com Tables